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| Curr Genet, 1998 Oct, 34(4), 280 - 6 Genetic and functional relationship between Rvsp, myosin and actin in Saccharomyces cerevisiae; Breton AM et al.; The rvs mutants display phenotypes close to those described for the actin mutants: disorganization of the actin cytoskeleton, random budding of the diploids, loss of polarity and sensitivity to salt . Mutations in the RVS genes lead to synthetic lethality with a set of mutations in the actin gene, ACT1 . This synthetic lethality is allele-specific regarding the act1 mutations, pointing to a region on the actin molecule where contacts with the myosin head have been described . The possible involvement of a myosin in a vital function fulfilled both by the Rvsp proteins and actin is strengthened further by the fact that the double mutants rvs167, myo1 and rvs167, myo2 are lethal and severely affected in growth respectively . These data support the idea that actin, myosin and Rvsp proteins are linked in a common functional pathway in yeast. Curr Genet, 1998 Oct, 34(4), 259 - 68 Involvement of the Saccharomyces cerevisiae UTH1 gene in the oxidative-stress response; Bandara PD et al.; The UTH1 gene was identified by screening a Saccharomyces cerevisiae promoter-probe gene bank for oxidative stress-responsive genes . Transcription of UTH1 was decreased by the superoxide anion and increased by hydrogen peroxide . Deletion of UTH1 did not affect the growth of grande cells, however in a rho0 background it caused retarded growth . The uth1 mutant showed increased resistance to peroxides and, in contrast, was sensitive to superoxide or the thiol oxidant diamide . Furthermore, the mutant exhibited increased survival under starvation conditions, with elevated levels of dormant cells in starved cell cultures . A multicopy plasmid containing the first half of the ORF could confer increased resistance to superoxide and increased sensitivity to peroxides/diamide/starvation on wild-type cells . The same plasmid in the uth1 background caused a highly increased mortality. Genetics, 1998 Nov, 150(3), 1019 - 35 Accumulation of mRNA coding for the ctf13p kinetochore subunit of Saccharomyces cerevisiae depends on the same factors that promote rapid decay of nonsense mRNAs; Dahlseid JN et al.; The CTF13 gene codes for a subunit of the kinetochore in Saccharomyces cerevisiae . The temperature-sensitive mutation ctf13-30, which confers reduced fidelity of chromosome transmission, is a G --> A transition causing an amino acid substitution of Lys for Glu146 . Strains carrying one chromosomal copy of ctf13-30 fail to grow at the restrictive temperature, whereas a haploid strain carrying two copies of ctf13-30 can grow . Four genes, UPF1, UPF2, UPF3, and ICK1, were represented among extragenic suppressors of ctf13-30 . The UPF genes encode proteins that promote rapid decay of pre-mRNAs and mRNAs containing a premature stop codon . Suppressor mutations in these genes restore kinetochore function by causing increased accumulation of ctf13-30 mRNA . They also cause increased accumulation of CYH2 pre-mRNA, which is a natural target of UPF-mediated decay . Mutations in ICK1 restore kinetochore function but have no effect on ctf13-30 mRNA or CYH2 pre-mRNA accumulation . Most importantly, loss of UPF1 function causes increased accumulation of wild-type CTF13 mRNA but has no effect on the mRNA half-life . We propose that UPF-mediated decay modulates the mRNA level of one or more factors involved in CTF13 mRNA expression. Genetics, 1998 Nov, 150(3), 987 - 1005 Sth1p, a Saccharomyces cerevisiae Snf2p/Swi2p homolog, is an essential ATPase in RSC and differs from Snf/Swi in its interactions with histones and chromatin-associated proteins; Du J et al.; The essential Sth1p is the protein most closely related to the conserved Snf2p/Swi2p in Saccharomyces cerevisiae . Sth1p purified from yeast has a DNA-stimulated ATPase activity required for its function in vivo . The finding that Sth1p is a component of a multiprotein complex capable of ATP-dependent remodeling of the structure of chromatin (RSC) in vitro, suggests that it provides RSC with ATP hydrolysis activity . Three sth1 temperature-sensitive mutations map to the highly conserved ATPase/helicase domain and have cell cycle and non-cell cycle phenotypes, suggesting multiple essential roles for Sth1p . The Sth1p bromodomain is required for wild-type function; deletion mutants lacking portions of this region are thermosensitive and arrest with highly elongated buds and 2C DNA content, indicating perturbation of a unique function . The pleiotropic growth defects of sth1-ts mutants imply a requirement for Sth1p in a general cellular process that affects several metabolic pathways . Significantly, an sth1-ts allele is synthetically sick or lethal with previously identified mutations in histones and chromatin assembly genes that suppress snf/swi, suggesting that RSC interacts differently with chromatin than Snf/Swi . These results provide a framework for understanding the ATP-dependent RSC function in modeling chromatin and its connection to the cell cycle. Genetics, 1998 Nov, 150(3), 977 - 86 Sir3p domains involved in the initiation of telomeric silencing in Saccharomyces cerevisiae; Park Y et al.; Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p . This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation . A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing . Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing . Curiously, the LexA-Sir3(N205) mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site . A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus . In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity . This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID . These data suggest that the CTD "active" site is under both positive and negative control mediated by multiple Sir3p domains. Genetics, 1998 Nov, 150(3), 963 - 76 Novel mutations in the RAD3 and SSL1 genes perturb genome stability by stimulating recombination between short repeats in Saccharomyces cerevisiae; Maines S et al.; Maintaining genome stability requires that recombination between repetitive sequences be avoided . Because short, repetitive sequences are the most abundant, recombination between sequences that are below a certain length are selectively restricted . Novel alleles of the RAD3 and SSL1 genes, which code for components of a basal transcription and UV-damage-repair complex in Saccharomyces cerevisiae, have been found to stimulate recombination between short, repeated sequences . In double mutants, these effects are suppressed, indicating that the RAD3 and SSL1 gene products work together in influencing genome stability . Genetic analysis indicates that this function is independent of UV-damage repair and mutation avoidance, supporting the notion that RAD3 and SSL1 together play a novel role in the maintenance of genome integrity. Yeast, 1998 Sep 30, 14(13), 1209 - 21 Transcriptional profiling on all open reading frames of Saccharomyces cerevisiae; Hauser NC et al.; Open reading frames (6116) of the budding yeast Saccharomyces cerevisiae were PCR-amplified from genomic DNA using 12,232 primers specific to the ends of the coding sequences; the success rate of amplification was 97% . PCR-products were made accessible to hybridization by being arrayed at very high density on solid support media using various robotic devices . Probes made from total RNA preparations were hybridized for the analysis of the transcriptional activity of yeast under various growth conditions and of different strains . Experimental factors that proved critical to the performance, such as different RNA isolation procedures and the assessment of hybridization results, for example, were investigated in detail . Various software tools were developed that permit convenient handling and sound analysis of the large data quantities obtained from transcriptional profiling studies . Comprehensive arrays are being distributed within the European Yeast Functional Analysis Network (EUROFAN) and beyond. Yeast, 1998 Sep 30, 14(13), 1199 - 208 Disruption of six Saccharomyces cerevisiae genes from chromosome IV and basic phenotypic analysis of deletion mutants; Lopez MC et al.; We report here the construction of six deletion mutants and the analysis of their basic phenotype . Deletion cassettes containing the KanMX4 marker module and long flanking regions homologous to the target locus were constructed for each of the six open reading-frames (ORFs YDL088c, YDL087c, YDL086w, YDL085w, YDL084w and YDL082w) located on chromosome IV . Sporulation and tetrad analysis of heterozygous deletant strains revealed that, in the FY1679 genetic background, ORFs YDL088c, YDL087c and YDL084w are essential genes for vegetative growth whereas YDL086w, YDL085w and YDL082w are non-essential . ydl088c delta and ydl084w delta haploid strains are viable in the CEN . PK2 genetic background although ydl084w delta grows at a slower rate than the wild type . Complementation tests by corresponding cognate genes confirmed that gene inactivation was responsible for these growth defects. Yeast, 1998 Sep 30, 14(13), 1189 - 97 Fluorescent probing of membrane potential in walled cells: diS-C3(3) assay in Saccharomyces cerevisiae; Gaskova D et al.; Membrane-potential-dependent accumulation of diS-C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, lambda max, caused by a readily reversible probe binding to cell constituents . Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift . As the potential-reporting parameter, the lambda max shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity . The magnitude of the potential-dependent red lambda max shift depends on relative cell-to-probe concentration ratio, a maximum shift (572-->582 nm) being found in very thick suspensions and in cell lysates . The potential therefore has to be assessed at reasonably low cell (< or = 5 x 10(6) cells/ml) and probe (10(-7)M) concentrations at which a clearly defined relationship exists between the lambda max shift and the potential-dependent accumulation of the dye in the cells . The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10-30 min because the cell wall acts as a barrier, hampering probe penetration into the cells . The barrier properties of the cell wall correlate with its thickness: cells grown in 0.2% glucose (cell wall thickness 0.175 +/- 0.015 micron, n = 30) are stained much faster and the lambda max is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0.260 +/- 0.043 micron, n = 44) . At a suitable cell and probe concentration and under standard conditions, the lambda max shift of diS-C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae. J Biol Chem, 1998 Nov 6, 273(45), 30046 - 55 Role of the putative zinc finger domain of Saccharomyces cerevisiae DNA polymerase epsilon in DNA replication and the S/M checkpoint pathway; Dua R et al.; It has been proposed that C-terminal motifs of the catalytic subunit of budding yeast polymerase (pol) epsilon (POL2) couple DNA replication to the S/M checkpoint (Navas, T . A., Zheng, Z., and Elledge, S . J . (1995) Cell 80, 29-39) . Scanning deletion analysis of the C terminus reveals that 20 amino acid residues between two putative C-terminal zinc fingers are essential for DNA replication and for an intact S/M cell cycle checkpoint . All mutations affecting the inter-zinc finger amino acids or the zinc fingers themselves are sensitive to methylmethane sulfonate and have reduced ability to induce RNR3, showing that the mutants are defective in the transcriptional response to DNA damage as well as the cell cycle response . The mutations affect the assembly of the pol epsilon holoenzyme . Two-hybrid assays show that the POL2 subunit interacts with itself, and that the replication and checkpoint mutants are specifically defective in the interaction, suggesting (but not proving) that direct or indirect dimerization may be important for the normal functions of pol epsilon . The POL2 C terminus is sufficient for interaction with DPB2, the essential and phylogenetically conserved subunit of pol epsilon, but not for interaction with DPB3 . Neither Dpb3p nor Dpb2p homodimerizes in the two-hybrid assay. J Biol Chem, 1998 Nov 6, 273(45), 30018 - 23 Complementation of the Saccharomyces cerevisiae plasma membrane H+-ATPase by a plant H+-ATPase generates a highly abundant fusicoccin binding site; Piotrowski M et al.; Accumulating evidence suggests that the H+-ATPase of the plant plasma membrane is activated by a direct, reversible interaction with 14-3-3 proteins involving the displacement of the C-terminal autoinhibitory domain of the enzyme . The fungal phytotoxin fusicoccin (FC) appears to stabilize this H+-ATPase.14-3-3 complex, thus leading to a persistent activation of the H+-ATPase in vivo . In this study we show that functional replacement of the Saccharomyces cerevisiae H+-ATPase genes by a Nicotiana plumbaginifolia H+-ATPase (pma2) results in the generation of a high affinity fusicoccin binding site that is exceptionally abundant . Acquisition of FC binding capacity is accompanied by a significant increase in the amount of plasma membrane-associated yeast 14-3-3 homologs . The existence of a (plant) PMA2.(yeast)14-3-3 complex was demonstrated using two-dimensional gel systems (native/denaturing) . After expression of PMA2 lacking most of its C-terminal region, neither H+-ATPase.14-3-3 complex formation nor FC binding activity could be observed . Furthermore, we obtained direct biochemical evidence for a minimal FC binding complex consisting of the C-terminal PMA2 domain and yeast 14-3-3 homologs . Thus we demonstrated unambiguously the relevance of this regulatory ATPase domain for 14-3-3 interaction as well as its requirement for FC binding. J Biol Chem, 1998 Nov 6, 273(45), 29915 - 22 A novel membrane-bound glutathione S-transferase functions in the stationary phase of the yeast Saccharomyces cerevisiae; Choi JH et al.; The glutathione S-transferases (GSTs) represent a significant group of detoxification enzymes that play an important role in drug resistance in all eukaryotic species . In this paper we report an identification and characterization of the two Saccharomyces cerevisiae genes, GTT1 and GTT2 (glutathione transferase 1 and 2), coding for functional GST enzymes . Despite only limited similarity with GSTs from other organisms (approximately 50%), recombinant Gtt1p and Gtt2p exhibit GST activity with 1-chloro-2, 4-dinitrobenzene as a substrate . Both Gtt1p and Gtt2p are able to form homodimers, as determined by two hybrid assay . Subcellular fractionation demonstrated that Gtt1p associates with the endoplasmic reticulum . Expression of GTT1 is induced after diauxic shift and remains high throughout the stationary phase . Strains deleted for GTT1 and/or GTT2 are viable but exhibit increased sensitivity to heat shock in stationary phase and limited ability to grow at 39 degreesC. J Bacteriol, 1998 Nov, 180(21), 5718 - 26 The highly conserved, coregulated SNO and SNZ gene families in Saccharomyces cerevisiae respond to nutrient limitation; Padilla PA et al.; SNZ1, a member of a highly conserved gene family, was first identified through studies of proteins synthesized in stationary-phase yeast cells . There are three SNZ genes in Saccharomyces cerevisiae, each of which has another highly conserved gene, named SNO (SNZ proximal open reading frame), upstream . The DNA sequences and relative positions of SNZ and SNO genes have been phylogenetically conserved . This report details studies of the expression of the SNZ-SNO gene pairs under various conditions and phenotypic analysis of snz-sno mutants . An analysis of total RNA was used to determine that adjacent SNZ-SNO gene pairs are coregulated . SNZ2/3 and SNO2/3 mRNAs are induced prior to the diauxic shift and decrease in abundance during the postdiauxic phase, when SNZ1 and SNO1 are induced . In snz2 snz3 mutants, SNZ1 mRNA is induced prior to the diauxic shift, when SNZ2/3 mRNAs are normally induced . Under nitrogen-limiting conditions, SNZ1 mRNAs accumulate in tryptophan, adenine, and uracil auxotrophs but not in prototrophic strains, indicating that induction occurs in response to the limitation of specific nutrients . Strains carrying deletions in all SNZ-SNO gene pairs are viable, but snz1 and sno1 mutants are sensitive to 6-azauracil (6-AU), an inhibitor of purine and pyrimidine biosynthetic enzymes, and methylene blue, a producer of singlet oxygen . The conservation of sequence and chromosomal position, the coregulation and pattern of expression of SNZ1 and SNO1 genes, and the sensitivity of snz1 and sno1 mutants to 6-AU support the hypothesis that the associated proteins are part of an ancient response to nutrient limitation. J Bacteriol, 1998 Nov, 180(21), 5682 - 8 The Saccharomyces cerevisiae GATA factors Dal80p and Deh1p can form homo- and heterodimeric complexes; Svetlov VV et al.; GATA family proteins Gln3p, Gat1p, Dal80p, and Deh1p mediate the regulation of nitrogen catabolite repression (NCR)-sensitive gene expression in Saccharomyces cerevisiae . Thus far, Gln3p, Dal80p, and Deh1p have been shown to bind to GATA sequences in NCR-sensitive promoters, in some cases to exactly the same GATA sequences . A minimal Gln3p binding site consists of a single GATA sequence, whereas a Dal80p binding site consists of two GATA sequences in specific orientation, 15 to 35 bp apart, suggesting that Dal80p may bind to DNA as a dimer . Additionally, both Dal80p and Deh1p are predicted to contain a leucine zipper motif near their C termini . Therefore, we tested whether they could form homo- and/or heterodimers in two-hybrid assays . We show that Dal80p-Dal80p, Dal80p-Dal80pLZ (leucine zipper), Dal80pLZ-Dal80pLZ, Dal80p-Deh1pLZ, Dal80pLZ-Deh1pLZ, and Deh1pLZ-Deh1pLZ complexes can form . Dal80p-Dal80p and Dal80pLZ-Dal80pLZ complexes yield 5- to 10-fold stronger signals than the other possible dimers . If Dal80p and Deh1p bind to DNA only after dimerization, then the difference in ability to form complexes could significantly affect their affinity for binding DNA and thus the degree of regulation exerted by each of the two factors. Biochem Biophys Res Commun, 1998 Oct 29, 251(3), 732 - 6 Shs1p: a novel member of septin that interacts with spa2p, involved in polarized growth in saccharomyces cerevisiae; Mino A et al.; The Rho family small G proteins regulate various cell functions including cytokinesis . We have shown that Bni1p, a potential target of Rho1p, interacts with Spa2p and that Spa2p is required for the localization of Bni1p at the growth sites in Saccharomyces cerevisiae . We isolated here a novel member of the septin family, implicated in cytokinesis, as a Spa2p-binding protein by the yeast two-hybrid method . We named this gene SHS1 (Seventh Homolog of Septin) . The shs1 mutant cells showed cytokinesis deficiency and Shs1p was localized at the bud neck in budded cells . The Spa2p-Shs1p interactions may play an important role in cytokinesis . Mol Gen Genet, 1998 Sep, 259(4), 404 - 13 Isolation of an extragenic suppressor of the rna1-1 mutation in Saccharomyces cerevisiae; Hong SJ et al.; The small GTPase Ran is essential for nucleocytoplasmic transport of macromolecules . In the yeast Saccharomyces cerevisiae, Rna1p functions as a Ran-GTPase activating protein (RanGAP1) . Strains carrying the rna1-1 mutation exhibit defects in nuclear transport and, as a consequence, accumulate precursor tRNAs . We have isolated two recessive suppressors of the rna1-1 mutation . Further characterization of one of the suppressor mutations, srn10-1, reveals that the mutation (i) can not bypass the need for Rna1p function and (ii) suppresses the accumulation of unspliced pre-tRNA caused by rna1-1 . The SRN10 gene is not essential for cell viability and encodes an acidic protein (pI = 5.27) of 24.8 kDa . Srn10p is located in the cytoplasm, as determined by indirect immunofluorescence microscopy . Two-hybrid analysis reveals that there is a physical interaction between Srn10p and Rna1p in vivo . Our results identify a protein that interacts with the yeast RanGAP1. Mol Gen Genet, 1998 Sep, 259(5), 541 - 8 Involvement of histidine permease (Hip1p) in manganese transport in Saccharomyces cerevisiae; Farcasanu IC et al.; In a search for components involved in Mn2+ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn2+ transport . The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine . The mutation, designated hip1-272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF . This mutation led to Mn2+ resistance, whereas the corresponding null mutation did not . Both hip1-272 cells and the null mutant exhibited low tolerance to divalent cations such as Co2+, Ni2+, Zn2+, and Cu2+ . The Mn2+ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine . The cellular Mn2+ content of the hip1-272 mutant was lower than that of wild type or null mutant, due to increased rates of Mn2+ efflux . We propose that Hiplp is involved in Mn2+ transport, carrying out a function related to Mn2+ export. Genomics, 1998 Oct 1, 53(1), 90 - 6 Characterization of a human homologue of the Saccharomyces cerevisiae transcription factor spt3 (SUPT3H); Yu J et al.; Spt3 is a Saccharomyces cerevisiae transcription factor that is required in vivo for the transcription of a number of RNA polymerase II-transcribed genes . We report the cloning of the gene encoding the human homologue of Spt3, SUPT3H, and its initial functional analysis . The human and yeast Spt3 homologues share an overall identity of 30% that defines three conserved regions, suggesting possible functional domains . To determine whether SUPT3H is a true functional Spt3 homologue, we tested for complementation of an spt3Delta mutation in yeast . While expression of the full-length SUPT3H is unable to complement an spt3Delta mutation, expression of a human-yeast chimeric gene that contains 42% human sequences can partially complement an spt3Delta mutation . These data suggest that the transcriptional control carried out by Spt3 has been conserved from yeast to human . J Cell Biol, 1998 Oct 19, 143(2), 375 - 89 Identification of Kel1p, a kelch domain-containing protein involved in cell fusion and morphology in Saccharomyces cerevisiae; Philips J et al.; We showed previously that protein kinase C, which is required to maintain cell integrity, negatively regulates cell fusion (Philips, J., and I . Herskowitz . 1997 . J . Cell Biol . 138:961-974) . To identify additional genes involved in cell fusion, we looked for genes whose overexpression relieved the defect caused by activated alleles of Pkc1p . This strategy led to the identification of a novel gene, KEL1, which encodes a protein composed of two domains, one containing six kelch repeats, a motif initially described in the Drosophila protein Kelch (Xue, F., and L . Cooley . 1993 . Cell . 72:681- 693), and another domain predicted to form coiled coils . Overexpression of KEL1 also suppressed the defect in cell fusion of spa2Delta and fps1Delta mutants . KEL2, which corresponds to ORF YGR238c, encodes a protein highly similar to Kel1p . Its overexpression also suppressed the mating defect associated with activated Pkc1p . Mutants lacking KEL1 exhibited a moderate defect in cell fusion that was exacerbated by activated alleles of Pkc1p or loss of FUS1, FUS2, or FPS1, but not by loss of SPA2 . kel1Delta mutants form cells that are elongated and heterogeneous in shape, indicating that Kel1p is also required for proper morphology during vegetative growth . In contrast, kel2Delta mutants were not impaired in cell fusion or morphology . Both Kel1p and Kel2p localized to the site where cell fusion occurs during mating and to regions of polarized growth during vegetative growth . Coimmunoprecipitation and two-hybrid analyses indicated that Kel1p and Kel2p physically interact . We conclude that Kel1p has a role in cell morphogenesis and cell fusion and may antagonize the Pkc1p pathway. Biochim Biophys Acta, 1998 Oct 21, 1405(1-3), 147 - 54 The PLC1 encoded phospholipase C in the yeast Saccharomyces cerevisiae is essential for glucose-induced phosphatidylinositol turnover and activation of plasma membrane H+-ATPase; Coccetti P et al.; Addition of glucose to glucose-deprived cells of the yeast Saccharomyces cerevisiae triggers rapid turnover of phosphatidylinositol, phosphatidylinositol-phosphate and phosphatidylinositol 4,5-bisphosphate . Glucose stimulation of PI turnover was measured both as an increase in the specific ratio of 32P-labeling and as an increase in the level of diacylglycerol after addition of glucose . Glucose also causes rapid activation of plasma membrane H+-ATPase . We show that in a mutant lacking the PLC1 encoded phospholipase C, both processes were strongly reduced . Compound 48/80, a known inhibitor of mammalian phospholipase C, inhibits both processes . However, activation of the plasma membrane H+-ATPase is only inhibited by concentrations of compound 48/80 that strongly inhibit phospholipid turnover . Growth was inhibited by even lower concentrations . Our data suggest that in yeast cells, glucose triggers through activation of the PLC1 gene product a signaling pathway initiated by phosphatidylinositol turnover and involved in activation of the plasma membrane H+-ATPase. Yeast, 1998 Sep 15, 14(12), 1105 - 13 A highly conserved intraspecies homolog of the Saccharomyces cerevisiae elongation factor-3 encoded by the HEF3 gene; Maurice TC et al.; A paralog (intraspecies homolog) of the Saccharomyces cerevisiae YEF3 gene, encoding elongation factor-3, has been sequenced in the course of the yeast genome project, and identified by database searching; this gene has been designated HEF3 . Bioinformatic and Northern blot analysis indicate that the HEF3 gene is not expressed during vegetative growth . Deletion of the HEF3 gene reveals no growth defects, nor any defects in mating or sporulation . A high copy 2 mu clone of HEF3 was constructed, and was shown to be unable to complement a null allele of yef3 . Finally, an in vitro assay for ribosome-stimulated ATPase activity was performed with isogenic HEF3 and delta hef3 strains; no difference in biochemical activity could be detected in these strains . From these results, we conclude that the HEF3 gene does not encode a functional homolog of YEF3. Mol Cell Biol, 1998 Nov, 18(11), 6548 - 59 Poly(A) tail length control in Saccharomyces cerevisiae occurs by message-specific deadenylation; Brown CE et al.; We report that newly synthesized mRNA poly(A) tails are matured to precise lengths by the Pab1p-dependent poly(A) nuclease (PAN) of Saccharomyces cerevisiae . These results provide evidence for an initial phase of mRNA deadenylation that is required for poly(A) tail length control . In RNA 3'-end processing extracts lacking PAN, transcripts are polyadenylated to lengths exceeding 200 nucleotides . By contrast, in extracts containing PAN, transcripts were produced with the expected wild-type poly(A) tail lengths of 60 to 80 nucleotides . The role for PAN in poly(A) tail length control in vivo was confirmed by the finding that mRNAs are produced with longer poly(A) tails in PAN-deficient yeast strains . Interestingly, wild-type yeast strains were found to produce transcripts which varied in their maximal poly(A) tail length, and this message-specific length control was lost in PAN-deficient strains . Our data support a model whereby mRNAs are polyadenylated by the 3'-end processing machinery with a long tail, possibly of default length, and then in a PAN-dependent manner, the poly(A) tails are rapidly matured to a message-specific length . The ability to control the length of the poly(A) tail for newly expressed mRNAs has the potential to be an important posttranscriptional regulatory step in gene expression. Mol Cell Biol, 1998 Nov, 18(11), 6273 - 80 Snf1 protein kinase regulates phosphorylation of the Mig1 repressor in Saccharomyces cerevisiae; Treitel MA et al.; In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucose-repressed promoters in the yeast Saccharomyces cerevisiae . Previous work showed that Mig1 is differentially phosphorylated in response to glucose . Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function . Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 and reg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1 . We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo . In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction . Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1 . These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose. J Biol Chem, 1998 Oct 23, 273(43), 28019 - 24 Coiled-coil interaction of N-terminal 36 residues of cyclase-associated protein with adenylyl cyclase is sufficient for its function in Saccharomyces cerevisiae ras pathway; Nishida Y et al.; In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins . We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to confer RAS2(Val-19)-dependent heat shock sensitivity to yeast cells . The CAP-binding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus . Both of these regions contained tandem repetitions of a heptad motif alphaXXalphaXXX (where alpha represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction . When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats . Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP . These results indicate the coiled-coil mechanism as a basis of the CAP-adenylyl cyclase interaction. J Biol Chem, 1998 Oct 23, 273(43), 27945 - 52 MTO1 codes for a mitochondrial protein required for respiration in paromomycin-resistant mutants of Saccharomyces cerevisiae; Colby G et al.; Mutations in MTO1 express a respiratory defect only in the context of a mitochondrial genome with a paromomycin-resistance allele . This phenotype is similar to that described previously for mss1 mutants by Decoster, E., Vassal, A., and Faye, G . (1993) J . Mol . Biol . 232, 79-88 . We present evidence that Mto1p and Mss1p are mitochondrial proteins and that they form a heterodimer complex . In a paromomycin-resistant background, mss1 and mto1 mutants are inefficient in processing the mitochondrial COX1 transcript for subunit 1 of cytochrome oxidase . The mutants also fail to synthesize subunit 1 and show a pleiotropic absence of cytochromes a, a3, and b . In vivo pulse labeling of an mto1 mutant, however, indicate increased rates of synthesis of other mitochondrial translation products . The respiratory defective phenotype of mto1 and mss1 mutants is not seen in a paromomycin-sensitive genetic background . The visible absorption spectra of such strains indicate a higher ratio of cytochromes b/a and elevated NADH- and succinate-cytochrome c reductase activities . To explain these phenotypic characteristics, we proposed that the Mto1p.Mss1p complex plays a role in optimizing mitochondrial protein synthesis in yeast, possibly by a proofreading mechanism. FEBS Lett, 1998 Sep 25, 436(1), 35 - 40 Telomerase from Saccharomyces cerevisiae contains several protein subunits and may have different activities depending on the protein content; Petrov AV et al.; Telomerase is a ribonucleoprotein responsible for maintaining telomeres during the cell cycle {1,2} . Here we describe a two-step purification procedure for the Saccharomyces cerevisiae telomerase complex . We have found that the properties (processivity, nuclease activity) of telomerase depend on the isolation procedure . Using a cross-linking approach, we have revealed several proteins that could be components of the telomerase complex . Furthermore, spectra of cross-linked proteins differ in processive and non-processive telomerase complexes. Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12438 - 43 Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae; Miret JJ et al.; A quantitative and selective genetic assay was developed to monitor expansions of trinucleotide repeats (TNRs) in yeast . A promoter containing 25 repeats allows expression of a URA3 reporter gene and yields sensitivity to the drug 5-fluoroorotic acid . Expansion of the TNR to 30 or more repeats turns off URA3 and provides drug resistance . When integrated at either of two chromosomal loci, expansion rates were 1 x 10(-5) to 4 x 10(-5) per generation if CTG repeats were replicated on the lagging daughter strand . PCR analysis indicated that 5-28 additional repeats were present in 95% of the expanded alleles . No significant changes in CTG expansion rates occurred in strains deficient in the mismatch repair gene MSH2 or the recombination gene RAD52 . The frequent nature of CTG expansions suggests that the threshold number for this repeat is below 25 in this system . In contrast, expansions of the complementary repeat CAG occurred at 500- to 1,000-fold lower rates, similar to a randomized (C,A,G) control sequence . When the reporter plasmid was inverted within the chromosome, switching the leading and lagging strands of replication, frequent expansions were observed only when CTG repeats resided on the lagging daughter strand . Among the rare CAG expansions, the largest gain in tract size was 38 repeats . The control repeats CTA and TAG showed no detectable rate of expansions . The orientation-dependence and sequence-specificity data support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments. Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12404 - 9 The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations; Flores-Rozas H et al.; The Saccharomyces cerevisiae genome encodes four MutL homologs . Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches . We have investigated the role of MLH3 in mismatch repair . Mutations in MLH3 increased the rate of reversion of the hom3-10 allele by increasing the rate of deletion of a single T in a run of 7 Ts . Combination of mutations in MLH3 and MSH6 caused a synergistic increase in the hom3-10 reversion rate, whereas the hom3-10 reversion rate in an mlh3 msh3 double mutant was the same as in the respective single mutants . Similar results were observed when the accumulation of mutations at frameshift hot spots in the LYS2 gene was analyzed, although mutation of MLH3 did not cause the same extent of affect at every LYS2 frameshift hot spot . MLH3 interacted with MLH1 in a two-hybrid system . These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex. C R Acad Sci III, 1998 Jun, 321(6), 453 - 62 First characterization of the gene RGD1 in the yeast Saccharomyces cerevisiae; Barthe C et al.; We identified the ORF YBR260c during systematic sequencing of one region of chromosome II of Saccharomyces cerevisiae . This ORF encodes a putative protein of 666 aa, of which the C-terminal part of the deduced amino acid sequence resembles human and yeast Rho/Rac GTPase activating proteins (GAP) . An initial study is reported in the paper . This gene was expressed in haploid and diploid cells and was called RGD1 for related GAP domain 1 . Inactivation of RGD1 was carried out and phenotypic analysis of the mutant strain revealed only a slight viability defect when cells grown in minimal medium were close to stationary phase . Northern and western analyses showed that the RGD1 transcript and the corresponding protein were still abundant in cells cultivated in YNB during the stationary phase . No functional link seems to exist with the highly conserved GTPase Cdc42 involved in cytoskeletal polarization and cell polarity. RNA, 1998 Oct, 4(10), 1268 - 81 Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae; de la Cruz J et al.; Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits . Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an underaccumulation of 60S ribosomal subunits . Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs . At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, Al, and A2 were also inhibited . Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle . An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area . On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes . We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase. RNA, 1998 Oct, 4(10), 1239 - 50 Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae; Dix I et al.; We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae . Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site . Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop . Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly . The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions . Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome . This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome. Biochim Biophys Acta, 1998 Oct 2, 1394(1), 23 - 32 Incorporation of extracellular phospholipids and their effect on the growth and lipid metabolism of the Saccharomyces cerevisiae cho1/pss mutant; Yon JO et al.; The cho1/pss mutant of Saccharomyces cerevisiae, which is auxotrophic for choline or ethanolamine because of the deficiency in phosphatidylserine synthesis, grew in the presence of 0.05 mM phosphatidylcholine (PC) with octanoic acids (diC8PC) or decanoic acids (diC10PC), but not in the presence of PC with longer acyl residues . It did not grow in the presence of the soluble hydrolytic products of PC, phosphorylcholine or glycerophosphorylcholine, at comparable concentrations . Addition of 10 mM hemicholinium-3, a choline transport inhibitor, or disruption of the CTR gene, which encodes a choline transporter, inhibited the growth of the cho1/pss mutant in the presence of choline, but not in the presence of 0.1 mM diC8PC . Under diC8PC-supported growth conditions, octanoic acid was barely detectable in the cellular phospholipid fraction, but was recovered in the culture medium as the free acid, and the phosphatidylethanolamine (PE) content was low in comparison to the choline-supported conditions . These results suggest that PCs with short acyl residues were taken up by the cho1/pss mutant and remodeled as they were used, and that PCs with short acyl residues do not inhibit conversion of PE to PC . The current results provide a new direction in the analysis of intracellular phospholipid movement and metabolism in yeast. Genes Dev, 1998 Oct 1, 12(19), 3137 - 43 Identification of APN2, the Saccharomyces cerevisiae homolog of the major human AP endonuclease HAP1, and its role in the repair of abasic sites; Johnson RE et al.; Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bases . APN1 encodes the major AP-endonuclease of Saccharomyces cerevisiae . Human HAP1 (REF1) encodes the major AP endonuclease which, in addition to its role in DNA repair, functions as a redox regulatory protein . We identify APN2, the yeast homolog of HAP1 and provide evidence that Apn1 and Apn2 represent alternate pathways for repairing AP sites . The apn1Delta apn2Delta strain displays a highly elevated level of MMS-induced mutagenesis, which is dependent on the REV3, REV7, and REV1 genes . Our findings indicate that AP sites are highly cytotoxic and mutagenic in eukaryotes, and that the REV3, REV7-encoded DNA polymerase zeta mediates the mutagenic bypass of AP sites. Appl Microbiol Biotechnol, 1998 Aug, 50(2), 187 - 92 Efficient production of intact human parathyroid hormone in a Saccharomyces cerevisiae mutant deficient in yeast aspartic protease 3 (YAP3); Kang HA et al.; When human parathyroid hormone (hPTH) is expressed as a secretory product in yeast, the main problem is the aberrant proteolytic cleavage that reduces the yield of intact protein . To overcome this problem, we developed an hPTH expression system using a host strain in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted . After 48 h of culture, most of the hPTH secreted by the yap3 disruptant remained intact, whereas more than 90% of the hPTH secreted by the wild-type strain was cleaved . When the authentic hPTH was incubated in each of the culture supernatants of untransformed yap3 disruptant and wild-type strain, the proteolysis proceeded much more slowly in the culture supernatant of yap3 disruptant than in that of the wild type . The extent of hPTH proteolysis was also significantly reduced by the addition of pepstatin A, a specific aspartic protease inhibitor . The results suggest that YAP3 is involved in the internal cleavage of hPTH expressed in yeast . The correct processing of the intact hPTH secreted in the yap3 disruptant demonstrates that the yeast mutant lacking the YAP3 activity is a suitable host for the high-level expression of intact hPTH. J Cell Sci, 1998 Nov, 111 ( Pt 21), 3235 - 44 N-terminal domain of Gpa1 (G protein alpha) subunit) is sufficient for plasma membrane targeting in yeast Saccharomyces cerevisiae; Gillen KM et al.; G proteins play a central role in transmitting signals from cell surface receptors to effector proteins inside the cell . Signaling can only occur, however, if all these protein components are properly assembled and localized at the plasma membrane . Past studies have shown that certain segments within the N-terminal region of the G protein alpha subunit are necessary for membrane attachment . Here we identify a region within the yeast G alpha (Gpa1) that is sufficient for membrane attachment, as well as for specific targeting to the plasma membrane . Initially, we constructed chimeric proteins that replace the N terminus of mammalian Gsalpha with the corresponding sequence from Gpa1 . Gsalpha is inefficiently targeted to the yeast plasma membrane and therefore cannot fully complement the loss of Gpa1 . Gpa1-Gsaplha chimeras were assayed for proper membrane localization by functional complementation of a gpa1Delta ;) mutant, and by sucrose density gradient fractionation of cell membranes . Most of the chimeras tested, including one with only the N-terminal 7 amino acids from Gpa1, exhibited normal membrane targeting and complementing activity . We also fused various lengths of N-terminal Gpa1 sequence to glutathione-S-transferase (GST), a heterologous protein normally expressed in the cytoplasm . The first 67- 36- or 9-amino acids of Gpa1 were all sufficient to direct GST specifically to the plasma membrane in yeast . This analysis defines the extreme N terminus of Gpa1 as the primary determinant of proper membrane targeting, and represents an essential step towards isolating and identifying G protein-targeting proteins within the plasma membrane. Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 413 - 5 Crystallization and preliminary X-ray diffraction studies of homoserine dehydrogenase from Saccharomyces cerevisiae; DeLaBarre B et al.; Recombinant homoserine dehydrogenase from Saccharomyces cerevisiae has been crystallized in three different forms . Crystals of the apo-enzyme belong to the tetragonal space group P4 and have unit-cell-dimensions a = b = 130 and c = 240 A . The resolution limit for these crystals is 3.9 A . Crystals of homoserine dehydrogenase grown in the presence of the co-factor NAD+ have the tetragonal space group P41212 or its enantiomorph P43212 . The unit-cell dimensions for these crystals are a = b = 80.4 and c = 250.2 A, and the observed resolution limit is 2.2 A . Protein crystals grown in the presence of the product L-homoserine and the inert NAD+ analogue 3-aminopyridine adenine dinucleotide belong to the monoclinic space group P21 with unit-cell parameters a = 58.8, b = 104.2, c = 120.7 A, beta = 91.9 degrees . This last crystal form has a diffraction limit of 2.7 A resolution. Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 96 - 8 Crystallization and preliminary X-ray analysis of the 12S form of phosphofructokinase from Saccharomyces cerevisiae; Obmolova G et al.; The tetrameric 12S form of yeast phosphofructokinase, obtained by limited proteolytic cleavage of the native enzyme, was crystallized under a variety of conditions . The crystals have been characterized in the X-ray beam and are suitable for crystallographic studies. Mol Biol Cell, 1998 Oct, 9(10), 2803 - 17 A late mitotic regulatory network controlling cyclin destruction in Saccharomyces cerevisiae; Jaspersen SL et al.; Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase-cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis . Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC) . In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15, cdc5, cdc14, dbf2, and tem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins . We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis . We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, and SIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2 . When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1 . Mutant cells arrested in G1 contain normal APC activity . We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1. Mol Biol Cell, 1998 Oct, 9(10), 2767 - 84 Ste6p mutants defective in exit from the endoplasmic reticulum (ER) reveal aspects of an ER quality control pathway in Saccharomyces cerevisiae; Loayza D et al.; We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins . In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast . We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation . By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes . The single member of Class I, Ste6-166p, is highly unstable . We show that its degradation involves the ubiquitin-proteasome system, as indicated by its in vivo stabilization in certain ubiquitin-proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132 . The two Class II mutant proteins, Ste6-13p and Ste6-90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane . This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system . We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast . In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane. J Cell Biol, 1998 Oct 5, 143(1), 135 - 45 Clb5-associated kinase activity is required early in the spindle pathway for correct preanaphase nuclear positioning in Saccharomyces cerevisiae; Segal M et al.; In Saccharomyces cerevisiae, a single cyclin-dependent kinase, Cdc28, regulates both G1/S and G2/M phase transitions by associating with stage-specific cyclins . During progression through S phase and G2/M, Cdc28 is activated by the B-type cyclins Clb1-6 . Because of functional redundancy, specific roles for individual Clbs have been difficult to assign . To help genetically define such roles, strains carrying a cdc28(ts) allele, combined with single CLB deletions were studied . We assumed that by limiting the activity of the kinase, these strains would be rendered more sensitive to loss of individual Clbs . By this approach, a novel phenotype associated with CLB5 mutation was observed . Homozygous cdc28-4(ts) clb5 diploids were inviable at room temperature . Cells were defective in spindle positioning, leading to migration of undivided nuclei into the bud . Occasionally, misplaced spindles were observed in cdc28-4 clb5 haploids; additional deletion of CLB6 caused full penetrance . Thus, CLB5 effects proper preanaphase spindle positioning, yet the requirement differs in haploids and diploids . The execution point for the defect corresponded to the time of Clb5-dependent kinase activation . Nevertheless, lethality of cdc28-4 clb5 diploids was not rescued by CLB2 or CLB4 overexpression, indicating a specificity of Clb5 function beyond temporality of expression. J Cell Biol, 1998 Oct 5, 143(1), 23 - 34 Mutational analysis of the structure and localization of the nucleolus in the yeast Saccharomyces cerevisiae; Oakes M et al.; The nucleolus in Saccharomyces cerevisiae is a crescent-shaped structure that makes extensive contact with the nuclear envelope . In different chromosomal rDNA deletion mutants that we have analyzed, the nucleolus is not organized into a crescent structure, as determined by immunofluorescence microscopy, fluorescence in situ hybridization, and electron microscopy . A strain carrying a plasmid with a single rDNA repeat transcribed by RNA polymerase I (Pol I) contained a fragmented nucleolus distributed throughout the nucleus, primarily localized at the nuclear periphery . A strain carrying a plasmid with the 35S rRNA coding region fused to the GAL7 promoter and transcribed by Pol II contained a rounded nucleolus that often lacked extensive contact with the nuclear envelope . Ultrastructurally distinct domains were observed within the round nucleolus . A similar rounded nucleolar morphology was also observed in strains carrying the Pol I plasmid in combination with mutations that affect Pol I function . In a Pol I-defective mutant strain that carried copies of the GAL7-35S rDNA fusion gene integrated into the chromosomal rDNA locus, the nucleolus exhibited a round morphology, but was more closely associated with the nuclear envelope in the form of a bulge . Thus, both the organization of the rDNA genes and the type of polymerase involved in rDNA expression strongly influence the organization and localization of the nucleolus. Cell Mol Biol (Noisy-le-grand), 1998 Sep, 44(6), 919 - 25 Elevated expression of chitinase 1 and chitin synthesis in myosin II-deficient Saccharomyces cerevisiae; Rodriguez-Medina JR et al.; To determine if the attached cells formed in Myosin II-deficient Saccharomyces cerevisiae result from deficient chitinase 1 (CTS1) expression, the activity of chitinase 1 was assayed . Secretion of this enzyme was not prevented by a MYO1 gene deficiency, and soluble and cell wall-associated Cts1p activity were increased approximately 5-fold and 20-fold, respectively, in these cells . The increase in soluble activity was correlated with an increase in enzyme levels . Likewise, intracellular chitinase activity was increased approximately 22-fold, and the chitin content of cell walls was elevated 2-fold . These data suggest that the origin of myo1-associated phenotypes is not due to deficient chitinase expression and may instead be due to a deregulation of cell wall metabolism in these cells. Biochem Mol Biol Int, 1998 Sep, 45(6), 1081 - 7 Constitutive nitric oxide synthase in Saccharomyces cerevisiae; Kanadia RN et al.; After removing nonspecific immunoreactivities from crude extract by immunoaffinity chromatography, an immunoreactive-band at 60 kDa of constitutive nitric oxide synthase (cNOS) from Saccharomyces cerevisiae was detected by Western blot using mouse monoclonal anti-neuronal NOS (cNOS) . The activity of yeast cNOS, which was prepared by either histone-agarose chromatography or anti-neuronal NOS immunoprecipitation, was monitored by the formation of citrulline . Yeast cNOS was activated in the presence of calmodulin and arginine, whereas it was inhibited by L-NAME, a mammalian NOS inhibitor . Moreover, actinomycin-D decreased the extracellular and the intracellular levels of nitrate and nitrite which had been converted from NO . The results suggest that cNOS occurs in unicellular eukaryotes and the enzyme activity can be regulated. Microbiol Res, 1998 Aug, 153(2), 179 - 84 Polyamine metabolism in Saccharomyces cerevisiae exposed to ethanol; Walters D et al.; Growth of the yeast Saccharomyces cerevisiae was unaffected by up to 24 h exposure to ethanol concentrations ranging from 1% to 9%, but was reduced following exposure to 12% ethanol . Concentrations of the polyamines putrescine, cadaverine and spermidine were not affected by a 24 h exposure to 12% ethanol, although there was a significant increase in spermine level . These changes were accompanied by significant increases in the activities of the polyamine biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) and in the flux of label from ornithine into the polyamines . Formation of the cadaverine derivatives aminopropylcadaverine and N,N-bis(3-aminopropyl)cadaverine was greatly increased in yeast exposed to 12% ethanol for 24 h, probably via the action of ODC, AdoMetDC and the aminopropyltransferases . Exposure to 12% ethanol also led to substantial reductions in the uptake of putrescine and spermidine and the amino acid methionine. Eur J Biochem, 1998 Sep 1, 256(2), 427 - 35 Molecular and pharmacological characterization of recombinant rat/mice N-methyl-D-aspartate receptor subtypes in the yeast Saccharomyces cerevisiae; Becker J et al.; The genes encoding the ionotropic N-methyl-D-aspartate (NMDA) receptor subunits NR1a, NR2B and NR2D were cloned in the multi-copy yeast-Escherichia coli shuttle vectors pMBO1 and pMB02 . The protease-deficient yeast Saccharomyces cerevisiae c13-ABYS-86 (leu-, ura-, his-) was transformed with the recombinant plasmids pMBNR1a (leu+), pMBNR1a/pMBNR2D (ura+), pMBNR1a/pMBNR2D/ pMBNR2B (his+) or pMBNR1a/pMBNR2A/pMBNR2B, respectively, and was used to express the different NMDA receptor subunit genes . Western-blotting analysis with the specific NMDA receptor antibodies showed a clear but differently strong expression of the recombinant receptor proteins which were found to be only partially glycosylated in the cell membranes of the recombinant yeast strains . By immunofluorescence microscopy using the specific subunit antibodies and fluorescence-labeled secondary antibodies, the distinctly expressed NR1a and NR2D subunits could be located in the plasma membrane of the transformed yeast cells . Pharmacological characterization of crude membrane preparations of the recombinant yeast cells expressing 1-3 NMDA receptor subunits showed saturable binding of the glycine antagonist {3H}MDL105,519 with different Kd values of 56.88+/-5.38 nM (NR1a), 1365.11+/-76 nM (NR1a/NR2D), 22.97+/-3.37 nM for NR1a/NR2B/NR2D and 7.4+/-1.2 nM for NR1a/NR2A/NR2B . The bound capacities were 13.07+/-0.92 (NRla), 14.63+/-0.50 (NR1a/NR2D), 12.85+/-1.68 (NR1a/NR2B/NR2D) and 8.3+/-0.7 (NR1a/NR2A/NR2B) pmol/mg membrane protein . The {3H}MDL105,519 binding was inhibited by the glycine antagonist 5,7-dichlorokynurenate (DCKA), ethyl-2-carboxy-4.6-dichloro-3-indoleacetate (ECDI) and itself, but not by glycine, D-serine and 1-amino-cyclopropanecarboxylic acid (ACPC) . Each of these recombinant receptor proteins consisting both of NR1 and NR2 subunits also showed a specific binding site for the NMDA agonist glutamate when using L-{3H}glutamate as a radioligand . Analysis of saturation experiments revealed that this ligand binds to a specific site with Kd values of 536+/-43, 688+/-60, and 856+/-48 nM for NR1a/NR2B, NR1a/NR2D, and NR1a/NR2B/NR2D respectively. Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1615 - 8 Expression of Aspergillus aculeatus No . F-50 cellobiohydrolase I (cbhI) and beta-glucosidase 1 (bgl1) genes by Saccharomyces cerevisiae; Takada G et al.; A cellobiohydrolase I (cbhI) and a beta-glucosidase 1 (bgl1) gene of Aspergillus aculeatus were expressed in Saccharomyces cerevisiae . The transformed cells secreted the enzymes efficiently in an active form . The recombinant CBHI gave two bands of different molecular mass (110 and 90 kDa) and the recombinant BGL1 gave one band (180 kDa) by SDS-PAGE . The recombinant CBHI and BGL1 had the same enzymatical properties as the native enzyme except for the specific activity toward cellulosic substrates . By the combination of three different types of cellulases, FI-CMCase, CBHI, and BGL1, we could hydrolyze Avicel up to 59% under our experimental conditions. J Biol Chem, 1998 Oct 9, 273(41), 26946 - 53 Amino acid sequence requirement for efficient incorporation of glycosylphosphatidylinositol-associated proteins into the cell wall of Saccharomyces cerevisiae; Hamada K et al.; During cell wall biogenesis in Saccharomyces cerevisiae, some glycosylphosphatidylinositol (GPI)-attached proteins are detached from GPI moieties and bound to beta-1,6-glucan of the cell wall . The amino acid sequence requirement for the incorporation of GPI-attached proteins into the cell wall was studied by using reporter fusion proteins . Only the short omega-minus region composed of five amino acids, which is located upstream of the omega site for GPI attachment, determined the cellular localization of the GPI-associated proteins . Within the omega-minus region, amino acid residues at the omega-4 or -5 and omega-2 sites were important for the cell wall incorporation . Yap3p, a well characterized GPI-anchored plasma membrane aspartic protease, was localized in the cell wall when the omega-minus region was mutated to sequences containing Val or Ile at the omega-4 or -5 site and Val or Tyr at the omega-2 site. J Biol Chem, 1998 Oct 9, 273(41), 26880 - 90 Dna2 of Saccharomyces cerevisiae possesses a single-stranded DNA-specific endonuclease activity that is able to act on double-stranded DNA in the presence of ATP; Bae SH et al.; To gain further insights into the biological functions of Dna2, previously known as a cellular replicative helicase in Saccharomyces cerevisiae, we examined biochemical properties of the recombinant Dna2 protein purified to homogeneity . Besides the single-stranded (ss) DNA-dependent ATPase activity as reported previously, we were able to demonstrate that ssDNA-specific endonuclease activity is intrinsically associated with Dna2 . Moreover, Dna2 was capable of degrading duplex DNA in an ATP-dependent fashion . ATP and dATP, the only nucleotides hydrolyzed by Dna2, served to stimulate Dna2 to utilize duplex DNA, indicating their hydrolysis is required . Dna2 was able to unwind short duplex only under the condition where the endonuclease activity was minimized . This finding implies that Dna2 unwinds only partially the 3'-end of duplex DNA and generates a stretch of ssDNA of limited length, which is subsequently cleaved by the ssDNA-specific endonuclease activity . A point mutation at the conserved ATP-binding site of Dna2 inactivated concurrently ssDNA-dependent ATPase, ATP-dependent nuclease, and helicase activities, indicating that they all reside in Dna2 itself . By virtue of its nucleolytic activities, the Dna2 protein may function in the maintenance of chromosomal integrity, such as repair or other related process, rather than in propagation of cellular replication forks. J Biol Chem, 1998 Oct 9, 273(41), 26426 - 31 Genetic probing of the stalk segments associated with M2 and M3 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae; Soteropoulos P et al.; The stalk region of the H+-ATPase from Saccharomyces cerevisiae has been proposed to play a role in coupling ATP hydrolysis to proton transport . Genetic probing was used to examine the role of stalk segments S2 and S3, associated with M2 and M3, respectively . Saturation mutagenesis was used to explore the role of side group character at position Ile183 in S2, at which an alanine substitution was shown previously to partially uncouple the enzyme (Wang, G., Tamas, M . J., Hall, M . J., Pascual-Ahuir, A., and Perlin, D . S . (1996) J . Biol . Chem . 271, 25438-25445) . Diverse side group substitutions were tolerated at this position, although three substitutions, I183N, I183R, and I183Y required second site mutations at the C terminus of the enzyme for stabilization . Substitution of glycine and proline at Ile183 resulted in lethal phenotypes, suggesting that the backbone may be more important than side group at this position . Proline/glycine mutagenesis was used to study additional sites in S2 and S3 . The substitution of proline at Gly186 resulted in a lethal phenotype, whereas substitutions in S3 of proline or serine at Gly270 and proline or glycine at Thr287 resulted in viable mutants . Mutations G270P and T287P resulted in mutant enzymes that produced pronounced growth defects and ATP hydrolysis rates that were 35% and 60% lower than wild type enzyme, respectively . The mutant enzymes transported protons at rates consistent with their ATPase activity, suggesting that the growth defects observed were due to a reduced rate of ATP hydrolysis and not to uncoupling of proton transport . The prominent growth phenotypes produced by mutations G270P and T287P permitted the isolation of suppressor mutations, which restored wild type growth . Most of the suppressors either replaced the primary site mutation with alanine or restored the wild type residue by ectopic recombination with PMA2, both of which restore alpha-helical tendency . This study suggests that maintaining alpha-helical character is essential to S2 and may play an important role in S3. FEBS Lett, 1998 Sep 11, 435(1), 89 - 95 Gos1p, a Saccharomyces cerevisiae SNARE protein involved in Golgi transport; McNew JA et al.; Specific transport between secretory compartments requires that vesicular carriers contain targeting proteins known as SNAREs . Ten v-SNAREs have been identified in the genome of the yeast Saccharomyces cerevisiae by sequence analysis . We report here the characterization of Gos1p, a v-SNARE localized to the Golgi compartment and likely homolog of the mammalian protein GOS-28/GS28 . Gos1p is a type II membrane protein with characteristic SNARE sequence hallmarks and is functionally a SNARE protein . Gos1p was originally identified as a 28 kDa protein in an immunoprecipitate of the cis-Golgi t-SNARE Sed5p . This interaction between Sed5p and Gos1p is direct as demonstrated by in vitro binding with recombinant proteins . Deletion of GOS1 results in viable haploids with modest growth and secretory defects . Close examination of the secretory phenotype of GOS1-disrupted cells suggests that Gos1p may play a role in multiple transport steps, specifically ER-Golgi and/or intra-Golgi transport. Genetics, 1998 Oct, 150(2), 613 - 32 Identification of high-copy disruptors of telomeric silencing in Saccharomyces cerevisiae; Singer MS et al.; The ends of chromosomes in Saccharomyces cerevisiae initiate a repressive chromatin structure that spreads internally and inhibits the transcription of nearby genes, a phenomenon termed telomeric silencing . To investigate the molecular basis of this process, we carried out a genetic screen to identify genes whose overexpression disrupts telomeric silencing . We thus isolated 10 DOT genes (disruptor of telomeric silencing) . Among these were genes encoding chromatin component Sir4p, DNA helicase Dna2p, ribosomal protein L32, and two proteins of unknown function, Asf1p and Ifh1p . The collection also included genes that had not previously been identified: DOT1, DOT4, DOT5, DOT6, and TLC1, which encodes the RNA template component of telomerase . With the exception of TLC1, all these genes, particularly DOT1 and DOT4, also reduced silencing at other repressed loci (HM loci and rDNA) when overexpressed . Moreover, deletion of the latter two genes weakened silencing as well, suggesting that DOT1 and DOT4 normally play important roles in gene repression . DOT1 deletion also affected telomere tract length . The function of Dot1p is not known . The sequence of Dot4p suggests that it is a ubiquitin-processing protease . Taken together, the DOT genes include both components and regulators of silent chromatin. Genetics, 1998 Oct, 150(2), 591 - 600 Alteration of N-terminal phosphoesterase signature motifs inactivates Saccharomyces cerevisiae Mre11; Bressan DA et al.; Saccharomyces cerevisiae Mre11, Rad50, and Xrs2 function in a protein complex that is important for nonhomologous recombination . Null mutants of MRE11, RAD50, and XRS2 are characterized by ionizing radiation sensitivity and mitotic interhomologue hyperrecombination . We mutagenized the four highly conserved phosphoesterase signature motifs of Mre11 to create mre11-11, mre11-2, mre11-3, and mre11-4 and assessed the functional consequences of these mutant alleles with respect to mitotic interhomologue recombination, chromosome loss, ionizing radiation sensitivity, double-strand break repair, and protein interaction . We found that mre11 mutants that behaved as the null were sensitive to ionizing radiation and deficient in double-strand break repair . We also observed that these null mutants exhibited a hyperrecombination phenotype in mitotic cells, consistent with previous reports, but did not exhibit an increased frequency of chromosome loss . Differential ionizing radiation sensitivities among the hypomorphic mre11 alleles correlated with the trends observed in the other phenotypes examined . Two-hybrid interaction testing showed that all but one of the mre11 mutations disrupted the Mre11-Rad50 interaction . Mutagenesis of the phosphoesterase signatures in Mre11 thus demonstrated the importance of these conserved motifs for recombinational DNA repair. Genetics, 1998 Oct, 150(2), 577 - 89 An arf1Delta synthetic lethal screen identifies a new clathrin heavy chain conditional allele that perturbs vacuolar protein transport in Saccharomyces cerevisiae; Chen CY et al.; ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles . To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1Delta allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1Delta) . Most of the swa mutants exhibit phenotypes comparable to arf1Delta mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects . Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3' end of the CHC1 open reading frame . This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly . Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants . The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y. EMBO J, 1998 Oct 1, 17(19), 5796 - 804 Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs; Sanchirico ME et al.; The essential products of the yeast mitochondrial translation system are seven hydrophobic membrane proteins and Var1p, a hydrophilic protein in the small ribosomal subunit . Translation of the membrane proteins depends on nuclearly encoded, mRNA-specific translational activators that recognize the 5'-untranslated leaders of their target mRNAs . These translational activators are themselves membrane associated and could therefore tether translation to the inner membrane . In this study, we tested whether chimeric mRNAs with the untranslated sequences normally present on the mRNA encoding soluble Var1p, can direct functional expression of coding sequences specifying the integral membrane proteins Cox2p and Cox3p . DNA sequences specifying these chimeric mRNAs were inserted into mtDNA at the VAR1 locus and expressed in strains containing a nuclearly localized plasmid that supplies a functional form of Var1p, imported from the cytoplasm . Although cells expressing these chimeric mRNAs actively synthesized both membrane proteins, they were severely deficient in cytochrome c oxidase activity and in the accumulation of Cox2p and Cox3p, respectively . These data strongly support the physiological importance of interactions between membrane-bound mRNA-specific translational activators and the native 5'-untranslated leaders of the COX2 and COX3 mRNAs for localizing productive synthesis of Cox2p and Cox3p to the inner membrane. Biochem Biophys Res Commun, 1998 Sep 18, 250(2), 335 - 41 The subcellular location of the yeast Saccharomyces cerevisiae homologue of the protein defective in the juvenile form of Batten disease; Croopnick JB et al.; The mutation responsible for the juvenile form of Batten disease was mapped to a single gene, Cln3 (T . J . Lerner et al . (1995) Cell 82:949-957) . Yeast Saccharomyces cerevisiae has an open reading frame, BTN1 (YHC3), that encodes the putative homologue of Cln3p . Primary structure comparison indicates that the human Cln3p and yeast Btn1p are 59% similar and 39% identical and they have similar hydropathy profiles . Gene disruption of BTN1 in yeast has no apparent effect on growth or viability of the cells under a variety of conditions . Gene fusion protein constructs of green fluorescent protein (GFP) and Btn1p, with GFP at the amino and carboxyl ends of Btn1p, localize to the vacuole in yeast . These data indicate that BTN1 is a nonessential gene under most growth conditions which functions in the vacuole in yeast Saccharomyces cerevisiae. Ann Fr Anesth Reanim, 1998, 17(2), 130 - 2 {Four hours for a record, or a severe fuminating cellulitis: can Saccharomyces cerevisiae be the causal agent?}; Almanza L et al.; A 31-year-old woman presented with a subcutaneous cellulitis which occurred within four hours following a minor wound of a knee . This very short delay could be explained neither by the health state, nor the mechanism of injury, nor the bacteria usually responsible for such a cellulitis . Considering the clinical characteristics (high gas production) and the professional context (wine cellar employee), Saccharomyces cerevisiae, a yeast used for wine or bread production, may explain this complication. Mol Gen Genet, 1998 Aug, 259(3), 246 - 55 Synthesis of glutamine, glycine and 10-formyl tetrahydrofolate is coregulated with purine biosynthesis in Saccharomyces cerevisiae; Denis V et al.; Glutamine, glycine and 10-formyl tetrahydrofolate are consumed during de novo purine biosynthesis . We have found that, in Saccharomyces cerevisiae, synthesis of these cosubstrates is coregulated with synthesis of enzymes of the purine biosynthetic pathway . Analysis of three genes required for synthesis of glutamine, glycine and 10-formyl tetrahydrofolate (GLN1, SHM2 and MTD1, respectively) shows that their expression is repressed by adenine and requires the transcription factors Baslp and Bas2p . Northern analysis reveals that regulation of SHM2 and MTD1 expression by adenine takes place at the transcriptional level . We also show that Bas1p and Bas2p bind in vitro to the promoters of the SHM2 and MTD1 genes, and that mutations in the consensus Bas1p binding sequences strongly affect expression of these genes in vivo . Finally, we have found that a SHM2-lacZ fusion is expressed at a significantly higher level in a bas2-2 disrupted strain than in bas1-2 or bas1-2 bas2-2 mutant strains . The BAS1-dependent, BAS2-independent expression of SHM2-lacZ suggests that, in the absence of Bas2p, Bas1p can interact with another protein partner to activate SHM2 expression. J Bacteriol, 1998 Oct, 180(19), 5030 - 7 New potential cell wall glucanases of Saccharomyces cerevisiae and their involvement in mating; Cappellaro C et al.; Biotinylation of intact Saccharomyces cerevisiae cells with a nonpermeant reagent (Sulfo-NHS-LC-Biotin) allowed the identification of seven cell wall proteins that were released from intact cells by dithiothreitol (DTT) . By N-terminal sequencing, three of these proteins were identified as the known proteins beta-exoglucanase 1 (Exg1p), beta-endoglucanase (Bgl2p), and chitinase (Cts1p) . One protein was related to the PIR protein family, whereas the remaining three (Scw3p, Scw4p, and Scw10p {for soluble cell wall proteins}) were found to be related to glucanases . Single knockouts of these three potential glucanases did not result in dramatic phenotypes . The double knockout of SCW4 and the homologous gene SCW10 resulted in slower growth, significantly increased release of proteins from intact cells by DTT, and highly decreased mating efficiency when these two genes were disrupted in both mating types . The synergistic behavior of the disruption of SCW4 and SCW10 was partly antagonized by the disruption of BGL2 . The data are discussed in terms of a possible counterplay of transglucosidase and glucosidase activities. J Biol Chem, 1998 Oct 2, 273(40), 25864 - 74 A role for Saccharomyces cerevisiae fatty acid activation protein 4 in regulating protein N-myristoylation during entry into stationary phase; Ashrafi K et al.; Saccharomyces cerevisiae contains four known acyl-CoA synthetases (fatty acid activation proteins, Faaps) . Faa1p and Faa4p activate exogenously derived fatty acids . Acyl-CoA metabolism plays a critical role in regulating protein N-myristoylation by the essential enzyme, myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) . In this report, we have examined whether Faa1p and Faa4p have distinct roles in affecting protein N-myristoylation as cells transition from growth in rich media to a growth-arrested state during nutrient deprivation (stationary phase) . The colony-forming potential of 10 isogenic strains was defined as a function of time spent in stationary phase . These strains contained either a wild type or mutant NMT1 allele, and wild type or null alleles of each FAA . Only the combination of the Nmt mutant (nmt451Dp; reduced affinity for myristoyl-CoA) and loss of Faa4p produced a dramatic loss of colony-forming units (CFU) . The progressive millionfold reduction in CFU was associated with a deficiency in protein N-myristoylation that first appeared during logarithmic growth, worsened through the post-diauxic phase, and became extreme in stationary phase . Northern and Western blot analyses plus N-myristoyltransferase assays showed that Nmt is normally present only during the log and diauxic/post-diauxic periods, indicating that N-myristoylproteins present in stationary phase are "inherited" from these earlier phases . Moreover, FAA4 is the only FAA induced during the critical diauxic/early post-diauxic transition . Although substitution of nmt1-451D for NMT1 results in deficiencies in protein N-myristoylation, these deficiencies are modest and limited by compensatory responses that include augmented expression of nmt1-451D and precocious induction of FAA4 in log phase . Loss of Faa4p from nmt1-451D cells severely compromises their capacity to adequately myristoylate Nmt substrates prior to entry into stationary phase since none of the other Faaps are able to functionally compensate for its absence . To identify Nmt1p substrates that may affect maintenance of proliferative potential during stationary phase, we searched the yeast genome for known and putative N-myristoylproteins . Of the 64 genes found, 48 were successfully deleted in NMT1 cells . Removal of any one of the following nine substrates produced a loss of CFU similar to that observed in nmt1-451Dfaa4Delta cells: Arf1p, Arf2p, Sip2p, Van1p, Ptc2p, YBL049W (homology to Snf7p), YJR114W, YKR007W, and YMR077C . These proteins provide opportunities to further define the molecular mechanisms that regulate survival during stationary phase. J Biol Chem, 1998 Oct 2, 273(40), 25751 - 6 An investigation of the metabolism of valine to isobutyl alcohol in Saccharomyces cerevisiae; Dickinson JR et al.; The metabolism of valine to isobutyl alcohol in yeast was examined by 13C nuclear magnetic resonance spectroscopy and combined gas chromatography-mass spectrometry . The product of valine transamination, alpha-ketoisovalerate, had four potential routes to isobutyl alcohol . The first, via branched-chain alpha-ketoacid dehydrogenase to isobutyryl-CoA is not required for the synthesis of isobutyl alcohol because abolition of branched-chain alpha-ketoacid dehydrogenase activity in an lpd1 disruption mutant did not prevent the formation of isobutyl alcohol . The second route, via pyruvate decarboxylase, is the one that is used because elimination of pyruvate decarboxylase activity in a pdc1 pdc5 pdc6 triple mutant virtually abolished isobutyl alcohol production . A third potential route involved alpha-ketoisovalerate reductase, but this had no role in the formation of isobutyl alcohol from alpha-hydroxyisovalerate because cell homogenates could not convert alpha-hydroxyisovalerate to isobutyl alcohol . The final possibility, use of the pyruvate decarboxylase-like enzyme encoded by YDL080c, seemed to be irrelevant, because a strain with a disruption in this gene produced wild-type levels of isobutyl alcohol . Thus there are major differences in the catabolism of leucine and valine to their respective "fusel" alcohols . Whereas in the catabolism of leucine to isoamyl alcohol the major route is via the decarboxylase encoded by YDL080c, any single isozyme of pyruvate decarboxylase is sufficient for the formation of isobutyl alcohol from valine . Finally, analysis of the 13C-labeled products revealed that the pathways of valine catabolism and leucine biosynthesis share a common pool of alpha-ketoisovalerate. Mol Gen Genet, 1998 Aug, 259(2), 142 - 9 The chromatin structure of the GAL1 promoter forms independently of Reb1p in Saccharomyces cerevisiae; Reagan MS et al.; Positive and negative regulation of the GAL1 promoter of the yeast Saccharomyces cerevisiae results from a network of interactions between transcription factors and chromatin . In this study we used footprinting procedures to characterize these interactions in vivo . DNase I analysis of the GAL1 upstream activating sequence (UAS(GAL1/10)) showed expected Gal4 activator protein binding during growth in galactose, and also revealed binding of the Reb1 protein (Reb1p) during growth in glucose . In addition, we mapped to nucleotide resolution a positioned nucleosome that, in the inactive promoter, packages DNA between the UAS(GAL1/10) and the GAL1 TATA sequence, leaving both of these elements nucleosome free . The nucleosome footprint was lost when the promoter was activated . Surprisingly, mutation of the Reb1p binding site had no effect on nucleosome positioning or on the kinetics or extent of activation or repression of either the GAL1 or GAL10 promoters under any of the conditions assayed. Curr Genet, 1998 Sep, 34(3), 183 - 91 Ace2p, a regulator of CTS1 (chitinase) expression, affects pseudohyphal production in Saccharomyces cerevisiae; King L et al.; Some diploid strains of Saccharomyces cerevisiae can grow both as a spherical yeast form and as a filamentous pseudohyphal form . Most yeasts capable of forming pseudohyphae possess a functional FLO8 gene . We show that disrupting the ACE2 transcription factor results in the production of pseudohyphae in a flo8-1 background . Disrupting the CTS1 (chitinase) gene also produces pseudohyphal growth in this background, but at a reduced level . Invasion of solid media by haploid and diploid cells is increased in ACE2 disruptions, but the diploids adhere poorly to the agar . Sigma1278b-derived strains, which generally produce pseudohyphae, have about 30-fold lower chitinase activity than other strains. Genes Dev, 1998 Sep 15, 12(18), 2943 - 55 Saccharomyces cerevisiae cAMP-dependent protein kinase controls entry into stationary phase through the Rim15p protein kinase; Reinders A et al.; The Saccharomyces cerevisiae protein kinase Rim15p was identified previously as a stimulator of meiotic gene expression . Here, we show that loss of Rim15p causes an additional pleiotropic phenotype in cells grown to stationary phase on rich medium; this phenotype includes defects in trehalose and glycogen accumulation, in transcriptional derepression of HSP12, HSP26, and SSA3, in induction of thermotolerance and starvation resistance, and in proper G1 arrest . These phenotypes are commonly associated with hyperactivity of the Ras/cAMP pathway . Tests of epistasis suggest that Rim15p may act in this pathway downstream of the cAMP-dependent protein kinase (cAPK) . Accordingly, deletion of RIM15 suppresses the growth defect of a temperature-sensitive adenylate-cyclase mutant and, most importantly, renders cells independent of cAPK activity . Conversely, overexpression of RIM15 suppresses phenotypes associated with a mutation in the regulatory subunit of cAPK, exacerbates the growth defect of strains compromised for cAPK activity, and partially induces a starvation response in logarithmically growing wild-type cells . Biochemical analyses reveal that cAPK-mediated in vitro phosphorylation of Rim15p strongly inhibits its kinase activity . Taken together, these results place Rim15p immediately downstream and under negative control of cAPK and define a positive regulatory role of Rim15p for entry into both meiosis and stationary phase. Genes Dev, 1998 Sep 15, 12(18), 2874 - 86 The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae; O'Rourke SM et al.; The MAPKKK Ste11p functions in three Saccharomyces cerevisiae MAPK cascades {the high osmolarity glycerol (HOG), pheromone response, and pseudohyphal/invasive growth pathways}, but its activation in response to high osmolarity stimulates only the HOG pathway . To determine what restricts cross-activation of MAPK cascades (cross talk), we have studied mutants in which the pheromone response pathway is activated by high osmolarity (1 M sorbitol) . We found that mutations in the HOG1 gene, encoding the p38-type MAPK of the HOG pathway, and in the PBS2 gene, encoding the activating kinase for Hog1p, allowed osmolarity-induced activation of the pheromone response pathway . This cross talk required the osmosensor Sho1p, as well as Ste20p, Ste50p, the pheromone response MAPK cascade (Ste11p, Ste7p, and Fus3p or Kss1p), and Ste12p but not Ste4p or the MAPK scaffold protein, Ste5p . The cross talk in hog1 mutants induced multiple responses of the pheromone response pathway: induction of a FUS1::lacZ reporter, morphological changes, and mating in ste4 and ste5 mutants . We suggest that Hog1p may prevent osmolarity-induced cross talk by inhibiting Sho1p, perhaps as part of a feedback control on the HOG pathway . We have also shown that Ste20p and Ste50p function in the Sho1p branch of the HOG pathway and that a second osmosensor in addition to Sho1p may activate Ste11p . Finally, we have found that pseudohyphal growth exhibited by wild-type (HOG1) strains depends on SHO1, suggesting that Sho1p may be a receptor that feeds into the pseudohyphal growth pathway. Science, 1998 Sep 18, 281(5384), 1854 - 7 Regulation of meiotic S phase by Ime2 and a Clb5,6-associated kinase in Saccharomyces cerevisiae; Dirick L et al.; Cyclin-dependent kinase (Cdk) mutations that prevent entry into the mitotic cell cycle of budding yeast fail to block meiotic DNA replication, suggesting there may be fundamental differences between these pathways . However, S phase in meiosis was found to depend on the same B-type cyclins (Clb5 and Clb6) as it does in mitosis . Meiosis differs instead in the mechanism that controls removal of the Cdk inhibitor Sic1 . Destruction of Sic1 and activation of a Clb5-dependent kinase in meiotic cells required the action of the meiosis-specific protein kinase Ime2, thereby coupling early meiotic gene expression to control of DNA replication for meiosis. FEBS Lett, 1998 Sep 4, 434(3), 406 - 8 Effect of squalene synthase gene disruption on synthesis of polyprenols in Saccharomyces cerevisiae; Grabowska D et al.; Biosynthesis of polyprenols was investigated in a wild-type strain of Saccharomyces cerevisiae and a squalene synthase deficient strain auxotrophic for ergosterol . The quantitative data showed that disruption of squalene synthase gene caused a 6-fold increase in the synthesis of polyprenols in vitro in comparison with the wild-type strain . Microsomal preparation from the deleted strain only slightly reacted to the additional exogenous FPP, while that from the wild-type strain presented a 4-fold increase of polyprenol synthesis . Restoration of ergosterol synthesis, by introducing ERG9 functional allele into the deleted strain resulted in a significant lowering of polyprenol synthesis, indicating the immediate shift of the common substrate (FPP) to the sterol pathway . The role of squalene synthase in the regulation of polyprenol synthesis and 'flow diversion hypothesis' is discussed. FEBS Lett, 1998 Sep 4, 434(3), 219 - 25 Distribution and functional diversification of the ras superfamily in Saccharomyces cerevisiae; Garcia-Ranea JA et al.; The recent availability of the full Saccharomyces cerevisiae genome sequence offers a first opportunity to analyze the composition, function and evolution of GTPases in the ras-p21 superfamily . This superfamily in yeast is composed of 29 proteins divided into five families: ras with four sequences implicated in cell signalling; rho, six genes related to the cell shape machinery; ypt-rab, ten proteins with different roles in intracellular trafficking; arf-sar, seven proteins related to vesicular trafficking in secretory pathways; and ran, two proteins acting as components of the nuclear transport system . The superfamily covers a wide range of cellular functions from signalling to intracellular trafficking, while conserving the structural framework and a common mechanism of GTP hydrolysis. Mol Cell Biol, 1998 Oct, 18(10), 5771 - 9 Mutations in RNA polymerase II and elongation factor SII severely reduce mRNA levels in Saccharomyces cerevisiae; Lennon JC 3rd et al.; Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro . The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood . Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2 . An SII gene disruption or the rpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates . Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity . In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe . Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not . This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied. Mol Cell Biol, 1998 Oct, 18(10), 6102 - 9 Sld2, which interacts with Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication; Kamimura Y et al.; The DPB11 gene, which genetically interacts with DNA polymerase II (epsilon), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint in Saccharomyces cerevisiae . To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal with dpb11-1) mutations which fall into six complementation groups (sld1 to -6) . In this study, we characterized SLD2, encoding an essential 52-kDa protein . High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1 . Conversely, high-copy DPB11 suppressed the temperature-sensitive growth defect caused by sld2-6 . The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay . This interaction was evident at 25 degreesC but not at 34 degreesC when Sld2-6 or Dpb11-1 replaced its wild-type protein . No interaction between Sld2-6 and Dpb11-1 could be detected even at 25 degreesC . Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2 . sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1 cells . Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift . These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication. Mol Cell Biol, 1998 Oct, 18(10), 5970 - 80 Transcriptional regulation of the SMK1 mitogen-activated protein kinase gene during meiotic development in Saccharomyces cerevisiae; Pierce M et al.; Meiotic development (sporulation) in Saccharomyces cerevisiae is characterized by an ordered pattern of gene expression, with sporulation-specific genes classified as early, middle, mid-late, or late depending on when they are expressed . SMK1 encodes a mitogen-activated protein kinase required for spore morphogenesis that is expressed as a middle sporulation-specific gene . Here, we identify the cis-acting DNA elements that regulate SMK1 transcription and characterize the phenotypes of mutants with altered expression patterns . The SMK1 promoter contains an upstream activating sequence (UASS) that specifically interacts with the transcriptional activator Abf1p . The Abf1p-binding sites from the early HOP1 and the middle SMK1 promoters are functionally interchangeable, demonstrating that these elements do not play a direct role in their differential transcriptional timing . Timing of SMK1 expression is determined by another cis-acting DNA sequence termed MSE (for middle sporulation element) . The MSE is required not only for activation of SMK1 transcription during middle sporulation but also for its repression during vegetative growth and early meiosis . In addition, the SMK1 MSE can repress vegetative expression in the context of the HOP1 promoter and convert HOP1 from an early to a middle gene . SMK1 function is not contingent on its tight transcriptional regulation as a middle sporulation-specific gene . However, promoter mutants with different quantitative defects in SMK1 transcript levels during middle sporulation show distinct sporulation phenotypes. Mol Cell Biol, 1998 Oct, 18(10), 5942 - 51 Processivity of the Saccharomyces cerevisiae poly(A) polymerase requires interactions at the carboxyl-terminal RNA binding domain; Zhelkovsky A et al.; The interaction of the Fip1 subunit of polyadenylation factor I with the Saccharomyces cerevisiae poly(A) polymerase (PAP) was assayed in vivo by two-hybrid analysis and was found to involve two separate regions on PAP, located at opposite ends of the protein sequence . In vitro, Fip1 blocks access of the RNA primer to an RNA binding site (RBS) that overlaps the Fip1 carboxy-terminal interaction region and, in doing so, shifts PAP to a distributive mode of action . Partial truncation of this RBS has the same effect, indicating that this site is required for processivity . A comparison of the utilization of ribo- and deoxyribonucleotides as substrates indicates the existence on PAP of a second RBS which recognizes the last three nucleotides at the 3' end of the primer . This site discriminates against deoxyribonucleotides at the 3' end, and interactions at this site are not affected by Fip1 . Further analysis revealed that the specificity of PAP for adenosine is not simply a function of the ATP binding site but also reflects interactions with bases at the 3' end of the primer and at another contact site 14 nucleotides upstream of the 3' end . These results suggest that the unique specificity of PAP for ribose and base, and thus the extent and type of activity with different substrates, depends on interactions at multiple nucleotide binding sites. Mol Cell Biol, 1998 Oct, 18(10), 5750 - 61 NDT80 and the meiotic recombination checkpoint regulate expression of middle sporulation-specific genes in Saccharomyces cerevisiae; Hepworth SR et al.; Distinct classes of sporulation-specific genes are sequentially expressed during the process of spore formation in Saccharomyces cerevisiae . The transition from expression of early meiotic genes to expression of middle sporulation-specific genes occurs at about the time that cells exit from pachytene and form the meiosis I spindle . To identify genes encoding potential regulators of middle sporulation-specific gene expression, we screened for mutants that expressed early meiotic genes but failed to express middle sporulation-specific genes . We identified mutant alleles of RPD3, SIN3, and NDT80 in this screen . Rpd3p, a histone deacetylase, and Sin3p are global modulators of gene expression . Ndt80p promotes entry into the meiotic divisions . We found that entry into the meiotic divisions was not required for activation of middle sporulation genes; these genes were efficiently expressed in a clb1 clb3 clb4 strain, which fails to enter the meiotic divisions due to reduced Clb-dependent activation of Cdc28p kinase . In contrast, middle sporulation genes were not expressed in a dmc1 strain, which fails to enter the meiotic divisions because a defect in meiotic recombination leads to a RAD17-dependent checkpoint arrest . Expression of middle sporulation genes, as well as entry into the meiotic divisions, was restored to a dmc1 strain by mutation of RAD17 . Our studies also revealed that NDT80 was a temporally distinct, pre-middle sporulation gene and that its expression was reduced, but not abolished, on mutation of DMC1, RPD3, SIN3, or NDT80 itself . In summary, our data indicate that Ndt80p is required for expression of middle sporulation genes and that the activity of Ndt80p is controlled by the meiotic recombination checkpoint . Thus, middle genes are expressed only on completion of meiotic recombination and subsequent generation of an active form of Ndt80p. Mol Cell Biol, 1998 Oct, 18(10), 5712 - 23 A novel DNA-binding protein bound to the mitochondrial inner membrane restores the null mutation of mitochondrial histone Abf2p in Saccharomyces cerevisiae; Cho JH et al.; The yeast mitochondrial HMG-box protein, Abf2p, is essential for maintenance of the mitochondrial genome . To better understand the role of Abf2p in the maintenance of the mitochondrial chromosome, we have isolated a multicopy suppressor (YHM2) of the temperature-sensitive defect associated with an abf2 null mutation . The function of Yhm2p was characterized at the molecular level . Yhm2p has 314 amino acid residues, and the deduced amino acid sequence is similar to that of a family of mitochondrial carrier proteins . Yhm2p is localized in the mitochondrial inner membrane and is also associated with mitochondrial DNA in vivo . Yhm2p exhibits general DNA-binding activity in vitro . Thus, Yhm2p appears to be novel in that it is a membrane-bound DNA-binding protein . A sequence that is similar to the HMG DNA-binding domain is important for the DNA-binding activity of Yhm2p, and a mutation in this region abolishes the ability of YHM2 to suppress the temperature-sensitive defect of respiration of the abf2 null mutant . Disruption of YHM2 causes a significant growth defect in the presence of nonfermentable carbon sources such as glycerol and ethanol, and the cells have defects in respiration as determined by 2,3,5,-triphenyltetrazolium chloride staining . Yhm2p may function as a member of the protein machinery for the mitochondrial inner membrane attachment site of mitochondrial DNA during replication and segregation of mitochondrial genomes. FEMS Microbiol Lett, 1998 Sep 1, 166(1), 95 - 101 Unique morphogenesis in Saccharomyces cerevisiae strain GS1731; Singh VK et al.; During the lag and early exponential phase of growth, 50-60% of budded cells of Saccharomyces cerevisiae strain GS1731 were multiply budded . During subsequent culture growth, the frequency of multiply budded cells decreased until by stationary phase multiply budded cells were rare . Data from renewed growth of a culture after hydroxyurea treatment indicated that GS1731 mother cells could assemble up to three pre-bud sites and begin bud growth and development in each . Light and scanning electron microscopy showed two or three very small buds emerging simultaneously on a mother cell and either reaching full size at the same time or enlarging sequentially . Immunofluorescence studies revealed that these multiply budded cells had multiple bundles of cytoplasmic microtubules . DAPI staining of nuclei revealed that some of the unbudded mother cells were multinucleate and completed cytokinesis giving rise to normal daughter cells. Genomics, 1998 Aug 15, 52(1), 90 - 4 Isolation and characterization of human SGT and identification of homologues in Saccharomyces cerevisiae and Caenorhabditis elegans; Kordes E et al.; We have recently isolated a rat cDNA encoding a novel cellular protein able to interact with the major nonstructural protein NS1 of parvovirus H-1 and have termed this protein SGT, for small glutamine-rich tetratricopeptide repeat (TPR)-containing protein . Here we report the isolation of a cDNA from human placenta encoding the human homologue, human SGT . SGT from rat and human contain 314 and 313 amino acids, respectively, and share 91% sequence identity at the protein level . The highest degree of similarity is present within the central region containing three TPR motifs in tandem array . The similarities, however, also extend beyond this region . Human SGTtranscript was found to be ubiquitously present in all human tissues tested . By fluorescence in situ hybridization analysis we have mapped the human gene to chromosome 19p13 . The SGT-coding sequences are evolutionarily conserved, since we could identify genes encoding proteins of similar size and structure in the genomes of Saccharomyces cerevisiae and Caenorhabditis elegans . RNA, 1998 Sep, 4(9), 1096 - 110 Intronic snoRNA biosynthesis in Saccharomyces cerevisiae depends on the lariat-debranching enzyme: intron length effects and activity of a precursor snoRNA; Ooi SL et al.; The eukaryotic small nucleolar RNAs (snoRNAs) are involved in processing of pre-rRNA and modification of rRNA nucleotides . Some snoRNAs are derived from mono- or polycistronic transcription units, whereas others are encoded in introns of protein genes . The present study addresses the role of the RNA lariat-debranching enzyme (Dbr1p) in the synthesis and function of intronic snoRNAs in the yeast Saccharomyces cerevisiae . Intronic snoRNA production was determined to depend on Dbr1p . Accumulation of mature intronic snoRNAs is reduced in a dbr1 mutant; instead, intronic snoRNAs are "trapped" within host intron lariats . Interestingly, the extent of intronic snoRNA accumulation in the form of lariats in dbr1 cells varied among different intronic snoRNAs . Intronic snoRNAs encoded within shorter introns, such as U24 and snR38, accumulate more unprocessed lariat precursors than those encoded within longer introns, e.g., U18 and snR39 . This correlation was corroborated by experiments conducted with model intron:U24 snoRNA constructs . These results support a splicing-dependent exonucleolytic pathway for the biosynthesis of intronic snoRNAs . Curiously, U24 in a lariat may be functional in directing methylation of ribosomal RNA. Biochemistry, 1998 Sep 15, 37(37), 12899 - 908 Functional analysis of conserved domains in the phosphotyrosyl phosphatase activator . Molecular cloning of the homologues from Drosophila melanogaster and Saccharomyces cerevisiae; Van Hoof C et al.; Phosphotyrosyl phosphatase activator (PTPA), a 37 kDa cytosolic protein that specifically activates the phosphotyrosyl phosphatase activity of the dimeric form of PP2A, was cloned from Drosophila melanogaster and Saccharomyces cerevisiae . Sequence alignment of PTPA from yeast to human revealed highly conserved regions including the type B fragment of the putative PTPA ATP binding site . We generated PTPA deletion mutants of these conserved regions as well as point mutations within regions that were suggested to be functionally important . The recombinant proteins were expressed in E . coli and subsequently purified . Activity measurements, linked with immunological detection, revealed that most of the well-conserved regions are essential for PTPA activity . However, neither the type A fragment of the putative ATP binding site nor the cysteine-rich region, present in all but the Drosophila and yeast homologues, appeared to be essential for PTPA activity . Moreover, we observed that PTPA truncated at glycine266 behaves as a dominant negative mutant since it is inhibitory to the wild-type PTPA. Biochemistry, 1998 Sep 15, 37(37), 12761 - 71 Temperature-jump and potentiometric studies on recombinant wild type and Y143F and Y254F mutants of Saccharomyces cerevisiae flavocytochrome b2: role of the driving force in intramolecular electron transfer kinetics; Tegoni M et al.; The kinetics of intramolecular electron transfer between flavin and heme in Saccharomyces cerevisiae flavocytochrome b2 were investigated by performing potentiometric titrations and temperature-jump experiments on the recombinant wild type and Y143F and Y254F mutants . The midpoint potential of heme was determined by monitoring redox titrations spectrophotometrically, and that of semiquinone flavin/reduced flavin (Fsq/Fred) and oxidized flavin (Fox)/Fsq couples by electron paramagnetic resonance experiments at room temperature . The effects of pyruvate on the kinetic and thermodynamic parameters were also investigated . At room temperature, pH 7.0 and I = 0.1 M, the redox potential of the Fsq/Fred, Fox/Fsq, and oxidized heme/reduced heme (Hox/Hred) couples were -135, -45, and -3 mV, respectively, in the wild-type form . Although neither the mutations nor excess pyruvate did appreciably modify the potential of the heme or that of the Fsq/Fred couple, they led to variable positive shifts in the potential of the Fox/Fsq couple, thus modulating the driving force that characterizes the reduction of heme by the semiquinone in the -42 to +88 mV range . The relaxation rates measured at 16 degreesC in temperature-jump experiments were independent of the protein concentrations, with absorbance changes corresponding to the reduction of the heme . Two relaxation processes were clearly resolved in wild-type flavocytochrome b2 (1/tau1 = 1500 s-1, 1/tau2 = 200 +/- 50 s-1) and were assigned to the reactions whereby the heme is reduced by Fred and Fsq, respectively . The rate of the latter reaction was determined in the whole series of proteins . Its variation as a function of the driving force is well described by the expression obtained from electron-transfer theories, which provides evidence that the intramolecular electron transfer is not controlled by the dynamics of the protein. EMBO J, 1998 Sep 15, 17(18), 5400 - 8 Identification of a copper-induced intramolecular interaction in the transcription factor Mac1 from Saccharomyces cerevisiae; Jensen LT et al.; Mac1 mediates copper (Cu)-dependent expression of genes involved in high-affinity uptake of copper ions in Saccharomyces cerevisiae . Mac1 is a transcriptional activator in Cu-deficient cells, but is inhibited in Cu-replete cells . Mac1 resides within the nucleus in both Cu-deficient and Cu-loaded cells . Cu inhibition of Mac1 appears to result from binding of eight copper ions within a C-terminal segment consisting of two Cys-rich motifs . In addition, two zinc ions are bound within the N-terminal DNA-binding domain . Only 4-5 mol . eq . Cu are bound to a mutant Mac1 (His279Gln substitution) that is impervious to Cu inhibition . The CuMac1 complex is luminescent, indicative of copper bound in the Cu(I) state . Cu binding induces a molecular switch resulting in an intramolecular interaction in Mac1 between the N-terminal DNA-binding domain and the C-terminal activation domain . This allosteric interaction is Cu dependent and is not observed when Mac1 contained the mutant His279Gln substitution . Fusion of the minimal DNA-binding domain of Mac1 (residues 1-159) to the minimal Cu-binding activation domain (residues 252-341) yields a functional Cu-regulated transcriptional activator . These results suggest that Cu repression of Mac1 arises from a Cu-induced intramolecular interaction that inhibits both DNA binding and transactivation activities. Nat Genet, 1998 Sep, 20(1), 74 - 7 HIRA, a mammalian homologue of Saccharomyces cerevisiae transcriptional co-repressors, interacts with Pax3; Magnaghi P et al.; HIRA maps to the DiGeorge/velocardiofacial syndrome critical region (DGCR) at 22q11 (refs 1,2) and encodes a WD40 repeat protein similar to yeast Hir1p and Hir2p . These transcriptional co-repressors regulate cell cycle-dependent histone gene transcription, possibly by remodelling local chromatin structure . We report an interaction between HIRA and the transcription factor Pax3 . Pax3 haploinsufficiency results in the mouse splotch and human Waardenburg syndrome (WSI and WSIII) phenotypes . Mice homozygous for Pax3 mutations die in utero with a phenocopy of DGS, or neonatally with neural tube defects . HIRA was also found to interact with core histones . Thus, altered stoichiometry of complexes containing HIRA may be important for the development of structures affected in WS and DGS. Biochemistry, 1998 Sep 8, 37(36), 12465 - 76 Synthesis, biological activity, and conformational analysis of peptidomimetic analogues of the Saccharomyces cerevisiae alpha-factor tridecapeptide; Zhang YL et al.; Biochemical and biophysical investigations on the Saccharomyces cerevisiae alpha-factor indicate that this tridecapeptide mating pheromone (WHWLQLKPGQPMY) might adopt a type II beta-turn in the center of the peptide when it binds to its G protein-coupled receptor . To test this hypothesis we synthesized analogues of alpha-factor incorporating a (R or S)-gamma-lactam conformational constraint {3-(R or S)-amino-2-oxo-1-pyrrolidineacetamido} in place of the Pro-Gly at residues 8 and 9 of the peptide and tested their biological activities and receptor binding . Analogues were purified to >99% homogeneity as evidenced by high-performance liquid chromatography and capillary electrophoresis and characterized by amino acid analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy . The restricted alpha-factor analogue WHWLQLK{(R)-gamma-lactam}QP{Nle}Y was more active than its lactam-containing diastereomeric homologue WHWLQLK{(S)-gamma-lactam}QP{Nle}Y and about equally active with the {Nle12}-alpha-factor in growth arrest and FUS1-lacZ gene induction assays . Both lactam analogues competed with tritiated {Nle12}-alpha-factor for binding to the alpha-factor receptor (Ste2p) with the (R)-gamma-lactam-containing peptide having 7-fold higher affinity than the (S)-gamma-lactam-containing homologue . Two-dimensional NMR spectroscopy and modeling analysis gave evidence that the (R)-gamma-analogue is a flexible peptide that assumes a transient gamma-turn structure around the lactam moiety . The results represent the first example of an alpha-factor analogue containing a peptidomimetic constraint that is as active as the native pheromone . The correlation between activity and structure provides further evidence that the biologically active conformation of the molecule contains a turn in the middle of the pheromone . This study provides new insights into the structural basis of alpha-factor activity and adds to the repertoire of conformationally biasing constraints that can be used to maintain and even enhance biological activity in peptide hormones. Yeast, 1998 Aug, 14(11), 1041 - 50 A search in the genome of Saccharomyces cerevisiae for genes regulated via stress response elements; Moskvina E et al.; Stress response elements (STREs, core consensus AG4 or C4T) have been demonstrated previously to occur in the upstream region of a number of genes responsive to induction by a variety of stress signals . This stress response is mediated by the homologous transcription factors Msn2p and Msn4p, which bind specifically to STREs . Double mutants (msn2 msn4) deficient in these transcription factors have been shown to be hypersensitive to severe stress conditions . To obtain a more representative overview of the set of yeast genes controlled via this regulon, a computer search of the Saccharomyces cerevisiae genome was carried out for genes, which, similar to most known STRE-controlled genes, exhibit at least two STREs in their upstream region . In addition to the great majority of genes previously known to be controlled via STREs, 69 open reading-frames were detected . Expression patterns of a set of these were examined by grid filter hybridization, and 14 genes were examined by Northern analysis . Comparison of the expression patterns of these genes demonstrates that they are all STRE-controlled although their detailed expression patterns differ considerably. Int J Food Microbiol, 1998 Jul 21, 42(3), 225 - 30 Modification of Saccharomyces cerevisiae thermotolerance following rapid exposure to acid stress; Carmelo V et al.; Thermotolerance was induced in cells of Saccharomyces cerevisiae YPH499 pre-exposed, during 10 min and in the presence of glucose, to a mild acid-stress with HCl at pH 3.5 . Thermotolerance was not induced in cells exposed to a severe acid stress by 50 mM acetic acid at pH 3.5, or HCl at pH 2.5 or pH 2.0 . Yeast cells pre-incubated under glucose starvation were found to be more tolerant to a lethal heat stress than cells pre-incubated in glucose-supplemented media, despite the pH value of the media (range 2.0-6.5) and the type of acidulant used (HCl or acetic acid) . Moreover, the high thermotolerance exhibited by cells pre-incubated at pH 6.5 for 10 min under glucose starvation was not significantly modified by the acidification of the pre-incubation medium . Results are discussed based on the effect that glucose and a mild or severe acid stress have on plasma membrane H+-ATPase activity and on cytosolic pH values, estimated in a previous work. J Pept Res, 1998 Aug, 52(2), 95 - 106 Structure-function analysis of the Saccharomyces cerevisiae tridecapeptide pheromone using alanine-scanned analogs; Abel MG et al.; Twenty-six peptide analogs of the Saccharomyces cerevisiae alpha-factor, a tridecapeptide mating pheromone (W1H2W3L4Q5L6K7p8G9Ql0P11M12Y13) with either L- or D-alanine replacement of each amino acid residue (Ala-scanned) and with the isosteric replacement of methionine at position 12 by norleucine, were synthesized, purified to homogeneity and assayed for biological activity and receptor binding . Two new and effective antagonists, {D-Ala3,Nle12}alpha-factor and {D-Ala4,Nle12}alpha-factor, were found among the series, and the {D-Ala10,Nle12}alpha-factor demonstrated a marked ability to increase the biological activity of {Nle12}alpha-factor without having any effect by itself . One analog, the {L-Ala1 alpha-factor, showed a 3-fold increase in bioactivity over the {Nle12}alpha-factor, although its binding to the alpha-factor receptor was about 70-fold less than {Nle12}alpha-factor . Residues near the carboxyl terminus contributed more strongly to receptor binding than other residues, whereas those near the amine terminus of the alpha-factor played an important role in signal transduction . The effect of insertion of D-Ala residues at positions 7, 8, 9 and 10 on bioactivity and receptor binding of the peptide suggested a specific positioning role of the central loop in establishing optimal contacts between the receptor and the ends of the pheromone . We conclude that the alpha-factor may be divided into segments with dominant roles in forming the biologically active pheromone conformation, in receptor binding and in initiating signal transduction . The discovery of such relationships was made possible by the systematic variation of each residue in the peptide and by the testing of each analog in highly defined biological and binding assays. J Biol Chem, 1998 Sep 11, 273(37), 24102 - 7 Glucose repression in Saccharomyces cerevisiae is related to the glucose concentration rather than the glucose flux; Meijer MM et al.; Glucose plays an important regulatory role in the yeast Saccharomyces cerevisiae, which is mostly reflected at the transcriptional level by glucose repression . The signal that initiates glucose repression is unknown, but data indicate that it is located at or above the level of glucose 6-phosphate, suggesting the involvement of either the intracellular or extracellular glucose concentration or the glucose flux in triggering glucose repression . We have investigated the role of the glucose flux and the extracellular glucose concentration in glucose repression by growing the cells in continuous culture under nitrogen limitation . By a step-wise increase in the glucose feed concentration, the glucose flux and extracellular glucose concentrations were modulated in an accurate way . Furthermore, the glucose flux and glucose concentrations were modulated independently of each other by increasing the dilution rate or by the use of fructose as a substrate . Using these approaches we demonstrate that glucose repression is related to the extracellular (or intracellular) glucose concentration rather than the glucose flux . At external glucose concentrations lower than 14 mM, glucose repression of SUC2 gene transcription was not triggered, whereas glucose repression of this gene was activated when the glucose concentration exceeded 18 mM . A comparable effect was observed for the glucose-repressible carbon source fructose. J Biol Chem, 1998 Sep 11, 273(37), 23805 - 11 Mapping of the DNA binding domain of the copper-responsive transcription factor Mac1 from Saccharomyces cerevisiae; Jensen LT et al.; Mac1 from Saccharomyces cerevisiae activates transcription of genes, including CTR1 in copper-deficient cells . N-terminal fusions of Mac1 with the herpes simplex VP16 activation domain were used to show that residues 1-159 in Mac1 constitute the minimal DNA binding domain . Mac1-(1-159) purified from Escherichia coli contains two bound Zn(II) ions . Electrophoretic mobility shift assays showed direct and specific binding by Mac1-(1-159) to a DNA duplex containing the copper-responsive element TTTGCTCA . The DNA binding affinity of Mac1-(1-159) for a duplex containing a single promoter element or an inverted repeat was 5 nM for the 1:1 complex . The N-terminal 40-residue segment of Mac1 is homologous to the DNA binding zinc module found in the copper-activated transcription factors Ace1 and Amt1 . A MAC1 mutation yielding a Cys11 --> Tyr substitution at the first candidate zinc ligand position relative to Ace1 resulted in a loss of in vivo function . Two TTTGCTCA promoter elements are necessary for efficient Mac1-mediated transcriptional activation . The elements appear to function synergistically . Increasing the number of elements yields more than additive enhancements in CTR1 expression. Mol Biol Cell, 1998 Sep, 9(9), 2439 - 61 A novel fluorescence-based genetic strategy identifies mutants of Saccharomyces cerevisiae defective for nuclear pore complex assembly; Bucci M et al.; Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm . Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism . To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used . This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells . By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutant nup120 and gle2 alleles that result in clusters of NPCs . Interestingly, a third group was a novel temperature-sensitive allele of nup57 . The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57pE17 and wt Nup49p . Interestingly, the nup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC . Decreased levels of NPC-associated Nsp1p and Nup116p were observed . In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished . Coincidentally, nuclear import capacity was inhibited . Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis. Genetics, 1998 Sep, 150(1), 95 - 101 Roles of prenyl protein proteases in maturation of Saccharomyces cerevisiae a-factor; Boyartchuk VL et al.; In eukaryotes small secreted peptides are often proteolytically cleaved from larger precursors . In Saccharomyces cerevisiae multiple proteolytic processing steps are required for production of mature 12-amino-acid a-factor from its 36-amino-acid precursor . This study provides additional genetic data supporting a direct role for Afc1p in cleavage of the carboxyl-terminal tripeptide from the CAAX motif of the prenylated a-factor precursor . In addition, Afc1p had a second role in a-factor processing that was independent of, and in addition to, its role in the carboxyl-terminal processing in vivo . Using ubiquitin-a-factor fusions we confirmed that the pro-region of the a-factor precursor was not required for production of the mature pheromone . However, the pro-region of the a-factor precursor contributed quantitatively to a-factor production. Genetics, 1998 Sep, 150(1), 75 - 93 The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage; Paulovich AG et al.; In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent . Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA . In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA . We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction . We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange . Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication. Genetics, 1998 Sep, 150(1), 59 - 73 Spe3, which encodes spermidine synthase, is required for full repression through NRE(DIT) in Saccharomyces cerevisiae; Friesen H et al.; We previously identified a transcriptional regulatory element, which we call NRE(DIT), that is required for repression of the sporulation-specific genes, DIT1 and DIT2, during vegetative growth of Saccharomyces cerevisiae . Repression through this element is dependent on the Ssn6-Tup1 corepressor . In this study, we show that SIN4 contributes to NRE(DIT)-mediated repression, suggesting that changes in chromatin structure are, at least in part, responsible for regulation of DIT gene expression . In a screen for additional genes that function in repression of DIT (FRD genes), we recovered alleles of TUP1, SSN6, SIN4, and ROX3 and identified mutations comprising eight complementation groups of FRD genes . Four of these FRD genes appeared to act specifically in NRE(DIT)mediated repression, and four appeared to be general regulators of gene expression . We cloned the gene complementing the frd3-1 phenotype and found that it was identical to SPE3, which encodes spermidine synthase . Mutant spe3 cells not only failed to support complete repression through NRE(DIT) but also had modest defects in repression of some other genes . Addition of spermidine to the medium partially restored repression to spe3 cells, indicating that spermidine may play a role in vivo as a modulator of gene expression . We suggest various mechanisms by which spermidine could act to repress gene expression. Genetics, 1998 Sep, 150(1), 31 - 42 Identification of a calcineurin-independent pathway required for sodium ion stress response in Saccharomyces cerevisiae; Ganster RW et al.; The calcium-dependent protein phosphatase calcineurin plays an essential role in ion homeostasis in yeast . In this study, we identify a parallel ion stress response pathway that is independent of the calcineurin signaling pathway . Cells with null alleles in both STD1 and its homologue, MTH1, manifest numerous phenotypes observed in calcineurin mutants, including sodium, lithium, manganese, and hydroxyl ion sensitivity, as well as alpha factor toxicity . Furthermore, increased gene dosage of STD1 suppresses the ion stress phenotypes in calcineurin mutants and confers halotolerance in wild-type cells . However, Std1p functions in a calcineurin-independent ion stress response pathway, since a std1 mth1 mutant is FK506 sensitive under conditions of ion stress . Mutations in other genes known to regulate gene expression in response to changes in glucose concentration, including SNF3, RGT2, and SNF5, also affect cell growth under ion stress conditions . Gene expression studies indicate that the regulation of HAL1 and PMR2 expression is affected by STD1 gene dosage . Taken together, our data demonstrate that response to ion stress requires the participation of both calcineurin-dependent and -independent pathways. Curr Genet, 1998 Aug, 34(2), 146 - 51 C-terminal truncation of the Sup35 protein increases the frequency of de novo generation of a prion-based {PSI+} determinant in Saccharomyces cerevisiae; Kochneva-Pervukhova NV et al.; The yeast non-Mendelian {PSI+} determinant is presumed to be the manifestation of the aggregated prion-like state of the Sup35 protein . Plasmid-mediated amplification of the SUP35 gene greatly increases the frequency of Sup35p transition to this prion-like state . Here we show that the 3'-deletions of plasmid SUP35, leading to the C-terminal truncation of Sup35p, further increase the frequency of {PSI+} induction despite a marked decrease in Sup35p expression levels . The data suggest that the presence of Sup35p N-terminal proteolytic fragments can cause {PSI+} appearance in wild-type yeast cells. Curr Genet, 1998 Aug, 34(2), 138 - 45 Molecular and biochemical analysis of Saccharomyces cerevisiae cox1 mutants; Lemaire C et al.; We report on the molecular and biochemical analysis of a set of 13 respiratory deficient mutants of Saccharomyces cerevisiae which are specifically altered in COX1, the gene encoding the subunit Cox1p of cytochrome c oxidase . DNA sequence analysis shows that three are due to frameshift mutations, two to nonsense mutations, and eight to missense mutations . All, except the missense mutant S157L, have impaired electron transfer and respiratory activity . Analysis of the mitochondrial translation products shows that when Cox1p is absent, Cox2p and Cox3p are still synthesized . In the missense mutants, the steady state levels in the mitochondrial membranes of the three mitochondrially encoded subunits Cox1p, Cox2p and Cox3p and the nuclear-encoded subunit Cox4p are reduced . In the frameshift and nonsense mutants, Cox1p is absent and Cox2p, Cox3p and Cox4p are considerably decreased or undetectable . A comparison of the steady state levels of Cox1p through Cox4p in the COX1, COX2, COX3 and COX4 mutants shows the interdependance of the accumulation of these four subunits in the mitochondrial membranes. Curr Genet, 1998 Aug, 34(2), 124 - 7 The Saccharomyces cerevisiae gene PSO5/RAD16 is involved in the regulation of DNA damage-inducible genes RNR2 and RNR3; Paesi-Toresan SO et al.; The expression of beta-galactosidase from DNA damage-inducible RNR2-lacZ and RNR3-lacZ fusion constructs was compared in wild-type (WT) and pso5/rad16 mutant strains after treatment with five mutagens/oxidative stressors . While exposure to the mutagens UVC, 4NQO and H2O2 induced expression of the RNR2-lacZ and RNR3-lacZ fusion constructs in two WT strains, treatment with the two oxidative stressors tBOOH and paraquat did not . In the pso5-1 mutant induction of RNR2-lacZ was largely reduced after UVC and H2O2 while there was no significant induction of beta-galactosidase expression after 4NQO treatment for this construct . For RNR3-lacZ there was strongly reduced expression of pso5-1 after UVC and 4NQO while H2O2 failed to induce expression of beta-galactosidase . In the WT strains the ranking of the inducing power of the mutagens at 90% survival (as measured in the pso5-1 mutant) was 4NQO>UVC>H2O2 . Though the WT strains were clearly more resistant that the pso5-1 mutant to the two oxidative stressors paraquat and tBOOH, these substances failed to significantly enhance expression of the RNR2-lacZ and RNR3-lacZ fusion constructs in both the WT and the pso5-1 mutant . Our data suggest that Pso5p/Rad16p has a function in the signal transducing pathway controlling DNA damage-inducible components of nucleotide excision repair. Curr Genet, 1998 Aug, 34(2), 93 - 9 A calcium-dependent ergosterol mutant of Saccharomyces cerevisiae; Crowley JH et al.; ERG24 is the structural gene for the C14-sterol reductase in yeast . A lack of activity in that enzyme, mediated either by the morpholine fungicides or the insertional inactivation of ERG24, causes the accumulation of the aberrant sterol ignosterol . Cells producing this sterol are unable to grow aerobically in the routine laboratory medium, YPD . However, growth does occur on a synthetic defined medium . A novel calcium-dependent phenotype associated with alterations in the ergosterol biosynthetic pathway in yeast is described . In addition, reduction of yeast growth with an azole inhibitor of the C-14 sterol de-methylase was also modulated by an excess of calcium ions in the culture medium . These results define a new effect of ergosterol deficiency and provide important practical implications for utilizing morpholine and azole sterol biosynthetic-inhibiting fungicides. Nucleic Acids Res, 1998 Sep 15, 26(18), 4137 - 45 Cooperative interaction of branch signals in the actin intron of Saccharomyces cerevisiae; Castanotto D et al.; In pre-mRNA splicing, specific spliceosomal components recognize key intron sequences, but the mechanisms by which splice sites are selected arenot completely understood . In the Saccharomyces cerevisiae actin intron a silent branch point-like sequence (UACUAAG) is located 7 nt upstream of the canonical sequence . Mutation of the canonicalUACUAAC sequence to UAAUAAC reduces utilization of this signal and activates the cryptic UACUAAG . Splicing-dependent beta-galactosidase assays have shown that these two splice signals cooperate to enhance splicing . Analyses of several variants of this double branch point intron demonstrate that the upstream UACUAAG sequence significantly increases usage of the UAAUAAC as a site of lariat formation . This activation is sequence-specific and unidirectional . However the ability of the UACUAAG signal to activate the downstream branch point is dependent on the presence of a short non-conserved sequence located a few nucleotides upstream of the UACUAAG . Mutation of this sequence leads to the disappearance of the cooperative interactions between the two branch signals . Our results show that this non-conserved sequence and the UACUAAG signal must both be present to achieve activation of the downstream branch point and suggest that a specific structure may be necessary to allow efficient recognition of the UAAUAAC. J Biol Chem, 1998 Sep 4, 273(36), 22921 - 8 The cytoplasmic Cu,Zn superoxide dismutase of saccharomyces cerevisiae is required for resistance to freeze-thaw stress . Generation of free radicals during freezing and thawing; Park JI et al.; The involvement of oxidative stress in freeze-thaw injury to yeast cells was analyzed using mutants defective in a range of antioxidant functions, including Cu,Zn superoxide dismutase (encoded by SOD1), Mn superoxide dismutase (SOD2), catalase A, catalase T, glutathione reductase, gamma-glutamylcysteine synthetase and Yap1 transcription factor . Only those affecting superoxide dismutases showed decreased freeze-thaw tolerance, with the sod1 mutant and the sod1 sod2 double mutant being most affected . This indicated that superoxide anions were formed during freezing and thawing . This was confirmed since the sod1 mutant could be made more resistant by treatment with the superoxide anion scavenger MnCl2, or by freezing in the absence of oxygen, or by the generation of a rho0 petite . Increased expression of SOD2 conferred freeze-thaw tolerance on the sod1 mutant indicating the ability of the mitochondrial superoxide dismutase to compensate for the lack of the cytoplasmic enzyme . Free radicals generated as a result of freezing and thawing were detected in cells directly using electron paramagnetic resonance spectroscopy with either alpha-phenyl-N-tert-butylnitrone or 5, 5-dimethyl-1-pyrroline-N-oxide as spin trap . Highest levels were formed in the sod1 and sod1 sod2 mutant strains, but lower levels were detected in the wild type . The results show that oxidative stress causes major injury to cells during aerobic freezing and thawing and that this is mainly initiated in the cytoplasm by an oxidative burst of superoxide radicals formed from oxygen and electrons leaked from the mitochondrial electron transport chain. J Bacteriol, 1998 Sep, 180(17), 4508 - 15 Transcriptional regulation of CLN3 expression by glucose in Saccharomyces cerevisiae; Parviz F et al.; In Saccharomyces cerevisiae, the transition from the G1 phase of the mitotic cycle into S phase is controlled by a set of G1 cyclins that regulate the activity of the protein kinase encoded by CDC28 . Yeast cells regulate progress through the G1/S boundary in response to nutrients, moving quickly through G1 in glucose medium and more slowly in poorer medium . We have examined connections between glucose and the level of the message encoding Cln3, a G1 cyclin . We found that glucose positively regulates CLN3 mRNA levels through a set of repeated AAGAAAAA (A2GA5) elements within the CLN3 promoter . Mutations in these sequences reduce both transcriptional activation and specific interaction between CLN3 promoter elements and proteins in yeast extracts . Creation of five point mutations, replacing the G's within these repeats with T's, in the CLN3 promoter substantially reduces CLN3 expression in glucose medium and inhibits the ability of the cells to maintain a constant size when shifted into glucose. J Bacteriol, 1998 Sep, 180(17), 4460 - 5 Farnesol-induced generation of reactive oxygen species via indirect inhibition of the mitochondrial electron transport chain in the yeast Saccharomyces cerevisiae; Machida K et al.; The mechanism of farnesol (FOH)-induced growth inhibition of Saccharomyces cerevisiae was studied in terms of its promotive effect on generation of reactive oxygen species (ROS) . The level of ROS generation in FOH-treated cells increased five- to eightfold upon the initial 30-min incubation, while cells treated with other isoprenoid compounds, like geraniol, geranylgeraniol, and squalene, showed no ROS-generating response . The dependence of FOH-induced growth inhibition on such an oxidative stress was confirmed by the protection against such growth inhibition in the presence of an antioxidant such as alpha-tocopherol, probucol, or N-acetylcysteine . FOH could accelerate ROS generation only in cells of the wild-type grande strain, not in those of the respiration-deficient petite mutant ({rho0}), which illustrates the role of the mitochondrial electron transport chain as its origin . Among the respiratory chain inhibitors, ROS generation could be effectively eliminated with myxothiazol, which inhibits oxidation of ubiquinol to the ubisemiquinone radical by the Rieske iron-sulfur center of complex III, but not with antimycin A, an inhibitor of electron transport that is functional in further oxidation of the ubisemiquinone radical to ubiquinone in the Q cycle of complex III . Cellular oxygen consumption was inhibited immediately upon extracellular addition of FOH, whereas FOH and its possible metabolites failed to directly inhibit any oxidase activities detected with the isolated mitochondrial preparation . A protein kinase C (PKC)-dependent mechanism was suggested to exist in the inhibition of mitochondrial electron transport since FOH-induced ROS generation could be effectively eliminated with a membrane-permeable diacylglycerol analog which can activate PKC . The present study supports the idea that FOH inhibits the ability of the electron transport chain to accelerate ROS production via interference with a phosphatidylinositol type of signal. Arch Biochem Biophys, 1998 Sep 1, 357(1), 58 - 66 Purification and characterization of type 1 protein phosphatase from Saccharomyces cerevisiae: effect of the R73C mutation; Dignam SS et al.; Type 1 protein phosphatase encoded by the GLC7 gene was purified from Saccharomyces cerevisiae as a 1:1 complex with mammalian inhibitor 2 fused to glutathione S-transferase . The complex was inactive and required treatment with Co2+ and trypsin for maximal activity . The specific activity toward phosphorylase a was about 1.8 units/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 7.3, 81, and 0.30 nM, respectively . The complex could be activated by glycogen synthase kinase-3 in the presence of Mg2+ and ATP to 20% of the activity seen with Co2+ and trypsin . Thus, the catalytic properties of the yeast type 1 phosphatase are similar to those of the mammalian protein phosphatase 1 . The R73C mutant phosphatase from the glycogen-deficient strain, glc7-1, purified as a 1:1 complex with the inhibitor 2 fusion, had a specific activity toward phosphorylase a of 0.9 unit/mg of Glc7p, and IC50's for inhibitor 2, okadaic acid, and microcystin-LR were 13 . 1, 113, and 0.37 nM, respectively . The R73C mutation slightly decreases the specific activity and sensitivity to inhibitors, suggesting that changes in biochemical properties may affect glycogen levels . However, the modest changes are consistent with our previous proposal (E . M . Reimann et al., 1993, Adv . Protein Phosphatases 7,173-182) and with the results of Stuart et al . (1994, Mol . Cell . Biol . 14, 896-905) that the mutation may selectively alter the interaction of Glc7p with regulatory proteins . Biosci Biotechnol Biochem, 1998 Jul, 62(7), 1403 - 11 Molecular cloning and expression in Saccharomyces cerevisiae of tobacco NADPH-cytochrome P450 oxidoreductase cDNA; Yamada T et al.; We obtained information on the full length tobacco NADPH-cytochrome P450 oxidoreductase (P450 reductase) by a combination of the cDNA clone pCTR1 and the genomic DNA clone pGTR1 . The deduced primary structure consisting of 713 amino acid residues contained sequences corresponding to FMN, FAD, and NADPH-binding regions . Based on this information, we prepared the full-length cDNA pFTR of tobacco P450 reductase by RT-PCR and expressed it in the yeast Saccharomyces cerevisiae . The transformed yeast cells carrying pFTR produced the corresponding mRNA and protein, and had increased cytochrome c reductase activity in the microsomes . An in vitro reconstitution system of the yeast microsomal fractions expressed tobacco P450 reductase and rat P450 1A1 showed an increased 7-ethoxycoumarin O-deethylase activity . These results indicated that tobacco P450 reductase expressed in the yeast microsomes coupled with rat P450 1A1 resulting in an increased monooxygenase activity. Biosci Biotechnol Biochem, 1998 Jul, 62(7), 1376 - 80 Functional interaction of Isr1, a predicted protein kinase, with the Pkc1 pathway in Saccharomyces cerevisiae; Miyahara K et al.; Staurosporine is a potent inhibitor of protein kinase C . To identify the genes that functionally interact with the Pkc1 pathway of the yeast Saccharomyces cerevisiae, we screened for the genes that cause induced staurosporine sensitivity when overexpressed from a galactose-inducible promoter . The novel gene ISR1 encodes a predicted protein kinase with the highest sequence similarity to mammalian Raf in the kinase domain . Drug sensitivity induced by ISR1 overexpression is specific to staurosporine . Although ISR1 disruption causes no obvious phenotype, it does exacerbate the phenotypes of a temperature-sensitive allele (stt1-1) of PKC1, but not of the mpk1 and bck1 mutants of the Mpk1 MAP kinase pathway . These results suggest that Isr1 functions in an event important for growth in a manner redundant with a Mpk1-independent branch of the Pkc1 signalling pathways. J Cell Sci, 1998 Sep, 111 ( Pt 18), 2809 - 18 SPC72: a spindle pole component required for spindle orientation in the yeast Saccharomyces cerevisiae; Soues S et al.; The monoclonal antibody 78H6 recognises an 85 kDa component of the yeast spindle pole body . Here we identify and characterise this component as Spc72p, the product of YAL047C . The sequence of SPC72 contains potential coiled-coil domains; its overexpression induced formation of large polymers that were strictly localised at the outer plaque and at the bridge of the spindle pole body . Immunoelectron microscopy confirmed that Spc72p was a component of these polymers . SPC72 was found to be non-essential for cell growth, but its deletion resulted in abnormal spindle positioning, aberrant nuclear migration and defective mating capability . Precisely, deletion of SPC72 resulted in a decreased number of astral microtubules: early in the cell cycle only few were detectable, and these were unattached to the spindle pole body in small-budded cells . Later in the cell cycle few, if any, remained, and they were unable to align the spindle properly . We conclude that Spc72p is not absolutely required for nucleation per se, but is needed for normal abundance and stability of astral microtubules. Yeast, 1998 Jul, 14(10), 915 - 22 Genetic interaction between YPT6 and YPT1 in Saccharomyces cerevisiae; Li B et al.; Ypt6p, the yeast homologue of human RAB6, is required for protein trafficking at elevated temperatures . Biochemical data provide evidence that Ypt6p plays a role in an early step(s) of the secretory pathway: from ER to Golgi, or from cis to medial Golgi, or both . Here we show that overexpression of YPT1 suppresses the growth and secretion defects of a ypt6 temperature-sensitive (ts) strain . SLY1-20, encoding a dominant mutant allele that suppresses the lethal effect of YPT1, also suppresses the growth defect of a ypt6 ts strain . Conversely, SSD1, isolated as a suppressor of ypt6 ts, can suppress the growth defect of a ypt1 ts allele . These data suggest that Ypt6p has some redundant function with Ypt1p . However, overexpression of Ypt6p is toxic to a ypt1 ts strain, although it does not affect the growth of wild-type cells, suggesting that Ypt6p may sequester proteins shared with Ypt1p . This genetic evidence confirms the conclusion that Ypt6p is involved in an early step of the secretory pathway. Yeast, 1998 Jul, 14(10), 905 - 13 Control of division arrest and entry into meiosis by extracellular alkalisation in Saccharomyces cerevisiae; Hayashi M et al.; Limitation of nutrients allows yeast cells to arrest proliferation at G1 phase of the cell cycle and to enter the so-called stationary phase . We show here another pathway for cytostasis, which is associated with extracellular accumulation of bicarbonate and the resulting alkalisation of medium during the proliferation of cells respiring acetate . Alkalisation of medium by addition of bicarbonate or alkaline buffers ceased proliferation at G1 phase of logarithmically growing cells and caused a severe drop in G1-cyclin (CLN1 and CLN2) mRNAs . The arrested cells were heat-shock resistant, suggesting that the cells entered the stationary phase . Cells confluently grown on acetate re-entered into the cell cycle after acidification of the culture medium . These results indicate that external alkalisation is a primary cause of the cytostasis . The alkali-induced G1 arrest was shown to be cyclic AMP (cAMP)-independent using mutant cells which lack a functional Ras/cAMP signaling pathway . Alkalisation of medium also stimulated meiosis and sporulation in rich acetate medium, confirming our previous proposal that environmental alkalisation but not nitrogen limitation is a key condition for entry into meiosis and sporulation. Biochemistry, 1998 Aug 25, 37(34), 11989 - 95 Hexokinase 2 from Saccharomyces cerevisiae: regulation of oligomeric structure by in vivo phosphorylation at serine-14; Behlke J et al.; Homodimeric hexokinase 2 from Saccharomyces cerevisiae is known to have two sites of phosphorylation: for serine-14 the modification in vivo increases with glucose exhaustion {Kriegel et al . (1994) Biochemistry 33, 148-152}, while for serine-157 it occurs in vitro with ATP in the presence of nonphosphorylateable five-carbon analogues of glucose {Heidrich et al . (1997) Biochemistry 36, 1960-1964} . We show now by site-directed mutagenesis and sedimentation analysis that serine-14 phosphorylation affects the oligomeric state of hexokinase, its substitution by glutamate causing complete dissociation; glutamate exchange for serine-157 does not . Phosphorylation of wild-type hexokinase at serine-14 likewise causes dissociation in vitro . In view of the higher glucose affinity of monomeric hexokinase and the high hexokinase concentration in yeast {Womack, F., and Colowick, S . P . (1978) Arch . Biochem . Biophys . 191, 742-747; Mayes, E . L., Hoggett, J . G., and Kellett, G . L . (1983) Eur . J . Biochem . 133, 127-134}, we speculate that the in vivo phosphorylation at serine-14 as transiently occurring in glucose derepression might provide a mechanism to improve glucose utilization from low level and/or that nuclear localization of the monomer might be involved in the signal transduction whereby glucose causes catabolite repression. Antonie Van Leeuwenhoek, 1998 Feb, 73(2), 195 - 8 Effect of a Teucrium polium L . extract on the growth and fatty acid composition of Saccharomyces cerevisiae and Yarrowia lipolytica; Aggelis G et al.; Aqueous Teucrium polium extract slightly inhibits the growth of Saccharomyces cerevisiae (Ki = 0.029 {g/l}-1) and Yarrovia lipolytica (Ki = 0.06l {g/l}-1) . However, this extract causes important changes in the unsaturation degree (delta/mol) of the cellular lipids . It moreover favours the increase of the linolenic acid concentration and the decrease of the oleic one in both species. Indian J Exp Biol, 1998 May, 36(5), 493 - 6 Effect of sodium on Saccharomyces cerevisiae invertase activity; Raheja G et al.; Effect of Na+ ions on yeast invertase activity has been studied as a function of pH . At acidic pH, Na+ (5-100 mM) had no effect but invertase activity was inhibited (38-44%) by Na+ ions (100 mM) with an increase in pH (6.8 and 8.0) . Kinetic analysis revealed that invertase inhibition by Na+ ions was non-competitive and reversible in nature . Value of K(m) remained unaltered (33.3 mM) in presence of Na+ (20-100 mM) while Vmax decreased by 21-44% under these conditions . Value of Ki was of the order of 85 mM . Mechanism of observed inhibition of invertase activity as a consequence of Na+ ions interactions at the active site of the enzyme has been described. Biochem Mol Biol Int, 1998 Jul, 45(4), 663 - 71 De novo protein synthesis is essential for thermotolerance acquisition in a Saccharomyces cerevisiae trehalose synthase mutant; Gross C et al.; Heat shock (25 degrees C to 37 degrees C for 30 min) acquisition of thermotolerance (at 50 degrees C) was observed in a yeast trehalose synthase mutant and the corresponding control strain . The acquisition of thermotolerance in the control strain was maintained for a significantly longer time than in the trehalose synthase mutant . The heat shock was associated with the synthesis of specific heat shock proteins and, in the case of the control strain, also trehalose accumulation . Inhibition of protein synthesis during the heat shock totally abolished acquisition of thermotolerance in both strains but not trehalose accumulation in the control . It was concluded that trehalose may only be required for prolonged stress protection while heat shock proteins are required for heat shock acquisition of thermotolerance. J Biol Chem, 1998 Aug 28, 273(35), 22480 - 9 The H2O2 stimulon in Saccharomyces cerevisiae; Godon C et al.; The changes in gene expression underlying the yeast adaptive stress response to H2O2 were analyzed by comparative two-dimensional gel electrophoresis of total cell proteins . The synthesis of at least 115 proteins is stimulated by H2O2, whereas 52 other proteins are repressed by this treatment . We have identified 71 of the stimulated and 44 of the repressed targets . The kinetics and dose-response parameters of the H2O2 genomic response were also analyzed . Identification of these proteins and their mapping into specific cellular processes give a distinct picture of the way in which yeast cells adapt to oxidative stress . As expected, H2O2-responsive targets include an important number of heat shock proteins and proteins with reactive oxygen intermediate scavenging activities . Exposure to H2O2 also results in a slowdown of protein biosynthetic processes and a stimulation of protein degradation pathways . Finally, the most remarkable result inferred from this study is the resetting of carbohydrate metabolism minutes after the exposure to H2O2 . Carbohydrate fluxes are redirected to the regeneration of NADPH at the expense of glycolysis . This study represents the first genome-wide characterization of a H2O2-inducible stimulon in a eukaryote. J Biol Chem, 1998 Aug 28, 273(35), 22284 - 91 Apg14p and Apg6/Vps30p form a protein complex essential for autophagy in the yeast, Saccharomyces cerevisiae; Kametaka S et al.; Mutation in the Saccharomyces cerevisiae APG14 gene causes a defect in autophagy . Cloning and structural analysis of the APG14 gene revealed that APG14 encodes a novel hydrophilic protein with a predicted molecular mass of 40.5 kDa, and that Apg14p has a coiled-coil motif at its N terminus region . We found that overproduction of Apg14p partially reversed the defect in autophagy induced by the apg6-1 mutation . The apg6-1 mutant was found to be defective not only in autophagy but also in sorting of carboxypeptidase Y (CPY), a vacuolar-soluble hydrolase, to the vacuole . However, overexpression of APG14 did not alter the CPY sorting defect of the apg6-1 mutant, nor did the apg14 null mutation affect the CPY sorting pathway . Structural analysis of APG6 revealed that APG6 is identical to VPS30, which is involved in a retrieval step of the CPY receptor, Vps10p, to the late-Golgi from the endosome (Seaman, M . N . J., Marcusson, E . G., Cereghino, J . L., and Emr, S . D . (1997) J . Cell Biol . 137, 79-92) . Subcellular fractionation indicated that Apg14p and Apg6p peripherally associated with a membrane structure(s) . Apg14p was co-immunoprecipitated with Apg6p, suggesting that they form a stable protein complex . These results imply that Apg6/Vps30p has two distinct functions in the autophagic process and the vacuolar protein sorting pathway . Apg14p may be a component specifically required for the function of Apg6/Vps30p through the autophagic pathway. FEMS Microbiol Lett, 1998 Aug 1, 165(1), 111 - 6 Soluble fumarate reductase isoenzymes from Saccharomyces cerevisiae are required for anaerobic growth; Arikawa Y et al.; In Saccharomyces cerevisiae, the cytosolic and promitochondrial isoenzymes of fumarate reductase are encoded by the FRDS and OSM1 genes, respectively . The product of the OSM1 gene is reported to be required for growth in hypertonic medium . Simultaneous disruption of the FRDS and OSM1 genes resulted in the inability of the yeasts to grow anaerobically on glucose as a carbon source, and disruption of the OSM1 gene caused poor growth under anaerobic conditions . However, the disruption of both the FRDS and/or OSM1 genes had no effect on aerobic growth or growth under hypertonic conditions . These results suggest that the fumarate reductase isoenzymes in Saccharomyces cerevisiae are essential for anaerobic growth but not for growth under hypertonic conditions. Mol Cell Biol, 1998 Sep, 18(9), 5392 - 403 High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating type locus HMLalpha; Weiss K et al.; Genetic studies have suggested that chromatin structure is involved in repression of the silent mating type loci in Saccharomyces cerevisiae . Chromatin mapping at nucleotide resolution of the transcriptionally silent HMLalpha and the active MATalpha shows that unique organized chromatin structure characterizes the silent state of HMLalpha . Precisely positioned nucleosomes abutting the silencers extend over the alpha1 and alpha2 coding regions . The HO endonuclease recognition site, nuclease hypersensitive at MATalpha, is protected at HMLalpha . Although two precisely positioned nucleosomes incorporate transcription start sites at HMLalpha, the promoter region of the alpha1 and alpha2 genes is nucleosome free and more nuclease sensitive in the repressed than in the transcribed locus . Mutations in genes essential for HML silencing disrupt the nucleosome array near HML-I but not in the vicinity of HML-E, which is closer to the telomere of chromosome III . At the promoter and the HO site, the structure of HMLalpha in Sir protein and histone H4 N-terminal deletion mutants is identical to that of the transcriptionally active MATalpha . The discontinuous chromatin structure of HMLalpha contrasts with the continuous array of nucleosomes found at repressed a-cell-specific genes and the recombination enhancer . Punctuation at HMLalpha may be necessary for higher-order structure or karyoskeleton interactions . The unique chromatin architecture of HMLalpha may relate to the combined requirements of transcriptional repression and recombinational competence. FEBS Lett, 1998 Jul 31, 432(1-2), 59 - 64 NSC1: a novel high-current inward rectifier for cations in the plasma membrane of Saccharomyces cerevisiae; Bihler H et al.; The plasma membrane of the yeast Saccharomyces cerevisiae possesses a non-specific cation 'channel', tentatively dubbed NSC1, which is blocked by normal (mM) calcium and other divalent metal ions, is unblocked by reduction of extracellular free divalents below approximately 10 microM, and is independent of the identified potassium channel and porters in yeast, Duk1p, Trk1p, and Trk2p . Ion currents through NSC1, observed by means of whole-cell patch recording, have the following characteristics: Large amplitude, often exceeding 1 nA of K+/ cell at -200 mV, in tetraploid yeast, sufficient to double the normal intracellular K+ concentration within 10 s; non-saturation at large negative voltages; complicated activation kinetics, in which approximately 50% of the total current arises nearly instantaneously with a voltage-clamp step, while the remainder develops as two components, with time constants of approximately 100 ms and approximately 1.3 s; and voltage independence of both the activation time constants and the associated fractional current amplitudes. FEBS Lett, 1998 Jul 17, 431(2), 297 - 9 NO+, but not NO., inhibits respiratory oscillations in ethanol-grown chemostat cultures of Saccharomyces cerevisiae; Murray DB et al.; A continuous culture of Saccharomyces cerevisiae strain IFO 0233 growing aerobically at pH 3.4 shows persistent high-amplitude respiratory oscillations with a period of about 45 min . These robust autonomous cycles are accompanied by changes of product accumulation (acetaldehyde and acetic acid), intracellular pH, and intracellular redox state, as indicated by continuously monitored NADH fluorescence and the glutathione content of cell-free extracts . Perturbation of the oscillation of dissolved O2 was produced on addition of 100 microM glutathione, > 10 nM Na nitroprusside, 8 microM NaNO2, or 10 microM S-nitrosoglutathione . NO gas, putative NO.-releasing agents, or an inhibitor of NO synthase were ineffective . We suggest that nitrosation by NO+ of a component of a redox switch can account for these data, and we emphasise the different modes of action of the different redox forms of nitrogen monoxide. EMBO J, 1998 Aug 17, 17(16), 4771 - 9 Poly(A) signals control both transcriptional termination and initiation between the tandem GAL10 and GAL7 genes of Saccharomyces cerevisiae; Greger IH et al.; We have investigated transcriptional interactions between the GAL10 and GAL7 genes of Saccharomyces cerevisiae . Both genes are part of the galactose (GAL) gene cluster which is transcriptionally activated to high levels in the presence of galactose . Since GAL7 is positioned downstream of GAL10 and both genes are expressed co-ordinately at high levels, the possibility that GAL10 transcription influences GAL7 was analysed . Using transcriptional run-on assays, we show that high levels of polymerase are found in the 600 bp GAL10-7 intergenic region that accumulate over the GAL7 promoter . Furthermore, GAL7 transcription is enhanced when the GAL10 upstream activating sequence (UASG) is deleted, indicating that interference between GAL10 and GAL7 is likely to occur in the chromosomal locus . Deletions in the GAL10 poly(A) signal result in complete inactivation of the GAL7 promoter and cause a dramatic increase in bi-cistronic GAL10-7 mRNA, predominantly utilizing the downstream, GAL7 poly(A) site . These data demonstrate a pivotal role for the GAL10 poly(A) site in allowing the simultaneous expression of GAL10 and GAL7 . In effect, this RNA processing signal has a direct influence on both transcriptional termination and initiation. Biochem Biophys Res Commun, 1998 Jul 30, 248(3), 542 - 7 Hsp104 responds to heat and oxidative stress with different intracellular localization in Saccharomyces cerevisiae; Fujita K et al.; TPN (tetrachloroisophthalonitrile) affected the growth in yeast Saccharomyces cerevisiae and enhanced the superoxide dismutase and glutathione reductase activity under sublethal concentration . Conversely, mild heat-shock treatment had no effect on the enzyme activities . These show they inhibit the metabolism diversely: TPN is an oxidative stressor and mild heat-shock treatment leads to thermogenesis . We have earlier reported that on exposure to TPN under sublethal concentration, heat-shock protein Hsp104 was induced in the same way as in the mild heat-shock treatment (Fujita et al., Biochem . Biophys . Res . Commun . (1995) 216, 1041-1047) . However, intracellular localizations of Hsp104 showed different patterns in each treated cell according to immunoelectron microscopic observation . While Hsp104 was localized upon the circumference of the protein aggregates in mild heat-shocked cells, Hsp104 was distributed over the entire TPN-treated cells with no protein aggregates . These findings suggest Hsp104 adaptively responds to comprehensive stress and participates in an emergent rescue function as a molecular chaperone. Nat Biotechnol, 1998 Aug, 16(8), 773 - 7 Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments; Shusta EV et al.; We have produced single-chain antibody fragments (scFv) in Saccharomyces cerevisiae at levels up to 20 mg/L in shake flask culture by a combination of expression level tuning and overexpression of folding assistants . Overexpression of the chaperone BiP or protein disulfide isomerase (PDI) increases secretion titers 2-8 fold for five scFvs . The increases occur for scFv expression levels ranging from low copy to ER-saturating overexpression . The disulfide isomerase activity of PDI, rather than its chaperone activity, is responsible for the secretion increases . A synergistic increase in scFv production occurs upon cooverexpression of BiP and PDI. Mol Microbiol, 1998 Jul, 29(1), 297 - 310 PTR3, a novel gene mediating amino acid-inducible regulation of peptide transport in Saccharomyces cerevisiae; Barnes D et al.; We have isolated and characterized the Saccharomyces cerevisiae PTR3 gene by functional complementation of a mutant deficient for amino acid-inducible peptide transport . PTR3 is predicted to encode a protein of 678 amino acids that exhibits no similarity to any other protein in the database . Deletion of the PTR3 open reading frame pleiotropically reduced the sensitivity to toxic peptides and amino acid analogues . Initial rates of radiolabelled dipeptide uptake demonstrated that elimination of PTR3 resulted in the loss of amino acid-induced levels of peptide transport . PTR3 was required for amino acid-induced expression of PTR2, the gene encoding the dipeptide/tripeptide transport protein, but was not necessary for nitrogen catabolite repression of peptide import or PTR2 expression . It was determined that PTR3 also modulates expression of BAP2, the gene encoding the branched-amino acid permease . Furthermore, we present genetic evidence that suggests that PTR3 functions within a novel regulatory pathway that facilitates amino acid induction of the PTR system. Mol Microbiol, 1998 Jul, 29(1), 151 - 63 A nonameric core sequence is required upstream of the LYS genes of Saccharomyces cerevisiae for Lys14p-mediated activation and apparent repression by lysine; Becker B et al.; The expression of the structural genes for lysine (LYS) biosynthesis is controlled by a pathway-specific regulation mediated by the transcriptional activator Lys14 in the presence of alpha-aminoadipate semialdehyde, an intermediate of the pathway acting as a co-inducer . Owing to end product inhibition of the first step of the pathway, excess lysine reduces the production of the co-inducer and causes apparent repression of the LYS genes . Analysis of LYS promoters and insertions within an heterologous reporter gene have allowed the characterization of an upstream activating element (UASLYS) able to confer Lys14- and alpha-amino-adipate semialdehyde-dependent activation as well as apparent repression by lysine to another yeast gene . This DNA motif is present as one of several copies in the promoters of at least six LYS genes . The consensus sequence derived from the comparison of the UASLYS showing the highest activation capacities comprises the nonameric core sequence TCCRNYGGA . The RNY sequence of the 3 bp spacer as well as the presence of flanking AT-rich regions on both sides of the core sequence appear essential for optimal activation . Further evidence that this element is the target of Lys14p was provided by the demonstration that Lys14p binds to UASLYS in vitro . The binding is independent of the presence of the co-inducer and is not affected by lysine . It depends on the integrity of the putative Zn(II)2Cys6 binuclear cluster contained in the Lys14p. J Cell Sci, 1998 Sep, 111 ( Pt 17), 2689 - 96 Depolarization of the actin cytoskeleton is a specific phenotype in Saccharomyces cerevisiae; Karpova TS et al.; The yeast actin cytoskeleton is polarized during most of the cell cycle . Certain environmental factors and mutations are associated with depolarization of the actin cytoskeleton . Is depolarization of the actin cytoskeleton a specific response, or is it a nonspecific reaction to harsh conditions or poor metabolism? If depolarization is a nonspecific response, then any mutation that slows growth should induce depolarization . In addition, the number of genes with the depolarization phenotype should constitute a relatively large part of the genome . To address this question, we determined the effect of slow growth on the actin cytoskeleton, and we determined the frequency of mutations that affect the actin cytoskeleton . Eight mutants with slow growth showed no defect in actin polarization, indicating that slow growth alone is not sufficient to cause depolarization . Among 273 viable haploids disrupted for ORFs of chromosome I and VIII and 950 viable haploids with random genome disruptions, none had depolarization of the cytoskeleton . We conclude that depolarization of the actin cytoskeleton is a specific phenotype. J Cell Biol, 1998 Aug 10, 142(3), 711 - 22 Mlc1p is a light chain for the unconventional myosin Myo2p in Saccharomyces cerevisiae; Stevens RC et al.; In Saccharomyces cerevisiae, the unconventional myosin Myo2p is of fundamental importance in polarized growth . We explore the role of the neck region and its associated light chains in regulating Myo2p function . Surprisingly, we find that precise deletion of the six IQ sites in the neck region results in a myosin, Myo2-Delta6IQp, that can support the growth of a yeast strain at 90% the rate of a wild-type isogenic strain . We exploit this mutant in a characterization of the light chains of Myo2p . First, we demonstrate that the localization of calmodulin to sites of polarized growth largely depends on the IQ sites in the neck of Myo2p . Second, we demonstrate that a previously uncharacterized protein, Mlc1p, is a myosin light chain of Myo2p . MLC1 (YGL106w) is an essential gene that exhibits haploinsufficiency . Reduced levels of MYO2 overcome the haploinsufficiency of MLC1 . The mutant MYO2-Delta6IQ is able to suppress haploinsufficiency but not deletion of MLC1 . We used a modified gel overlay assay to demonstrate a direct interaction between Mlc1p and the neck of Myo2p . Overexpression of MYO2 is toxic, causing a severe decrease in growth rate . When MYO2 is overexpressed, Myo2p is fourfold less stable than in a wild-type strain . High copies of MLC1 completely overcome the growth defects and increase the stability of Myo2p . Our results suggest that Mlc1p is responsible for stabilizing this myosin by binding to the neck region. Biochemistry, 1998 Aug 11, 37(32), 11171 - 81 Structure determination and characterization of Saccharomyces cerevisiae profilin; Eads JC et al.; The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution . The overall fold of yeast profilin is similar to the fold observed for other profilin structures . The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics . The affinity of yeast profilin for rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 microM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins . The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to the Kd of 2.0 microM for the interaction between yeast profilin and pyrene-labeled yeast actin . The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface . The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar to the reported affinity of Acanthamoeba profilin for poly-L-proline . Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin . The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure. Nat Genet, 1998 Aug, 19(4), 384 - 9 Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae; Shimodaira H et al.; Hereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6) is a cancer-susceptibility syndrome linked to inherited defects in human mismatch repair (MMR) genes . Germline missense human MLH1 (hMLH1) mutations are frequently detected in HNPCC (ref . 3), making functional characterization of mutations in hMLH1 critical to the development of genetic testing for HNPCC . Here, we describe a new method for detecting mutations in hMLH1 using a dominant mutator effect of hMLH1 cDNA expressed in Saccharomyces cerevisiae . The majority of hMLH1 missense mutations identified in HNPCC patients abolish the dominant mutator effect . Furthermore, PCR amplification of hMLH1 cDNA from mRNA from a HNPCC patient, followed by in vivo recombination into a gap expression vector, allowed detection of a heterozygous loss-of-function missense mutation in hMLH1 using this method . This functional assay offers a simple method for detecting and evaluating pathogenic mutations in hMLH1. J Bacteriol, 1998 Aug, 180(16), 4177 - 83 A mutation in a purported regulatory gene affects control of sterol uptake in Saccharomyces cerevisiae; Crowley JH et al.; Aerobically growing wild-type strains of Saccharomyces cerevisiae are unable to take exogenously supplied sterols from media . This aerobic sterol exclusion is vitiated under anaerobic conditions, in heme-deficient strains, and under some conditions of impaired sterol synthesis . Mutants which can take up sterols aerobically in heme-competent cells have been selected . One of these mutations, designated upc2-1, gives a pleiotropic phenotype in characteristics as diverse as aerobic accumulation of sterols, total lipid storage, sensitivity to metabolic inhibitors, response to altered sterol structures, and cation requirements . During experiments designed to ascertain the effects of various cations on yeast with sterol alterations, it was observed that upc2-1 was hypersensitive to Ca2+ . Using resistance to Ca2+ as a screening vehicle, we cloned UPC2 and showed that it is YDR213W, an open reading frame on chromosome IV . This belongs to a fungal regulatory family containing the Zn(II)2Cys6 binuclear cluster DNA binding domain . The single guanine-to-adenine transition in upc2-1 gives a predicted amino acid change from glycine to aspartic acid . The regulatory defect explains the semidominance and pleiotropic effects of upc2-1. J Bacteriol, 1998 Aug, 180(16), 4051 - 5 Identification of a cytosolically directed NADH dehydrogenase in mitochondria of Saccharomyces cerevisiae; Small WC et al.; The reoxidation of NADH generated in reactions within the mitochondrial matrix of Saccharomyces cerevisiae is catalyzed by an NADH dehydrogenase designated Ndi1p (C . A . M . Marres, S . de Vries, and L . A . Grivell, Eur . J . Biochem . 195:857-862, 1991) . Gene disruption analysis was used to examine possible metabolic functions of two proteins encoded by open reading frames having significant primary sequence similarity to Ndi1p . Disruption of the gene designated NDH1 results in a threefold reduction in total mitochondrial NADH dehydrogenase activity in cells cultivated with glucose and in a fourfold reduction in the respiration of isolated mitochondria with NADH as the substrate . Thus, Ndh1p appears to be a mitochondrial dehydrogenase capable of using exogenous NADH . Disruption of a closely related gene designated NDH2 has no effect on these properties . Growth phenotype analyses suggest that the external NADH dehydrogenase activity of Ndh1p is important for optimum cellular growth with a number of nonfermentable carbon sources, including ethanol . Codisruption of NDH1 and genes encoding malate dehydrogenases essentially eliminates growth on nonfermentable carbon sources, suggesting that the external mitochondrial NADH dehydrogenase and the malate-aspartate shuttle may both contribute to reoxidation of cytosolic NADH under these growth conditions. Microbiology, 1998 Jul, 144 ( Pt 7), 1783 - 97 The aberrant positioning of nuclei and the microtubular cytoskeleton in Saccharomyces cerevisiae due to improper actin function; Kopecka M et al.; An excentric position of the nuclei, random orientation of mitoses, and multinuclear budding cells were identified in part of a population of temperature-sensitive (ts) Saccharomyces cerevisiae actin mutants at the permissive temperature of 23 degrees C by fluorescence and electron microscopy . The phenotype resembled that of mutants in beta-tubulin, dynein, JNM1, NUM1, ACT3, ACT5, myosins, profilin, tropomyosin 1, SLA2 and other genes . The question was addressed whether the cause was (i) defects in cell polarity in some ts actin mutants, manifested by lack of asymmetry of actin cortical patches, or (ii) lack of cytoplasmic or astral microtubules . The results indicated that in the cells with the nuclear defects, actin cortical patches showed the normal asymmetric distribution typical of undisturbed polarity . Cytoplasmic astral and spindle microtubules were also preserved . The principal difference found between the wild-type and actin mutant cells was in actin cables, which in the actin mutants were developed insufficiently . It is suggested that actin cables serve as a 'suspensory apparatus' and/or 'intracellular corridor', predetermining: the location of the nucleus in the central position in interphase; the axis of nuclear movement to the bud neck before mitosis; the direction of the elongating nucleus during mitosis; and the motion of each nucleus from an excentric to a central position during cytokinesis, in cooperation with the above-mentioned and other gene products. J Biol Chem, 1998 Aug 14, 273(33), 21332 - 41 DNA polymerase II (epsilon) of Saccharomyces cerevisiae dissociates from the DNA template by sensing single-stranded DNA; Maki S et al.; Two forms of DNA polymerase II (epsilon) of Saccharomyces cerevisiae, Pol II* and Pol II, were purified to near homogeneity from yeast cells . Pol II* is a four-subunit complex containing a 256-kDa catalytic polypeptide, whereas Pol II consists solely of a 145-kDa polypeptide derived from the N-terminal half of the 256-kDa polypeptide of Pol II* . We show that Pol II* and Pol II are indistinguishable with respect to the processivity and rate of DNA-chain elongation . The equilibrium dissociation constants of the complexes of Pol II* and Pol II with the DNA template showed that the stability of these complexes is almost the same . However, when the rates of dissociation of the Pol II* and Pol II from the DNA template were measured using single-stranded DNA as a trap for the dissociated polymerase, Pol II* dissociated 75-fold faster than Pol II . Furthermore, the rate of dissociation of Pol II* from the DNA template became faster as the concentration of the single-stranded DNA was increased . These results indicate that the rapid dissociation of Pol II* from the DNA template is actively promoted by single-stranded DNA . The dissociation of Pol II from the DNA template was also shown to be promoted by single-stranded DNA, although at a much slower rate . These results suggest that the site for sensing single-stranded DNA resides within the 145-kDa N-terminal portion of the catalytic subunit and that the efficiency for sensing single-stranded DNA by this site is positively modulated by either the C-terminal half of the catalytic subunit and/or the other subunits. J Biol Chem, 1998 Aug 14, 273(33), 21253 - 60 Rpg1, the Saccharomyces cerevisiae homologue of the largest subunit of mammalian translation initiation factor 3, is required for translational activity; Valasek L et al.; Eukaryotic initiation factor 3 (eIF3) consists of at least eight subunits and plays a key role in the formation of the 43 S preinitiation complex by dissociating 40 and 60 S ribosomal subunits, stabilizing the ternary complex, and promoting mRNA binding to 40 S ribosomal subunits . The product of the Saccharomyces cerevisiae RPG1 gene has been described as encoding a protein required for passage through the G1 phase of the cell cycle and exhibiting significant sequence similarity to the largest subunit of human eIF3 . Here we show that under nondenaturing conditions, Rpg1p copurifies with a known yeast eIF3 subunit, Prt1p . An anti-Rpg1p antibody co-immunoprecipitates Prt1p, and an antibody directed against the Myc tag of a tagged version of Prt1p co-immunoprecipitates Rpg1p, demonstrating that both proteins are present in the same complex . A cell-free translation system derived from the temperature-sensitive rpg1-1 mutant strain becomes inactivated by incubation at 37 degreesC, and its activity can be restored by the addition of the Rpg1-containing protein complex . Finally, the rpg1-1 temperature-sensitive mutant strain shows a dramatic reduction of the polysome/monosome ratio upon shift to the restrictive temperature . These data show that Rpg1p is an authentic eIF3 subunit and plays an important role in the initiation step of translation. Biochim Biophys Acta, 1998 Jun 10, 1365(1-2), 23 - 30 Phosphate permeases of Saccharomyces cerevisiae; Persson BL et al.; The PHO84 and PHO89 genes of Saccharomyces cerevisiae encode two high-affinity phosphate cotransporters of the plasma membrane . Hydropathy analysis suggests a secondary structure arrangements of the proteins in 12 transmembrane domains . The derepressible Pho84 and Pho89 transporters appear to have characteristic similarities with the phosphate transporters of Neurospora crassa . The Pho84 protein catalyzes a proton-coupled phosphate transport at acidic pH, while the Pho89 protein catalyzes a sodium-dependent phosphate uptake at alkaline pH . The Pho84 transporter can be stably overproduced in the cytoplasmic membrane of Escherichia coli, purified and reconstituted in a functional state into proteoliposomes. Protein Expr Purif, 1998 Aug, 13(3), 396 - 402 High-level production of human parathyroid hormone (hPTH) by induced expression in Saccharomyces cerevisiae; Vad R et al.; Saccharomyces cerevisiae was used as host for high-level production of intact human parathyroid hormone (hPTH) . The yield increased about 30-fold by changing from the constitutive MFalpha promoter to the inducible CUP1 promoter in the expression cassettes, use of another host strain, and optimization of growth conditions where especially the pH value was crucial . The secreted products consisted mainly of intact hormone, hPTH(1-84) . In addition, two C-terminally truncated forms that lacked the four or five last amino acid residues, hPTH(1-80) and hPTH(1-79), were identified . These hPTH forms migrated aberrantly by SDS-PAGE as 14-kDa proteins, while the real masses measured by mass spectrometry on HPLC-purified products were about 9 kDa . Availability of such easily purified truncated forms will be valuable for studies of how the C-terminal residues affect the structure and function of the hormone . Combination of mutations and disruptions of the host genes encoding proteinase A, B, carboxypeptidase Y, and Kex1p or Mkc7p did not influence the C-terminal deletions . The secretion of hPTH could be enhanced by overexpression of the yeast syntaxin gene SSO2, but the total level of the hormone was not improved due to impaired growth . Protein Expr Purif, 1998 Aug, 13(3), 291 - 300 Expression and purification of recombinant tick anticoagulant peptide (Y1W/D10R) double mutant secreted by Saccharomyces cerevisiae; Cook JC et al.; A double mutant of tick anticoagulant peptide (TAP) was cloned as a chimeric fusion with the yeast alpha-mating factor pre-proleader peptide . Expression in yeast (Saccharomyces cerevisiae) resulted in the secretion of the TAP mutein into the culture medium . An HPLC-based assay was used to screen yeast strains to find those giving highest expression levels . Efficiency of cleavage at the junction of the leader-TAP mutein varied from strain to strain, and a rapid purification method followed by N-terminal sequence analysis was used to identify a host strain that minimized undesirable cleavage products . A purification scheme was developed which separated the TAP mutein from improperly processed peptides present in the medium . This scheme employed cation-exchange chromatography and reversed-phase HPLC . Scale-up of the process was successful and produced 100 mg of fully functional TAP mutein of >96% homogeneity from a 50-L yeast culture . Genomics, 1998 Jul 1, 51(1), 128 - 31 Identification, expression, and chromosomal localization of ubiquitin conjugating enzyme 7 (UBE2G2), a human homologue of the Saccharomyces cerevisiae ubc7 gene; Katsanis N et al.; Protein degradation is an essential mechanism for the maintenance of cellular homeostasis, in which excess or aberrant proteins are eliminated from the cell . In eukaryotes, conjugation of target proteins to ubiquitin is an essential step in the proteasome-dependent degradation process and is mediated by a family of ubiquitin conjugating enzymes (UBC) . Several of these have been identified in a variety of organisms . Here we report the identification of UBE2G2, a human homologue of the yeast Ubc7 gene . We describe a 2.9-kb cDNA sequence encoding a 165-amino-acid protein that shares significant similarity with other members of the UBC family . We have found UBE2G2 to be ubiquitously expressed, with high levels of expression seen in adult muscle, and have detected two transcripts of 2.9 and 7.0 kb in all tissues . In addition, we have mapped UBC7 to human chromosome 21q22.3 close to 21qtel . Eur J Biochem, 1998 Jul 1, 255(1), 156 - 61 The Saccharomyces cerevisiae Pet309 protein is embedded in the mitochondrial inner membrane; Manthey GM et al.; The nuclear PET309 gene of Saccharomyces cerevisiae is necessary for expression of the mitochondrial COX1 gene, which encodes subunit I of cytochrome c oxidase . In a pet309 null mutant, there is a defect both in accumulation of COX1 pre-RNA, if it contains introns, and in translation of COX1 RNAs {Manthey, G . M . & McEwen, J . E . (1995) EMBO J . 14, 4031-4043} . To facilitate identification and intracellular localization of the protein Pet309p, that is encoded by the PET309 gene, Pet309p was tagged at the carboxy terminus with an epitope from the human c-myc protein . A monoclonal antibody against the c-myc epitope detected a 98-kDa protein in mitochondria of yeast cells that expressed the PET309-c-myc fusion protein from a high copy number plasmid . This protein was not detectable in cells that did not express the fusion protein, or that expressed it from a single copy centromeric vector . Additional analyses of mitochondrial subfractions demonstrated that the PET309-c-myc fusion protein is localized specifically in the inner mitochondrial membrane . It could not be extracted by alkaline sodium carbonate, yet it was susceptible to proteinase K digestion in mitoplasts (mitochondria with a disrupted outer membrane) . These results indicate that Pet309p spans the inner membrane, with domain(s) exposed to the intermembrane space side of the membrane . How Pet309p may function in concert with other gene products necessary for COX1 RNA translation or accumulation, such as Mss51p or Nam1p, respectively, is discussed. J Cancer Res Clin Oncol, 1998, 124(6), 321 - 5 Genotoxic effect of 4-aroyl-1-(2-chloroethyl)-1nitrosohydrazinecarboxamides on Saccharomyces cerevisiae cells; Staleva L et al.; The study of some 4-aroyl-1-(2-chloroethyl)-1-nitrosohydrazinecarboxamides with a Saccharomyces cerevisiae mutagenicity test of increased sensitivity defined two of them, 4-(4-bromobenzoyl)-1-(2-chloroethyl)-1-nitrosohydrazinecarboxam ide and 4-(4-fluorophenyl)-1-(2-chloroethyl)-1-nitrosohydrazine carboxamide as typical cytostatic agents . At concentrations of 2-5 microg/ml the substances kill up to 60%-70% of cells without having any detectable recombinogenic and mutagenic effects . At the same concentrations, lomustine, well known as a cytostatic reference, demonstrated recombinogenic and mutagenic activity on yeast cells . The advantage of the newly synthesized substances is that, in a certain concentration range, their biological activity is mainly cytotoxic without induction of recombinogenic and mutagenic events in surviving cells. Genetics, 1998 Aug, 149(4), 1717 - 27 Sro7p, a Saccharomyces cerevisiae counterpart of the tumor suppressor l(2)gl protein, is related to myosins in function; Kagami M et al.; Yeast SRO7 was identified as a multicopy suppressor of a defect in Rho3p, a small GTPase that maintains cell polarity . Sro7p and Sro77p, a homologue of Sro7p, possess domains homologous to the protein that are encoded by the Drosophila tumor suppressor gene lethal (2) giant larvae {l(2)gl} . sro7Delta sro77Delta mutants showed a partial defect of organization of the polarized actin cytoskeleton and a cold-sensitive growth phenotype . A human counterpart of l(2)gl could suppress the sro7Delta sro77Delta defect . Similar to the l(2)gl protein, Sro7p formed a complex with Myo1p, a type II myosin . These results indicate that Sro7p and Sro77p are the yeast counterparts of the l(2)gl protein . Our genetic analysis revealed that deletion of SRO7 and SRO77 showed reciprocal suppression with deletion of MYO1 (i.e., the sro7Delta sro77Delta defect was suppressed by myo1Delta and vice versa) . In addition, SRO7 showed genetic interactions with MYO2, encoding an essential type V myosin: Overexpression of SRO7 suppressed a defect in MYO2 and, conversely, overexpression of MYO2 suppressed the cold-sensitive phenotype of sro7Delta sro77Delta mutants . These results indicate that Sro7 function is closely related to both Myo1p and Myo2p . We propose a model in which Sro7 function is involved in the targeting of the myosin proteins to their intrinsic pathways. Genetics, 1998 Aug, 149(4), 1693 - 705 Conversion-type and restoration-type repair of DNA mismatches formed during meiotic recombination in Saccharomyces cerevisiae; Kirkpatrick DT et al.; Meiotic recombination in yeast is associated with heteroduplex formation . Heteroduplexes formed between nonidentical DNA strands contain DNA mismatches, and most DNA mismatches in wild-type strains are efficiently corrected . Although some patterns of mismatch correction result in non-Mendelian segregation of the heterozygous marker (gene conversion), one predicted pattern of correction (restoration-type repair) results in normal Mendelian segregation . Using a yeast strain in which a marker leading to a well-repaired mismatch is flanked by markers that lead to poorly repaired mismatches, we present direct evidence for restoration-type repair in yeast . In addition, we find that the frequency of tetrads with conversion-type repair is higher for a marker at the 5' end of the HIS4 gene than for a marker in the middle of the gene . These results suggest that the ratio of conversion-type to restoration-type repair may be important in generating gradients of gene conversion (polarity gradients). Nucleic Acids Res, 1998 Aug 15, 26(16), 3707 - 16 Mutational analysis of exoribonuclease I from Saccharomyces cerevisiae; Page AM et al.; Exoribonuclease I from yeast is a 175 kDa protein that is responsible for the majority of cytoplasmic mRNA degradation . Alignment of the Xrn1p sequence with homologs from yeast as well as from higher eukaryotes suggests that the protein is composed of several domains: two acidic N-terminal domains which likely contain the exonuclease, a basic middle domainand a basic C-terminal domain . Deletion analysisdemonstrated that the C-terminus is dispensable for most in vivo and in vitro functions but confers a dominant negative growth inhibition when expressed at high levels . This growth inhibition is not due to the exonuclease function of the protein . To identify specific residues responsible for in vivo function, a screen was carried out for non-complementing missense mutations . Fourteen single point mutations were identified that altered highly conserved amino acids within the first N-terminal domain of Xrn1p . All of the mutations reduced exonuclease activity measured in vivo and in vitro using affinity-purified proteins . The mutants fell into two phenotypic classes, those that reduced or abolished exonuclease activity without qualitatively changing the products of RNA degradation and those that gave rise to novel degradation intermediates on certain RNAs. J Biol Chem, 1998 Aug 7, 273(32), 20150 - 5 Fzo1p is a mitochondrial outer membrane protein essential for the biogenesis of functional mitochondria in Saccharomyces cerevisiae; Rapaport D et al.; Fzo1p is a novel component required for the biogenesis of functional mitochondria in the yeast Saccharomyces cerevisiae . The protein is homologous to Drosophila Fzo, the first known protein mediator of mitochondrial fusion . Deletion of the FZO1 gene results in a petite phenotype, loss of mitochondrial DNA, and a fragmented mitochondrial morphology . Fzo1p is an integral protein of the mitochondrial outer membrane exposing its major part to the cytosol . It is imported into the outer membrane in a receptor-dependent manner . Fzo1p is part of a larger protein complex of 800 kDa, and presumably is the first identified component of the yeast mitochondrial fusion machinery. Curr Genet, 1998 Jul, 34(1), 30 - 42 Mitotic recombination and localized DNA double-strand breaks are induced after 8-methoxypsoralen and UVA irradiation in Saccharomyces cerevisiae; Dardalhon M et al.; Mitotic recombination within the ARG4 gene of Saccharomyces cerevisiae was analysed after treatment of cells with the recombinogenic agent 8-methoxypsoralen (8-MOP) plus UVA . The appearance of DNA double-strand breaks (DSBs) in the ARG4 region during post-treatment incubation was also tested . The results obtained after 8-MOP plus UVA treatment indicate that in mitotic cells: (1) recombination at the ARG4 locus is increased 30 - 500 fold per survivor depending on the strains and the doses employed, (2) the increase of recombination results essentially from gene conversion events which involve the RV site located in the 5' region of the ARG4 gene twice as often as the Bgl site at the 3' end, (3) depending on 8-MOP/UVA dose, ectopic gene conversion is associated with reciprocal translocation, (4) DSBs occur preferentially in the ARG 5' region during post-treatment incubation, as well as in other intergenic regions containing both promoters or/and terminators of transcription, and (5) changes in sequence content in the 5' region of ARG4, which influences positions and frequencies of DSBs formed during repair, are correlated with a modification of the local chromatin structure. Carcinogenesis, 1998 Jul, 19(7), 1299 - 305 Opposite base-dependent excision of 7,8-dihydro-8-oxoadenine by the Ogg1 protein of Saccharomyces cerevisiae; Girard PM et al.; The yOgg1 protein of Saccharomyces cerevisiae is a DNA glycosylase/AP lyase that excises guanine lesions such as 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (me-Fapy-G) and incises apurinic/apyrimidinic sites (AP sites) in damaged DNA . The yOgg1 protein displays a marked preference for DNA duplexes containing 8-OxoG or AP sites placed opposite cytosine . In this paper, we show that yOgg1 can also excise an adenine lesion, 7,8-dihydro-8-oxoadenine (8-OxoA), when paired with cytosine or 5-methylcytosine . In contrast, yOgg1 does not release 8-OxoA when placed opposite thymine, adenine, guanine or uracil . The specificity constants (Kcat/Km) for repair of 8-OxoG/C and 8-OxoA/C duplexes are (50 +/- 18) x 10(-3) and (13 +/- 3) x 10(-3)/min/nM, respectively . The catalytic mechanism for strand cleavage at 8-OxoA/C involves excision of 8-OxoA by the DNA glycosylase activity of yOgg1, followed by incision at the newly formed AP site via a beta-elimination reaction . Furthermore, cleavage of 8-OxoA/C involves formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydride (NaBH4) . The yOgg1 protein binds strongly to the 8-OxoA/C duplex, as demonstrated by an apparent dissociation constant (Kdapp) value of 45 nM, as determined by gel mobility shift assay . In contrast, the yOgg1 protein has a very low binding affinity for the 8-OxoA/T duplex, a Kdapp value of 680 nM, which in turn can explain the lack of repair of 8-OxoA in this duplex . The capacity of other DNA glycosylases/AP lyases to repair 8-OxoA has also been investigated . The results show that human hOgg1 protein efficiently repairs 8-OxoA placed opposite cytosine or 5-methylcytosine . On the other hand, the Fpg protein of Escherichia coli cleaves 8-OxoA/C at a very slow rate as compared with yOgg1. FEMS Microbiol Lett, 1998 Jul 15, 164(2), 345 - 52 Regulatory regions in the promoters of the Saccharomyces cerevisiae PYC1 and PYC2 genes encoding isoenzymes of pyruvate carboxylase; Menendez J et al.; We have identified regions in the promoters of the PYC1 and PYC2 genes from Saccharomyces cerevisiae involved in their regulation in different culture conditions . In the case of PYC1, a UAS in the region between -330/-297 and three repressing sequences with the common central core CCGCC at positions -457, -432 and -399 were identified . Specific binding of nuclear proteins to the -330/-214 DNA fragment was abolished in rtg mutants suggesting a role for the RTG genes in the control of PYC1 expression . In the case of the PYC2 promoter, elimination of a fragment from -417 to -291 brings about a two-fold decrease in the expression in repressed conditions and a similar increase in derepression. Histochem Cell Biol, 1998 Jul, 110(1), 15 - 26 Fate and role of peroxisomes during the life cycle of the yeast Saccharomyces cerevisiae: inheritance of peroxisomes during meiosis; Gurvitz A et al.; Sporulation in the yeast Saccharormyces cerevisiae is a meiotic developmental process that occurs in MATa/MATalpha heterozygotes in response to nutrient deprivation . Here, the fate and role of peroxisomes during sporulation and germination has been examined by a combination of immunoelectron microscopy and the use of pex mutants defective in peroxisomal functions . Using a green fluorescent protein probe targeted to peroxisomes we show that peroxisomes are inherited through meiosis and that they do not increase in number either during sporulation or spore germination . In addition, there is no requirement for peroxisome degradation prior to spore packaging . Unlike the situation in filamentous fungi, peroxisomes do not proliferate during the yeast life cycle . Functional peroxisomes are dispensable for efficient meiotic development on acetate medium since homozygous delta pex6 diploids sporulated well and produced mature spores that were resistant to diethyl ether . Like haploids, diploid cells can proliferate their peroxisomes in response to oleate as sole carbon source in liquid medium, but under these conditions they do not sporulate . On solid oleate medium, homozygous pex5, delta pex6, and pex7 cells were unable to sporulate efficiently, whereas the wild type was . The results presented here are discussed in terms of the transmission of organelles to progeny cells. Extremophiles, 1997 May, 1(2), 89 - 93 Vacuolar acidification in Saccharomyces cerevisiae induced by elevated hydrostatic pressure is transient and is mediated by vacuolar H+-ATPase; Abe F et al.; We analyzed the vacuolar acidification in response to elevated hydrostatic pressure in Saccharomyces cerevisiae . The vacuolar pH, defined using 6-carboxyfluorescein, was directly measured in a hyperbaric chamber with a transparent window under high hydrostatic pressure . The vacuole of strain X2180 became acidified at the onset of pressurization to an extent dependent on the magnitude of pressure applied . A pressure of 40-60 MPa transiently reduced the vacuolar pH by about 0.33 within 4 min . The transient acidification was observed in the presence of D-glucose, D-fructose, or D-mannose as a carbon source, but not 3-o-methyl-D-glucose, ethanol, or glycerol, suggesting that the generation of CO2 was involved in the process . A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH when hydrostatic pressure was applied . This result indicates that the transient vacuolar acidification induced by elevated hydrostatic pressure is mediated through the function of the vacuolar H+-ATPase. FEBS Lett, 1998 Jun 23, 430(1-2), 116 - 25 Unified inventory of established and putative transporters encoded within the complete genome of Saccharomyces cerevisiae; Paulsen IT et al.; We present the complete inventory of currently recognized and putative transporters encoded within the genome of Saccharomyces cerevisiae . These 258 transporters are classified into 42 families according to phylogenetic and substrate specificity criteria . Twelve of these yeast families are found only in eukaryotic organisms, and four are so far unique to yeast . Putative yeast-specific families transport heavy metals, arsenite and calcium . The phylogenetic analyses reported allow classification of 139 functionally uncharacterized yeast transporters into families of known functions . The relative proportions of yeast transporters specific for different classes of substrates differ only slightly from those reported for Escherichia coli . However, the ratio of secondary transporters (uniporters, cation symporters and antiporters) to primary ATP-driven transporters is much higher for yeast than for bacteria. Genes Funct, 1997 Nov, 1(4), 273 - 84 Functional analysis of a 38 kilobase region on chromosome XVI in Saccharomyces cerevisiae; Ushinsky SC et al.; In this report we assess the functional importance of 16 open reading frames (ORFs) contained within a 38 780 base-pair region immediately adjacent to the centromere on the right arm of chromosome XVI in Saccharomyces cerevisiae . This analysis involved replacing one copy of each ORF in a diploid strain with a cassette encoding the green fluorescent protein from the jellyfish Aequorea victoria and HIS3 . Each replacement cassette was generated by PCR using oligonucleotide pairs with 45-base extensions complementary to sequences immediately upstream and downstream of the target gene's coding region . After replacement of the targeted genes, each gene-replacement strain was subjected to a series of genetic and phenotypic tests to assess the functional importance of the deleted gene . This analysis showed that two ORFs were essential, one for spores to germinate and another for vegetative growth . A third gene encoded a copper-fist-like transcription factor that was required for proper bud-site selection . One of the 16 ORFs was duplicated, a situation not observed in the strain used to sequence the yeast genome (S288C) . RNA analysis showed 11 of the 16 ORFs in this region expressed steady-state poly(A+) RNA levels that were greater than or equal to 2% of the level expressed from the yeast actin gene, ACT1. Cell Mol Biol (Noisy-le-grand), 1998 Jun, 44(4), 591 - 5 Regulatory role of ALA-S and ALA-D in a haem-deficient mutant of Saccharomyces cerevisiae; Araujo LS et al.; The biochemical characteristics of a haem-deficient mutant strain B231 of Saccharomyces cerevisiae isolated from D273-10B strain are described . B231 strain accumulates substantial amounts of 5-aminolevulinic acid (ALA) and uroporphyrin III (uro III) . This pattern of porphyrins accumulation is due to both a defect in uroporphyrinogen decarboxylase (Uro-D) activity and an enhancement of porphobilinogenase (PBGase) activity . ALA accumulation would indicate that feed-back control by haem is not operating in this strain . ALA synthase (ALA-S), ALA-dehydrase (ALA-D) and PBGase activities; intracellular content of ALA (I-ALA) and porphyrins (I-porphyrins) were examined during the different phases of growth . Both accumulation of metabolites and enzyme activities reached their maximum values at 20 hrs . of growth, when glucose concentration in the medium fell to zero . Evidence for negative feed-back control on ALA-S and ALA-D by heme are provided by the observations of both enhanced I-ALA accumulation and increased ALA-D activity (2.5 times) in the mutant strain . Our results would indicate that both ALA-S and ALA-D can be considered regulatory enzymes in yeast. J Biol Chem, 1998 Jul 31, 273(31), 19792 - 6 A family of Arf effectors defined as suppressors of the loss of Arf function in the yeast Saccharomyces cerevisiae; Zhang CJ et al.; Arf proteins are ubiquitous, eukaryotic regulators of virtually every step of vesicular membrane traffic . ADP-ribosylation factors are essential in yeast and the lethality resulting from either overexpression or underexpression (deletion) of Arf genes has previously been ascribed to dysregulation of the secretory process . We have identified a family of four genes (Suppressors of Arf ts, SAT) as high copy suppressors of a loss of function allele of ARF1 (arf1-3) . Those proteins with SAT activity were found to contain a minimal consensus motif, including a C2C2H2 cluster with a novel and specific spacing . Genetic interactions between members of this family and with ARF1 are consistent with each sharing a common cellular pathway . Included in this family is Gcs1, a protein previously described (Poon, P . P., Wang, X., Rotman, M., Huber, I., Cukierman, E., Cassel, D., Singer, R . A., and Johnston, G . C . (1996) Proc . Natl . Acad . Sci . U . S . A . 93, 10074-10077) to possess Arf GTPase-activating protein (GAP) activity, demonstrating a direct interaction between Arf and at least one of these suppressors . The suppression of the loss of Arf function by overexpression of Gcs1 and demonstration of direct, preferential binding of Gcs1 to the activated form of Arf (Arf.GTP) lead us to conclude that the biological role of Gcs1 is as an effector of the essential function of Arf in mitotic growth, rather than a down-regulator as implied by the biochemical (Arf GAP) activity . Suppression of the growth defect of arf1(-3) cells was observed under conditions that did not alter the secretory defect associated with arf1(-) mutation, indicating that the essential role of Arf in eukaryotes can be distinguished from role(s) in the secretory pathway and appear to employ distinct pathways and effectors. J Biol Chem, 1998 Jul 31, 273(31), 19756 - 62 Structure and processivity of two forms of Saccharomyces cerevisiae DNA polymerase delta; Burgers PM et al.; Yeast DNA polymerase delta (Poldelta) consists of three subunits encoded by the POL3, POL31, and POL32 genes . Each of these genes was cloned under control of the galactose-inducible GAL1-10 promoter and overexpressed in various combinations . Overexpression of all three genes resulted in a 30-fold overproduction of Poldelta, which was identical in enzymatic properties to Poldelta isolated from a wild-type yeast strain . Whereas overproduction of POL3 together with POL32 did not lead to an identifiable Pol3p.Pol32p complex, a chromatographically distinct and novel complex was identified upon overproduction of POL3 and POL31 . This two-subunit complex, designated Poldelta*, is structurally and functionally analogous to mammalian Poldelta . The properties of Poldelta* and Poldelta were compared . A gel filtration analysis showed that Poldelta* is a heterodimer (Pol3p.Pol31p) and Poldelta a dimer of a heterotrimer, (Pol3p.Pol31p.Pol32p)2 . In the absence of proliferating cell nuclear antigen (PCNA), Poldelta* showed a processivity of 2-3 on poly(dA) . oligo(dT) compared with 5-10 for Poldelta . In the presence of PCNA, both enzymes were fully processive on this template . DNA replication by Poldelta* on a natural DNA template was dependent on PCNA and on replication factor C . However, Poldelta*-mediated DNA synthesis proceeded inefficiently and was characterized by frequent pause sites . Reconstitution of Poldelta was achieved upon addition of Pol32p to Poldelta*. Yeast, 1998 Jun 15, 14(8), 711 - 21 Transcriptional co-regulation of Saccharomyces cerevisiae alcohol acetyltransferase gene, ATF1 and delta-9 fatty acid desaturase gene, OLE1 by unsaturated fatty acids; Fujiwara D et al.; The ATF1 gene encodes an alcohol acetyl transferase which catalyzes the synthesis of acetate esters from acetyl CoA and several kinds of alcohols . ATF1 expression is repressed by unsaturated fatty acids or oxygen . Analysis using ATF1-lacZ fusion plasmid revealed that ATF1 gene expression is widely repressed by a variety of unsaturated fatty acids, and the degree of ATF1 transcriptional repression varies according to the structure of the unsaturated fatty acids . Interestingly, it was noted that the degree of ATF1 transcriptional repression was related to the melting point of unsaturated fatty acids added to the medium . The OLE1 gene, which encodes delta-9 fatty acid desaturase, has been reported to be repressed by unsaturated fatty acids . Transcription of OLE1 was also repressed by a wide variety of unsaturated fatty acids under anaerobic conditions . The degree of transcriptional repression of OLE1 was also related to the melting point of the added unsaturated fatty acids . Therefore, it is considered that ATF1 and OLE1 transcription are regulated in response to cell membrane fluidity . As has been reported for OLE1, the repression of ATF1 by unsaturated fatty acids was relieved in a disruptant carrying a faa1 and faa4 double mutation, two fatty acid activation genes . However, the ATF1 transcript in this double gene disruptant was repressed by oxygen . These results suggested that ATF1 transcription was co-regulated by the same mechanism as the OLE1 gene and that unsaturated fatty acids and oxygen repressed the ATF1 transcript by a different regulation pathway. Biochim Biophys Acta, 1998 Jul 17, 1372(2), 153 - 62 Membrane-bound and soluble polyphosphatases of mitochondria of Saccharomyces cerevisiae: identification and comparative characterization; Lichko L et al.; Isolated mitochondria of Saccharomyces cerevisiae possess polyphosphatases insensitive to a number of inhibitors of ATPase and pyrophosphatase of the same organelles and differing from the last two by neutral pH optima and molecular masses . After subfractionation of mitochondria, the polyphosphatase activity is distributed among the membrane and soluble preparations . The membrane-bound and soluble polyphosphatase activities are represented by different enzymes distinguished by molecular masses, substrate specificity, Km values, and relation to mono- and divalent cations . The membrane-bound polyphosphatases have molecular masses of 120 and 76 kDa, and the soluble one of about 36 kDa . All three enzymes appear to have a monomeric structure . The soluble polyphosphatase activity is stimulated by divalent cations in contrast to the membrane-bound one which is inhibited by the same cations, including Mg2+ . Monovalent cations do not actually change the activity of the soluble enzyme, but stimulate it in the membrane preparation . Specific activities for the hydrolysis of polyphosphates with average chain lengths of 9-188 phosphate residues increase under increasing degree of substrate polymerization in the membrane preparation and are actually unchanged in the soluble one . The affinity of the soluble enzyme to polyphosphates is 5-10 times higher than that of the membrane-bound polyphosphatases. Protein Expr Purif, 1998 Jul, 13(2), 163 - 9 Secretory expression of human albumin domains in Saccharomyces cerevisiae and their binding of myristic acid and an acylated insulin analogue; Kjeldsen T et al.; Albumin is organized in three homologous domains formed by double loops stabilized by disulfide bonds . Utilizing a secretory expression system based on a synthetic secretory prepro-leader, the three human serum albumin domains were expressed in the yeast Saccharomyces cerevisiae . Human serum albumin domains I and III were efficiently expressed and secreted, indicating that these domains can form independent structural units capable of folding into stable tertiary structures . In contrast, albumin domain II was not secreted and disappeared early in the secretory pathway . Human serum albumin has the ability to bind a large number of small molecule ligands, including fatty acids, presumably due to its structure and structural flexibility . Purified albumin domain III bound myristic acid, whereas purified albumin domain I did not bind myristic acid . A new soluble long-acting insulin an alogue acylated with myristic acid (Markussen J., et al., Diabetologia 39, 281-288, 1996) bound to domain III and bound markedly more weakly to domain I . Genet Res, 1998 Feb, 71(1), 1 - 10 Mismatch repair and the accumulation of deleterious mutations influence the competitive advantage of MAT (mating type) heterozygosity in the yeast Saccharomyces cerevisiae; Selk E et al.; Competitions between matched pairs of diploid strains of Saccharomyces cerevisiae, one capable of undergoing sexual recombination (MAT-heterozygous) and the other not (MAT-homozygous), have proved useful for measuring the effects of mitotic and meiotic recombination and DNA repair on competitive ability in this organism . Overall competitive differences between the strains can be enhanced by converting them to petites (aerobic respiration incompetent) . Here we report the results of competitions between pairs of strains that also differ in their ability to undergo mismatch repair . In petite strains, the growth rates of mismatch-repair defective strains declined over time regardless of their MAT genotype . Mismatch-repair proficient MAT-heterozygous strains did not show a decline, while repair-deficient MAT-homozygous strains did . The decline appears to be due to the accumulation of deleterious mutations of small effect, which can be corrected by MAT-heterozygous strains having intact mismatch repair . The relative competitive abilities of MAT-heterozygous and MAT-homozygous strains diverged during the course of the competitions, and the variance of this divergence increased significantly when mismatch repair was defective . This large stochastic component indicates that a relatively small number of deleterious mutations may be involved . The accumulation of deleterious mutations and their subsequent repair may have a bearing on the origin of sex in this organism. Can J Microbiol, 1998 Apr, 44(4), 341 - 50 Transcriptional and translational regulation of major heat shock proteins and patterns of trehalose mobilization during hyperthermic recovery in repressed and derepressed Saccharomyces cerevisiae; Gross C et al.; Patterns of heat shock gene transcription and translation, as well as trehalose content, were investigated in both glucose (repressed) and acetate (derepressed) grown cells of Saccharomyces cerevisiae during heat shock and subsequent return of cells to 25 degrees C . Heat-shocked cells (37 degrees C for 30 min), grown in either glucose- or acetate-supplemented media, initially acquired high thermotolerance to a 50 degrees C heat stress, which was progressively lost when cultures were allowed to recover at 25 degrees C and subsequently exposed to a second heat stress . In all cases, with the notable exception of repressed cells of a relatively thermosensitive strain, inhibition of protein synthesis and coincident decrease in trehalose accumulation during the heat shock had little effect on the kinetics of loss of thermotolerance . Heat shock at 37 degrees C elicited a marked increase in transcription and translation of genes encoding major heat shock proteins (hsps) . During recovery at 25 degrees C, both metabolic activities were suppressed followed by a gradual increase in hsp mRNA transcription to levels observed prior to heat shock . De novo translation of hsp mRNAs, however, was no longer observed during the recovery phase, although immunodetection analyses demonstrated persistence of high levels of hsps 104, 90, 70, and 60 in cells throughout the 240-min recovery period . In addition, while heat shock induced trehalose was rapidly degraded during recovery in repressed cells, levels remained high in derepressed cells . Results therefore indicated that the progressive loss of induced thermotolerance exhibited by glucose- and acetate-grown cells was not closely correlated with levels of hsp or trehalose . It was concluded that both constitutive and de novo synthesized hsps require heat shock associated activation to confer thermotolerance and this modification is progressively reversed upon release from the heat-shocked state. Nucleic Acids Res, 1998 Aug 1, 26(15), 3577 - 83 Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids: expressionvectors with bi-directional promoters for use in Saccharomyces cerevisiae; Miller CA 3rd et al.; The pBEVY (bi-directional expression vectors for yeast) plasmids were designed with constitutive and galactose-induced bi-directional promoters to direct the expression of multiple proteins in Saccharomyces cerevisiae . Using human estrogen receptor as a test gene, relatively balanced expression levels from each side of a bi-directional promoter were observed . Expression of a functional heterodimeric transcription factor composed of human aryl hydrocarbon receptor (Ahr) and aryl hydrocarbon receptor nuclear translocator (Arnt) proteins was accomplished using a single pBEVY plasmid . Previous studies suggest that inhibitory cross-talk between the estrogen receptor and the Ahr/Arnt complex may occur and that Hsp90-Ahr complex formation is important for Ahr-mediated signal transduction . Evidence for functional interaction among these proteins was investigated using pBEVY plasmids in a yeast system . No inhibitory cross-talk was observed in signaling assays performed with yeast that co-expressed Ahr, Arnt and estrogen receptor . In contrast, Ahr/Arnt-mediated signal transduction was reduced by 80% in a temperature-sensitive Hsp90 strain grown under non-permissive conditions . We conclude that pBEVY plasmids facilitate the examination of multiple protein interactions in yeast model systems. Mol Cell Biol, 1998 Aug, 18(8), 4914 - 23 The RFC2 gene, encoding the third-largest subunit of the replication factor C complex, is required for an S-phase checkpoint in Saccharomyces cerevisiae; Noskov VN et al.; Replication factor C (RF-C), an auxiliary factor for DNA polymerases delta and epsilon, is a multiprotein complex consisting of five different polypeptides . It recognizes a primer on a template DNA, binds to a primer terminus, and helps load proliferating cell nuclear antigen onto the DNA template . The RFC2 gene encodes the third-largest subunit of the RF-C complex . To elucidate the role of this subunit in DNA metabolism, we isolated a thermosensitive mutation (rfc2-1) in the RFC2 gene . It was shown that mutant cells having the rfc2-1 mutation exhibit (i) temperature-sensitive cell growth; (ii) defects in the integrity of chromosomal DNA at restrictive temperatures; (iii) progression through cell cycle without definitive terminal morphology and rapid loss of cell viability at restrictive temperatures; (iv) sensitivity to hydroxyurea, methyl methanesulfonate, and UV light; and (v) increased rate of spontaneous mitotic recombination and chromosome loss . These phenotypes of the mutant suggest that the RFC2 gene product is required not only for chromosomal DNA replication but also for a cell cycle checkpoint . It was also shown that the rfc2-1 mutation is synthetically lethal with either the cdc44-1 or rfc5-1 mutation and that the restrictive temperature of rfc2-1 mutant cells can be lowered by combining either with the cdc2-2 or pol2-11 mutation . Finally, it was shown that the temperature-sensitive cell growth phenotype and checkpoint defect of the rfc2-1 mutation can be suppressed by a multicopy plasmid containing the RFC5 gene . These results suggest that the RFC2 gene product interacts with the CDC44/RFC1 and RFC5 gene products in the RF-C complex and with both DNA polymerases delta and epsilon during chromosomal DNA replication. Mol Cell Biol, 1998 Aug, 18(8), 4793 - 806 Hir proteins are required for position-dependent gene silencing in Saccharomyces cerevisiae in the absence of chromatin assembly factor I; Kaufman PD et al.; Chromatin assembly factor I (CAF-I) is a three-subunit histone-binding complex conserved from the yeast Saccharomyces cerevisiae to humans . Yeast cells lacking CAF-I (cacDelta mutants) have defects in heterochromatic gene silencing . In this study, we showed that deletion of HIR genes, which regulate histone gene expression, synergistically reduced gene silencing at telomeres and at the HM loci in cacDelta mutants, although hirDelta mutants had no silencing defects when CAF-I was intact . Therefore, Hir proteins are required for an alternative silencing pathway that becomes important in the absence of CAF-I . Because Hir proteins regulate expression of histone genes, we tested the effects of histone gene deletion and overexpression on telomeric silencing and found that alterations in histone H3 and H4 levels or in core histone stoichiometry reduced silencing in cacDelta mutants but not in wild-type cells . We therefore propose that Hir proteins contribute to silencing indirectly via regulation of histone synthesis . However, deletion of combinations of CAC and HIR genes also affected the growth rate and in some cases caused partial temperature sensitivity, suggesting that global aspects of chromosome function may be affected by the loss of members of both gene families. Mol Cell Biol, 1998 Aug, 18(8), 4707 - 18 Nuclear proteins Nut1p and Nut2p cooperate to negatively regulate a Swi4p-dependent lacZ reporter gene in Saccharomyces cerevisiae; Tabtiang RK et al.; The URS2 region of the Saccharomyces cerevisiae HO upstream region contains 10 binding sites for the Swi4p/Swi6p transcription factor and confers Swi4p dependence for transcription . Using a hybrid promoter, UASGAL (upstream activation sequence of GAL1)-URS2R, in which the GAL1-10 regulatory region is fused to the proximal 360 bp of URS2, we isolated mutants in which Swi4p is no longer required for transcription . Mutations of SIN4, ROX3, SRB8, SRB9, SRB10, SRB11, and two novel genes, NUT1 and NUT2, relieve the requirement of Swi4p for expression of this reporter . We found that NUT1 (open reading frame {ORF} YGL151w) is a nonessential gene, that NUT2 (ORF YPR168w) is essential, and that both Nut1p and Nut2p encode nuclear proteins . Deletion of NUT1 causes a constitutive, Swi4p-independent phenotype only in combination with the nut2-1 allele or an allele of CCR4 . In contrast, inactivation of a temperature-sensitive allele of NUT2, nut2-ts70, alone causes constitutivity . nut1Delta nut2-1 cells and sin4Delta cells exhibit Swi4p-independent expression of an ho-lacZ reporter but not of an intact ho gene . Likewise, a pPHO5-lacZ construct is constitutively expressed in nut1 nut2 mutants relative to their wild-type counterparts . These results suggest that Nut1p, Nut2p, Sin4p, and Ccr4p define a group of proteins that negatively regulate transcription in a subtle manner which is revealed by artificial reporter genes. Mol Cell Biol, 1998 Aug, 18(8), 4548 - 55 Snf1 kinase connects nutritional pathways controlling meiosis in Saccharomyces cerevisiae; Honigberg SM et al.; Glucose inhibits meiosis in Saccharomyces cerevisiae at three different steps (IME1 transcription, IME2 transcription, and entry into late stages of meiosis) . Because many of the regulatory effects of glucose in yeast are mediated through the inhibition of Snf1 kinase, a component of the glucose repression pathway, we determined the role of SNF1 in regulating meiosis . Deleting SNF1 repressed meiosis at the same three steps that were inhibited by glucose, suggesting that glucose blocks meiosis by inhibiting Snf1 . For example, the snf1Delta mutant completely failed to induce IME1 transcripts in sporulation medium . Furthermore, even when this block was bypassed by expression of IME1 from a multicopy plasmid, IME2 transcription and meiotic initiation occurred at only 10 to 20% of the levels seen in wild-type cells . The addition of glucose did not further inhibit IME2 transcription, suggesting that Snf1 is the primary mediator of glucose controls on IME2 expression . Finally, in snf1Delta cells in which both blocks on meiotic initiation were bypassed, early stages of meiosis (DNA replication and commitment to recombination) occurred, but later stages (chromosome segregation and spore formation) did not, suggesting that Snf1 controls later stages of meiosis independently from the two controls on meiotic initiation . Because Snf1 is known to activate the expression of genes required for acetate metabolism, it may also serve to connect glucose and acetate controls on meiotic differentiation. Mol Gen Genet, 1998 Jun, 258(6), 608 - 18 Phosphorylation of the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae does not affect its mating-specific signaling function; Li E et al.; The pheromone-responsive Gbeta subunit of Saccharomyces cerevisiae (encoded by STE4) is rapidly phosphorylated at multiple sites when yeast cells are exposed to mating pheromone . It has been shown that a mutant form of Ste4 lacking residues 310-346, ste4delta310-346, cannot be phosphorylated, and that its expression leads to defects in recovery from pheromone stimulation . Based on these observations, it was proposed that phosphorylation of Ste4 is associated with an adaptive response to mating pheromone . In this study we used site-directed mutagenesis to create two phosphorylation null (Pho-) alleles of STE4: ste4-T320A/S335A and ste4-T322A/S335A . When expressed in yeast, these mutant forms of Ste4 remained unphosphorylated upon pheromone stimulation . The elimination of Ste4 phosphorylation has no discernible effect on either signaling or adaptation . In addition, disruption of the FUS3 gene, which encodes a pheromone-specific MAP kinase, leads to partial loss of pheromone-induced Ste4 phosphorylation . Two-hybrid analysis suggests that the ste4delta310-346 deletion mutant is impaired in its interaction with Gpa1, the pheromone-responsive Galpha of yeast, whereas the Ste4-T320A/S335A mutant has normal affinity for Gpa1 . Taken together, these results indicate that pheromone-induced phosphorylation of Ste4 is not an adaptive mechanism, and that the adaptive defect exhibited by the 310-346 deletion mutant is likely to be due to disruption of the interaction between Ste4 and Gpa1. EMBO J, 1998 Jul 15, 17(14), 3981 - 9 The molecular chaperone Ssb from Saccharomyces cerevisiae is a component of the ribosome-nascent chain complex; Pfund C et al.; The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular chaperones . The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70 found associated with translating ribosomes . To understand better the function of Ssb in association with ribosomes, the Ssb-ribosome interaction was characterized . Incorporation of the aminoacyl-tRNA analog puromycin by translating ribosomes caused the release of Ssb concomitant with the release of nascent chains . In addition, Ssb could be cross-linked to nascent chains containing a modified lysine residue with a photoactivatable cross-linker . Together, these results suggest an interaction of Ssb with the nascent chain . The interaction of Ssb with the ribosome-nascent chain complex was stable, as demonstrated by resistance to treatment with high salt; however, Ssb interaction with the ribosome in the absence of nascent chain was salt sensitive . We propose that Ssb is a core component of the translating ribosome which interacts with both the nascent polypeptide chain and the ribosome . These interactions allow Ssb to function as a chaperone on the ribosome, preventing the misfolding of newly synthesized proteins. Mol Gen Genet, 1998 Jun, 258(5), 546 - 52 The involvement of the RAD6 gene in starvation-induced reverse mutation in Saccharomyces cerevisiae; Storchova Z et al.; The accumulation of Ade+ revertants during adenine starvation and Trp+ revertants during tryptophan starvation in haploid polyauxotrophic strains of Saccharomyces cerevisiae occurs in a time-dependent manner . Accumulation of revertants is enhanced in Rad6- strains, suggesting that starvation-induced reversion is influenced by some of the RAD6 gene functions . The higher frequency of adaptive reversions in Rad6- strains is somewhat influenced by, but does not totally depend on, the genetic background . Therefore, the RAD6 gene product is involved in maintaining a low level not only of spontaneous mutation but also of starvation-induced reversion . The starvation-induced Ade+ and Trp+ reversions both appear to be adaptive . The analysis of growth characteristics and the genotype of revertants shows a difference between early and late-appearing revertants . These results support the hypothesis that the adaptivity of starvation-induced reversion is based on the selective fixation of random mutations, and particularly on transcription-enhanced repair and/or mutagenesis processes. Mol Gen Genet, 1998 Jun, 258(5), 512 - 20 A physical assay for detection of early meiotic recombination intermediates in Saccharomyces cerevisiae; Bascom-Slack CA et al.; In most eukaryotic organisms, recombination events leading to exchanges between homologous chromosomes link the homologs in a manner that allows their proper attachment to the meiotic spindle . In the yeast Saccharomyces cerevisiae these exchanges are initiated in early prophase as double-strand breaks in the DNA . These breaks are processed through a series of intermediates to yield mature crossovers late in prophase . The following experiments were designed to monitor the appearance of the earliest recombinant DNA strands formed in this process . A polymerase chain reaction assay was devised that allows the detection of recombinant strands at a known initiation site for meiotic recombination . The time and rate of appearance of recombinant strands was found to coincide with commitment to recombination, demonstrating that DNA strands bearing sequences from both parental chromosomes are rapidly formed after the initiation of meiotic recombination. Biochemistry (Mosc), 1998 Jun, 63(6), 650 - 6 Adenylosuccinate synthetase of the yeast Saccharomyces cerevisiae: purification and properties; Ryzhova TA et al.; Adenylosuccinate synthetase (AS-synthetase) was purified from the yeast Saccharomyces cerevisiae . The purification procedure included chromatography on DEAE-cellulose, phosphocellulose, and heparin-agarose . The pH and temperature optima for the enzyme activity (7.0 and 35 degreesC, respectively) and also pH and thermostability of AS-synthetase were determined . The native form of the enzyme exists as a dimer . The Km values for IMP, GTP, and L-aspartate are 1.7, 0.16, and 6.7 mM, respectively . ATP cannot be used instead of substrate GTP, whereas 2'-dGTP and dd-GTP are able to substitute for GTP in the reaction . ITP also can be a substrate as an analog of GTP and as an analog of IMP . Two intermediates of purine nucleotide biosynthesis de novo, 5-amino-4-(N-succinocarboxamide)imidazole ribonucleotide (ASCIR) and 5-amino-4-carbamoyl-imidazole ribonucleotide (ACIR), inhibit AS-synthetase . Hydroxylamine and aspartate analogs also inhibit the enzyme . Effective binding requires a four-carbon-atom chain and unsubstituted amino group; the charge of the beta-carboxy group is not necessary . Comparison of primary structures and substrate specificity of yeast ASCIR- and AS-synthetases suggests independent origin of these proteins. J Biol Chem, 1998 Jul 24, 273(30), 18992 - 9001 Effect of CTP synthetase regulation by CTP on phospholipid synthesis in Saccharomyces cerevisiae; Ostrander DB et al.; CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition . Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition . The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu161 --> Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes . The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts . Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6-15-fold) levels of CTP when compared with cells expressing the wild-type enzymes . Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells . Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold) . These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene . Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1 . 3-fold) when compared with control cells . These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis. Gene, 1998 Jul 17, 215(1), 131 - 41 Amino acid residues in Std1 protein required for induction of SUC2 transcription are also required for suppression of TBPDelta57 growth defect in Saccharomyces cerevisiae; Zhang X et al.; The STD1 gene of Saccharomyces cerevisiae was isolated independently as a multicopy suppressor of a dominant negative mutation in the TATA-binding protein and of a mutation in the Snf1/Snf4 kinase complex, suggesting that Std1 might couple the Snf1 kinase signaling pathway to the transcriptional machinery . In order to identify the protein domains that specify these activities of the Std1 protein, a plasmid library of randomly mutagenized STD1 genes was screened for loss of function alleles using complementation of the raffinose growth defect of a std1-, mth1- strain as an assay . One missense allele (P236S) with complete loss of function at 30 degreesC and four missense alleles (L173F, E225K, S269L and E274K) that conferred a temperature sensitive phenotype were identified . The C-terminal 20 residues of Std1 were essential for SUC2 derepression, whereas the deletion of the N-terminal 96 residues did not affect SUC2 gene induction . Std1 mutants that lost the ability to induce SUC2, were also unable to suppress the growth defect caused by the expression of the dominant negative TBPDelta57 protein, suggesting that these two genetic screens may be detecting the same biological activity. Clin Diagn Lab Immunol, 1998 Jul, 5(4), 592 - 4 Expression of the extracellular domain of the human immunodeficiency virus type 1 envelope protein and its fusion with beta-galactosidase in Saccharomyces cerevisiae; Liu WF et al.; Two envelope glycoprotein gene fragments were cloned from the proviral genome of the HXB2 isolate of human immunodeficiency virus (HIV) . For the production of the two domains of the envelope gene product these cloned gene fragments were inserted into an Escherichia coli-yeast inducible shuttle vector fused to the galactokinase (GAL1) promoter . Cell extracts from strains of Saccharomyces cerevisiae harboring these two vectors (pYENV1 and pYENV2) were found to contain a specific protein with a size of 50 kDa when induced by galactose, while the protein could not be detected in extracts from control cells containing only the E . coli-yeast vector in the presence of galactose . Furthermore, another expression plasmid coding for fusion proteins from the majority of the external envelope glycoprotein (gp120) moiety and a large part of the beta-galactosidase was constructed . Antibodies from HIV type 1-positive sera could react with recombinant fusion polypeptides . Transformants could produce this fusion protein to a level of about 1.6% of the total protein content, as deduced from beta-galactosidase activity. J Mol Biol, 1998 Jul 17, 280(3), 355 - 63 Mutants with changes in different domains of yeast replication protein A exhibit differences in repairing the control region, the transcribed strand and the non-transcribed strand of the Saccharomyces cerevisiae MFA2 gene; Teng Y et al.; We have analysed the removal of UV-induced cyclobutane pyrimidine dimers (CPDs) at nucleotide resolution from the MFA2 gene of wild-type Saccharomyces cerevisiae and in strains harbouring mutations in one of the yeast replication protein A (RPA) genes, RFA1 . This gene codes for the 70 kDa subunit of RPA and it has previously been shown to have a role in nucleotide excision repair . Here two RFA1 mutants were examined: rfa1-M2 which is mutated in the protein interaction domain and rfa1-M4 which is mutated in the DNA-binding domain . A distinct difference in the removal of CPDs from the MFA2 sequence of these two mutants was observed . Compared to the parental strain, there was no defect in CPD removal in the rfa1-M2 mutant . Contrarily, the rfa1-M4 mutant was totally defective in the global repair of CPDs from the non-transcribed strand and the non-transcribed portions of the strand containing the transcribed sequence, yet it was able to perform reduced transcription coupled repair of the transcribed strand . These results indicate that the role of the DNA-binding domain of RPA is different for global repair versus transcription coupled nucleotide excision repair . J Cell Sci, 1998 Aug, 111 ( Pt 15), 2137 - 47 YEB3/VAC8 encodes a myristylated armadillo protein of the Saccharomyces cerevisiae vacuolar membrane that functions in vacuole fusion and inheritance; Pan X et al.; Armadillo (Arm) repeat proteins such as beta-catenin and alpha-karyopherin (importin) are thought to mediate the docking of cargo at membrane-associated cytoskeletal elements . YEB3 encodes an uncharacterized Saccharomyces cerevisiae protein that contains eleven tandem Arm repeats . While YEB3 is nonessential for growth, yeb3delta cells accumulated numerous small vacuoles and are defective in vacuolar inheritance . A functional Yeb3p-green fluorescent protein (GFP) chimera localized to vacuolar membranes . Confocal microscopy revealed that Yeb3p-GFP is localized over the surface of the vacuole, but is concentrated approximately 5- to 7-fold in bands located between clustered vacuoles . N-terminal myristylation of Yeb3p is required for vacuolar localization . The first 69 amino acids of Yeb3p were sufficient to target a GFP reporter protein to the vacuolar membrane; however, this fusion protein also localized to the plasma membrane, indicating that additional sequence is required for exclusive steady state vacuolar localization . By analogy to the function of beta-catenin in cell-cell adhesion, alpha-karyopherin in nuclear transport, and smgGDS in the control of ras-like GTPases, Yeb3p may provide a link between vacuoles and the actin cytoskeleton during vacuolar inheritance and fusion and perhaps mediate the assembly of a GTPase regulated docking complex. Mikrobiologiia, 1998 Mar-Apr, 67(2), 188 - 93 {Change in inorganic polyphosphate chain length depending on the stage of Saccharomyces cerevisiae growth}; Vagabov VM et al.; The dynamics of the content and the degree of polymerization of polyphosphates (PPs) during the growth of Saccharomyces cerevisiae VKM Y-1176 was studied by chemical methods and P NMR spectroscopy . The overall PP content of Saccharomyces cerevisiae cells growing in orthophosphate-sufficient medium was found to increase until glucose was exhausted in the medium . In the early logarithimic phase, the degree of polymerization of PPs (high-polymeric PPs in particular) fell drastically . Further cultivation of yeast in the glucose-depleted medium led to a decrease in the cellular content of all PP fractions without any considerable shortening of their chain length; by the 24th h, high-polymeric PPs accumulated in the cells . Shortening of PPs against a background of their intense synthesis in yeast cells growing in glucose-and orthophosphate-sufficient medium can be explained by the involvement of so far unknown mechanisms of activation of polyphosphate-depolymerizing enzymes. J Biol Chem, 1998 Jul 17, 273(29), 18573 - 85 Complex formation by all five homologues of mammalian translation initiation factor 3 subunits from yeast Saccharomyces cerevisiae; Asano K et al.; The PRT1, TIF34, GCD10, and SUI1 proteins of Saccharomyces cerevisiae were found previously to copurify with eukaryotic translation initiation factor 3 (eIF3) activity . Although TIF32, NIP1, and TIF35 are homologous to subunits of human eIF3, they were not known to be components of the yeast factor . We detected interactions between PRT1, TIF34, and TIF35 by the yeast two-hybrid assay and in vitro binding assays . Discrete segments (70-150 amino acids) of PRT1 and TIF35 were found to be responsible for their binding to TIF34 . Temperature-sensitive mutations mapping in WD-repeat domains of TIF34 were isolated that decreased binding between TIF34 and TIF35 in vitro . The lethal effect of these mutations was suppressed by increasing TIF35 gene dosage, suggesting that the TIF34-TIF35 interaction is important for TIF34 function in translation . Pairwise in vitro interactions were also detected between PRT1 and TIF32, TIF32 and NIP1, and NIP1 and SUI1 . Furthermore, PRT1, NIP1, TIF34, TIF35, and a polypeptide with the size of TIF32 were specifically coimmunoprecipitated from the ribosomal salt wash fraction . We propose that all five yeast proteins homologous to human eIF3 subunits are components of a stable heteromeric complex in vivo and may comprise the conserved core of yeast eIF3. J Biol Chem, 1998 Jul 17, 273(29), 18210 - 9 Disruption of the Saccharomyces cerevisiae FAT1 gene decreases very long-chain fatty acyl-CoA synthetase activity and elevates intracellular very long-chain fatty acid concentrations; Watkins PA et al.; Activation of fatty acids to their coenzyme A derivatives is necessary for subsequent metabolism . Very long-chain fatty acids, which accumulate in tissues of patients with X-linked adrenoleukodystrophy, are activated by very long-chain acyl-CoA synthetase (VLCS) normally found in peroxisomes and microsomes . We identified a candidate yeast VLCS gene (FAT1), previously identified as encoding a fatty acid transport protein, by its homology to rat liver peroxisomal VLCS . Disruption of this gene decreased, but did not abolish, cellular VLCS activity . Fractionation studies showed that VLCS activity, but not long-chain acyl-CoA synthetase activity, was reduced to about 40% of wild-type level in both 27,000 x g supernatant and pellet fractions . Separation of organelles in the pellet fraction by density gradient centrifugation revealed that VLCS activity was associated with peroxisomes and microsomes but not mitochondria . FAT1 deletion strains exhibited decreased growth on medium containing dextrose, oleic acid, and cerulenin, an inhibitor of fatty acid synthesis . FAT1 deletion strains grown on either dextrose or oleic acid medium accumulated very long-chain fatty acids . Compared with wild-type yeast, C22:0, C24:0, and C26:0 levels were increased approximately 20-, 18-, and 3-fold in deletion strains grown on dextrose, and 2-, 7-, and 5-fold in deletion strains grown on oleate . Long-chain fatty acid levels in wild-type and deletion strains were not significantly different . All biochemical defects in FAT1 deletion strains were restored to normal after functional complementation with the FAT1 gene . The level of VLCS activity measured in both wild-type and deletion yeast strains transformed with FAT1 cDNA paralleled the level of expression of the transgene . The extent of both the decrease in peroxisomal VLCS activity and the very long-chain fatty acid accumulation in the yeast FAT1 deletion model resembles that observed in cells from X-linked adrenoleukodystrophy patients . These studies suggest that the FAT1 gene product has VLCS activity that is essential for normal cellular very long-chain fatty acid homeostasis. J Biol Chem, 1998 Jul 17, 273(29), 18205 - 9 The human homolog of Saccharomyces cerevisiae CDC45; Saha P et al.; In budding yeast Saccharomyces cerevisiae CDC45 is an essential gene required for initiation of DNA replication . A structurally related protein Tsd2 is necessary for DNA replication in Ustilago maydis . We have identified and cloned the gene for a human protein homologous to the fungal proteins . The human gene CDC45L is 30 kilobases long and contains 15 introns . The 16 exons encode a protein of 566 amino acids . The human protein is 52 and 49.5% similar to CDC45p and Tsd2p, respectively . The level of CDC45L mRNA peaks at G1-S transition, but total protein amount remains constant throughout the cell cycle . Consistent with a role of CDC45L protein in the initiation of DNA replication it co-immunoprecipitates from cell extracts with a putative replication initiator protein, human ORC2L . In addition, subcellular fractionation indicates that the association of the protein with the nuclear fraction becomes labile as S phase progresses . The CDC45L gene is located to chromosome 22q11.2 region by cytogenetics and by fluorescence in situ hybridization . This region, known as DiGeorge syndrome critical region, is a minimal area of 2 megabases, which is consistently deleted in DiGeorge syndrome and related disorders . The syndrome is marked by parathyroid hypoplasia, thymic aplasia, or hypoplasia and congenital cardiac abnormalities . CDC45L is the first gene mapped to the DiGeorge syndrome critical region interval whose loss may negatively affect cell proliferation. Carbohydr Res, 1998 Feb, 307(1-2), 177 - 83 Structural analysis of the intact polysaccharide mannan from Saccharomyces cerevisiae yeast using 1H and 13C NMR spectroscopy at 750 MHz; Vinogradov E et al.; The mannan from Saccharomyces cerevisiae yeast was studied by high field NMR spectroscopy in an attempt to deduce the structure of the polysaccharide and to assess the ratio of different side chains . The results show that all structural information, agreeing with previously published data, can be extracted by analysis of the NMR spectra of the intact and modified mannan. Mol Biol Cell, 1998 Jul, 9(7), 1873 - 89 Multiple functions for actin during filamentous growth of Saccharomyces cerevisiae; Cali BM et al.; Saccharomyces cerevisiae is dimorphic and switches from a yeast form to a pseudohyphal (PH) form when starved for nitrogen . PH cells are elongated, bud in a unipolar manner, and invade the agar substrate . We assessed the requirements for actin in mediating the dramatic morphogenetic events that accompany the transition to PH growth . Twelve "alanine scan" alleles of the single yeast actin gene (ACT1) were tested for effects on filamentation, unipolar budding, agar invasion, and cell elongation . Some act1 mutations affect all phenotypes, whereas others affect only one or two aspects of PH growth . Tests of intragenic complementation among specific act1 mutations support the phenotypic evidence for multiple actin functions in filamentous growth . We present evidence that interaction between actin and the actin-binding protein fimbrin is important for PH growth and suggest that association of different actin-binding proteins with actin mediates the multiple functions of actin in filamentous growth . Furthermore, characterization of cytoskeletal structure in wild type and act1/act1 mutants indicates that PH cell morphogenesis requires the maintenance of a highly polarized actin cytoskeleton . Collectively, this work demonstrates that actin plays a central role in fungal dimorphism. J Bacteriol, 1998 Jul, 180(14), 3533 - 40 Regulation of expression of GLT1, the gene encoding glutamate synthase in Saccharomyces cerevisiae; Valenzuela L et al.; Saccharomyces cerevisiae glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by GLT1 . In this work, we analyzed GLT1 transcriptional regulation . GLT1-lacZ fusions were prepared and GLT1 expression was determined in a GDH1 wild-type strain and in a gdh1 mutant derivative grown in the presence of various nitrogen sources . Null mutants impaired in GCN4, GLN3, GAT1/NIL1, or UGA43/DAL80 were transformed with a GLT1-lacZ fusion to determine whether the above-mentioned transcriptional factors had a role in GLT1 expression . A collection of increasingly larger 5' deletion derivatives of the GLT1 promoter was constructed to identify DNA sequences that could be involved in GLT1 transcriptional regulation . The effect of the lack of GCN4, GLN3, or GAT1/NIL1 was also tested in the pertinent 5' deletion derivatives . Our results indicate that (i) GLT1 expression is negatively modulated by glutamate-mediated repression and positively regulated by Gln3p- and Gcn4p-dependent transcriptional activation; (ii) two cis-acting elements, a CGGN15CCG palindrome and an imperfect poly(dA-dT), are present and could play a role in GLT1 transcriptional activation; and (iii) GLT1 expression is moderately regulated by GCN4 under amino acid deprivation . Our results suggest that in a wild-type strain grown on ammonium, GOGAT constitutes an ancillary pathway for glutamate biosynthesis. FEBS Lett, 1998 Jun 5, 429(1), 83 - 8 The mating factor response pathway regulates transcription of TEC1, a gene involved in pseudohyphal differentiation of Saccharomyces cerevisiae; Oehlen L et al.; The transcription factor Tec1 is involved in pseudohyphal differentiation and agar-invasive growth of Saccharomyces cerevisiae cells . The sole element in the TEC1 promoter that has thus far been shown to control Tec1 function is the filament response element . We find that the TEC1 promoter also contains several pheromone response element sequences which are likely to be functional: TEC1 transcription is induced by mating factor, cell cycle regulated and dependent on the Ste4, Ste18 and Ste5 components of the mating factor signal transduction pathway . Using alleles of the transcription factor Ste12 that are defective in DNA binding, transcriptional induction or cooperativity with other transcription factors, we find little correlation between TEC1 transcript levels and agar-invasive growth. Immunol Lett, 1998 Apr, 61(2-3), 113 - 7 Humoral immune response to a 200-kDa glycoprotein antigen of Saccharomyces cerevisiae is common in man; Sander U et al.; According to Heelan et al . patients suffering from Crohn's disease (CD) produce antibodies against a cell wall associated glycoprotein antigen gp200 of the yeast Saccharomyces cerevisiae, while healthy people do not . Here the authors show, that antibodies against this glycoprotein gp200 can also be detected in the sera of healthy humans . The intensity of the antibody titer which is measured by immunoblot experiments is independent from the state of health . The Saccharomyces cerevisiae specific gp200 is a highly glycosylated protein localized not only in the cell wall but also accumulated in the culture medium . Some of the tested sera from CD patients, as well as from healthy adults, also reacted with a 120-kDa glycoprotein which is to be found in preparations containing secreted proteins . Because the binding of antibodies is greatly reduced by periodate treatment of gp200 and by the 120-kDa polypeptide, it is very likely that their carbohydrate moieties are the antigenic determinants against which the specific human antibodies are directed . The human humoral immune response applies only to Saccharomyces cerevisiae antigens, because no analogous immune responses could be detected against antigens derived from the yeast Arxula adeninivorans. Protein Sci, 1998 Jun, 7(6), 1380 - 7 Crystal structure of Saccharomyces cerevisiae cytosolic aspartate aminotransferase; Jeffery CJ et al.; The crystal structure of Saccharomyces cerevisiae cytoplasmic aspartate aminotransferase (EC 2.6.1.1) has been determined to 2.05 A resolution in the presence of the cofactor pyridoxal-5'-phosphate and the competitive inhibitor maleate . The structure was solved by the method of molecular replacement . The final value of the crystallographic R-factor after refinement was 23.1% with good geometry of the final model . The yeast cytoplasmic enzyme is a homodimer with two identical active sites containing residues from each subunit . It is found in the "closed" conformation with a bound maleate inhibitor in each active site . It shares the same three-dimensional fold and active site residues as the aspartate aminotransferases from Escherichia coli, chicken cytoplasm, and chicken mitochondria, although it shares less than 50% sequence identity with any of them . The availability of four similar enzyme structures from distant regions of the evolutionary tree provides a measure of tolerated changes that can arise during millions of years of evolution. FEBS Lett, 1998 May 29, 428(3), 281 - 5 Analysis of functional domains of Wilson disease protein (ATP7B) in Saccharomyces cerevisiae; Iida M et al.; Wilson disease is a genetic disorder of copper metabolism characterized by the toxic accumulation of copper in the liver . The ATP7B gene, which encodes a copper transporting P-type ATPase, is defective in patients with Wilson disease . To investigate the function of ATP7B, wild type or mutated ATP7B cDNA was introduced into a yeast strain lacking the CCC2 gene (delta ccc2), the yeast homologue of ATP7B . Wild type and the H1069Q mutant could rescue delta ccc2, however, the N1270S mutant could not, reflecting phenotypic variability of Wilson disease . In addition, the mutant containing only the sixth copper binding domain could rescue delta ccc2, indicating its functional importance. FEBS Lett, 1998 May 29, 428(3), 245 - 9 Saccharomyces cerevisiae mitochondria can synthesise FMN and FAD from externally added riboflavin and export them to the extramitochondrial phase; Pallotta ML et al.; Evidence is given that mitochondria isolated from Saccharomyces cerevisiae can take up externally added riboflavin and synthesise from it both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) probably due to the existence of the mitochondrial riboflavin kinase already reported and the novel mitochondria FAD synthetase . Moreover Saccharomyces cerevisiae mitochondria can export the newly synthesised flavin derivatives to the extramitochondrial phase . This has been proven to take place with 1:1 stoichiometry with riboflavin decrease outside mitochondria, thus showing that flavin traffic occurs across the mitochondrial membranes. Eur J Biochem, 1998 May 1, 253(3), 560 - 75 Identification of Man alpha1-3Man alpha1-2Man and Man-linked phosphate on O-mannosylated recombinant leech-derived tryptase inhibitor produced by Saccharomyces cerevisiae and determination of the solution conformation of the mannosylated polypeptide; Bergwerff AA et al.; The production of recombinant leech-derived tryptase inhibitor (rLDTI) by two different strains of Saccharomyces cerevisiae resulted in the secretion of non-glycosylated and glycosylated rLTDI . Monosaccharide analysis and a-mannosidase treatment demonstrated that glycosylated rLDTI was exclusively alpha-mannosylated . A trypsin digest of reduced and S-carboxymethylated glycosylated rLDTI was separated on a reverse-phase HPLC column . Glycopeptides identified by a combination of matrix-assisted laser desorption mass spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed the presence of different glycoforms . It was found that Ser24, Ser33 and Ser36 were partially glycosylated with a single mannose residue, whereas Thr42 in glycosylated rLDTI from both strains was fully occupied with manno-oligosaccharides with a degree of polymerization ranging over 1-3 and 1-13 depending on the yeast strain . In phosphorylated rLDTI a single phosphate group was predominantly located at the innermost Man residue of units of mannobiose, mannotriose, mannotetraose and mannopentaose at Thr42 . Oligosaccharides released by alkaline treatment were reduced by sodium borohydride and separated by high-pH anion-exchange chromatography on a CarboPac MA1 column, and analyzed by one- and two-dimensional 1H-NMR spectroscopy . Besides the major oligosaccharide Man alpha1-2Man-ol, the (for yeast protein O-glycosylation) unusual Man alpha1-3Man alpha1-2Man-ol was determined . The solution conformation of glycosylated rLDTI was investigated by two-dimensional NMR spectroscopy . Structure calculations by means of distance geometry showed that glycosylated rLDTI is compactly folded and contained small secondary structure elements . Analysis of the chemical shifts showed that amino acids Val32-Ser33, Ser36-Ser39 and Thr42 were affected by the O-mannosylation . In addition, changes in chemical shift were observed within the beta-hairpin peptide regions Val13-Ser16 and Gly18-Tyr21 attributed to direct interactions of the mannose residue at Ser36 . Furthermore, the protein-linked oligosaccharides were spatially grouped in a position opposite of the canonical binding loop. Eur J Cell Biol, 1998 May, 76(1), 43 - 52 Traffic into the prevacuolar/endosomal compartment of Saccharomyces cerevisiae: a VPS45-dependent intracellular route and a VPS45-independent, endocytic route; Bryant NJ et al.; The vps (vacuolar protein sorting) mutants have been used to dissect and characterize the vacuolar biogenesis pathway in the yeast Saccharomyces cerevisiae . The vps mutants were isolated through their loss of ability to correctly sort the vacuolar hydrolase CPY, which travels from Golgi membranes to the vacuole through a prevacuolar compartment . Over 50 VPS genes have been divided into 6 classes according to vacuolar morphology . Mutations in any one of the class E VPS genes, such as VPS27, lead to an exaggerated form of the prevacuolar compartment . This class E compartment contains endocytosed proteins as well as proteins en route to the vacuole, and is thus taken to represent an intersection point between the endocytic and biosynthetic pathways . Mutations in the class D gene VPS45 can be used to define a second transport intermediate along the vacuolar biogenesis pathway, Golgi-derived transport vesicles carrying vacuolar membrane proteins on their way to the vacuole . Here we demonstrate that the Sec1p-like protein Vps45p is required for the fusion of Golgi-derived vesicles with the prevacuolar compartment indicating that VPS45 functions before VPS27 in the vacuolar biogenesis pathway . In addition, we show that VPS45 function is not required for the delivery of endocytosed proteins to the prevacuolar compartment from the plasma membrane suggesting that the function of Vps45p is restricted to a single vesicular pathway. J Biotechnol, 1998 Mar 26, 61(1), 57 - 68 Expression in Saccharomyces cerevisiae of antigenically and enzymatically active recombinant glutamic acid decarboxylase; Law RH et al.; Glutamic acid decarboxylase (GAD) is one of the major autoantigens found in insulin-dependent (Type 1) diabetes mellitus (IDDM) . A novel hybrid form of GAD was created by fusing amino acids 1-101 of the human GAD67 protein to amino acids 96-585 of the human GAD65 protein . This hybrid GAD67/65 was expressed constitutively under the control of the phosphoglycerate kinase promoter (PGK1) in the yeast Saccharomyces cerevisiae . Enzymatically active GAD was prepared from yeast lysates by a one-step purification on an affinity column using GAD-1 antibody . The purified hybrid GAD67/65 was radiolabelled with iodine-125 and tested in an immunoprecipitation assay with IDDM sera . Results obtained using the recombinant yeast hybrid GAD67/65 were very similar to those obtained using 125I-labelled porcine GAD . Recombinant yeast hybrid GAD67/65 should have utility for diagnosis and presymptomatic detection of IDDM. J Biotechnol, 1998 Mar 26, 61(1), 15 - 31 Effect of the dilution rate on the mode of oscillation in continuous cultures of Saccharomyces cerevisiae; Beuse M et al.; The growth properties of the asymmetric budding yeast Saccharomyces cerevisiae were analysed during spontaneous oscillations in continuous cultures at varying dilution rates D . The length of the oscillation period changed between 1.4 and 14 h in response to the decrease of dilution rate from 0.15 to 0.05 h-1 . The distribution of parent and daughter cells in the population was determined microscopically after staining the bud scars and DNA . Most of the data obtained fits a theoretical population balance model assuming two-classes of subpopulations and integer ratios between the generation times of both classes . Some data has to be described by an extended population model assuming there is one parent and two daughter cell classes . How changes of dilution rate may cause an accidental switch of the mode of oscillation is demonstrated . Glucose consumption and metabolite production were measured off-line by enzymatic methods and gas exchange was monitored on-line . All these data of one period point to internal and external signals responsible for the synchronisation of the cell cycle. Genetics, 1998 Jul, 149(3), 1495 - 509 Isolation of the gene encoding the Drosophila melanogaster homolog of the Saccharomyces cerevisiae GCN2 eIF-2alpha kinase; Olsen DS et al.; Genomic and cDNA clones homologous to the yeast GCN2 eIF-2alpha kinase (yGCN2) were isolated from Drosophila melanogaster . The identity of the Drosophila GCN2 (dGCN2) gene is supported by the unique combination of sequence encoding a protein kinase catalytic domain and a domain homologous to histidyl-tRNA synthetase and by the ability of dGCN2 to complement a deletion mutant of the yeast GCN2 gene . Complementation of Deltagcn2 in yeast by dGCN2 depends on the presence of the critical regulatory phosphorylation site (serine 51) of eIF-2alpha . dGCN2 is composed of 10 exons encoding a protein of 1589 amino acids . dGCN2 mRNA is expressed throughout Drosophila development and is particularly abundant at the earliest stages of embryogenesis . The dGCN2 gene was cytogenetically and physically mapped to the right arm of the third chromosome at 100C3 in STS Dm2514 . The discovery of GCN2 in higher eukaryotes is somewhat unexpected given the marked differences between the amino acid biosynthetic pathways of yeast vs . Drosophila and other higher eukaryotes . Despite these differences, the presence of GCN2 in Drosophila suggests at least partial conservation from yeast to multicellular organisms of the mechanisms responding to amino acid deprivation. Genetics, 1998 Jul, 149(3), 1277 - 92 The PBN1 gene of Saccharomyces cerevisiae: an essential gene that is required for the post-translational processing of the protease B precursor; Naik RR et al.; The vacuolar hydrolase protease B in Saccharomyces cerevisiae is synthesized as an inactive precursor (Prb1p) . The precursor undergoes post-translational modifications while transiting the secretory pathway . In addition to N- and O-linked glycosylations, four proteolytic cleavages occur during the maturation of Prb1p . Removal of the signal peptide by signal peptidase and the autocatalytic cleavage of the large amino-terminal propeptide occur in the endoplasmic reticulum (ER) . Two carboxy-terminal cleavages of the post regions occur in the vacuole: the first cleavage is catalyzed by protease A and the second results from autocatalysis . We have isolated a mutant, pbn1-1, that exhibits a defect in the ER processing of Prb1p . The autocatalytic cleavage of the propeptide from Prb1p does not occur and Prb1p is rapidly degraded in the cytosol . PBN1 was cloned and is identical to YCL052c on chromosome III . PBN1 is an essential gene that encodes a novel protein . Pbn1p is predicted to contain a sub-C-terminal transmembrane domain but no signal sequence . A functional HA epitope-tagged Pbn1p fusion localizes to the ER . Pbn1p is N-glycosylated in its amino-terminal domain, indicating a lumenal orientation despite the lack of a signal sequence . Based on these results, we propose that one of the functions of Pbn1p is to aid in the autocatalytic processing of Prb1p. Mol Gen Genet, 1998 May, 258(4), 437 - 41 DNA replication is completed in Saccharomyces cerevisiae cells that lack functional Cdc14, a dual-specificity protein phosphatase; Fitzpatrick PJ et al.; The Cdc14 protein encodes a dual-specificity protein phosphatase which functions in late mitosis, and considerable genetic evidence suggests a role in DNA replication . We find that cdc14 mutants arrested in late mitosis maintain persistent levels of mitotic kinase activity, suggesting that Cdc14 controls inactivation of this kinase . Overexpression of Sicl, a cyclin-dependent protein kinase inhibitor, is able to suppress telophase mutants such as dbf2, cdc5 and cdc15, but not cdc14 . It does, however, force cdc14-arrested cells into the next cell cycle, in which an apparently normal S phase occurs as judged by FACS and pulsed-field gel electrophoretic analysis . Furthermore, in a promoter shut-off experiment, cells lacking Cdc14 appear to carry out a normal S phase . Thus Cdc14 functions mainly in late mitosis and it has no essential role in S phase. Mol Gen Genet, 1998 May, 258(3), 288 - 96 Identification and characterisation of an RPD3 homologue from maize (Zea mays L.) that is able to complement an rpd3 null mutant of Saccharomyces cerevisiae; Rossi V et al.; In mammals, yeast and Drosophila, the histone deacetylase RPD3 proteins can alter the expression of genes involved in fundamental biological processes by affecting the degree of acetylation of histones and changing chromatin structure . Here we report the isolation of a cDNA sequence encoding an RPD3 homologue from maize, which is able to complement the phenotype of an rpd3 null mutant of the yeast Saccharomyces cerevisiae . The expression of the corresponding gene(s) was assessed in different maize tissues . The number of homologous loci was estimated by Southern hybridisation to be in the range of two to three, and the chromosomal location of one of these loci was determined . Phylogenetic analysis and tests for relative divergence rates, using related RPD3 sequences from different species, were performed, and suggest that different polymorphic forms of RPD3-like proteins that evolve at distinct rates are present in the species considered. J Biochem (Tokyo), 1998 Jul, 124(1), 141 - 7 Substrate specificities and kinetic properties of proteinase A from the yeast Saccharomyces cerevisiae and the development of a novel substrate; Kondo H et al.; The substrate specificities and kinetic properties of proteinase A, an intracellular aspartic proteinase from the yeast Saccharomyces cerevisiae, were determined using a series of synthetic chromogenic peptides with the general structure P5-P4-P3-P2-Phe-(NO2)Phe-P2'-P3' {P5, P4, P3, P2, P2', P3' are various amino acids; (NO2)Phe is p-nitro-L-phenylalanine} . The nature of the residues occupying the NH2-terminal region of the substrate had a strong influence on the kinetic constants.Among those tested, Ala-Pro-Ala-Lys-Phe-(NO2)-Phe-Arg-Leu had the best kinetic constants (Km = 0.012 mM, kcat = 14.4 s-1, kcat/Km = 1,200 M-1.s-1) . Compared with such aspartic proteinases as pepsin, cathepsin D, and renin, the substrate specificity of proteinase A was unique . Based on these results, a novel fluorescent substrate, MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2, was developed for the sensitive measurement of proteinase A. Curr Genet, 1998 Jun, 33(6), 406 - 11 Functional analysis of upstream activating elements in the promoter of the FBP1 gene from Saccharomyces cerevisiae; de Mesquita JF et al.; We have investigated the effect of different carbon sources and of different mutations on the capacity of two elements, UAS1 and UAS2, from the promoter of the FBP1 gene to form specific DNA-protein complexes and to activate expression of a reporter gene . The complexes are observed with nuclear extracts from yeast derepressed on glycerol or ethanol . When hxk2 mutants are grown on glucose the nuclear extracts are able to complex UAS1 but not UAS2, while for wild-type cells grown on galactose only the complex with UAS2 is formed . In contrast, in vivo the operation of both UASs is high in ethanol, moderate to low in glycerol, and negligible in galactose; no expression is observed in glucose even in a hxk2 background . There is no effect of a MIG1 deletion, either in the formation of DNA-protein complexes or on the expression of reporter genes. J Bacteriol, 1998 Jul, 180(13), 3381 - 7 Sed1p is a major cell wall protein of Saccharomyces cerevisiae in the stationary phase and is involved in lytic enzyme resistance; Shimoi H et al.; A 260-kDa structural cell wall protein was purified from sodium dodecyl sulfate-treated cell walls of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme . Amino acid sequence analysis revealed that this protein is the product of the SED1 gene . SED1 was formerly identified as a multicopy suppressor of erd2, which encodes a protein involved in retrieval of luminal endoplasmic reticulum proteins from the secretory pathway . Sed1p is very rich in threonine and serine and, like other structural cell wall proteins, contains a putative signal sequence for the addition of a glycosylphosphatidylinositol anchor . However, the fact that Sed1p, unlike other cell wall proteins, has six cysteines and seven putative N-glycosylation sites suggests that Sed1p belongs to a new family of cell wall proteins . Epitope-tagged Sed1p was detected in a beta-1,3-glucanase extract of cell walls by immunoblot analysis, suggesting that Sed1p is a glucanase-extractable cell wall protein . The expression of Sed1p mRNa increased in the stationary phase and was accompanied by an increase in the Sed1p content of cell walls . Disruption of SED1 had no effect on exponentially growing cells but made stationary-phase cells sensitive to Zymolyase . These results indicate that Sed1p is a major structural cell wall protein in stationary-phase cells and is required for lytic enzyme resistance. Biochem J, 1998 Jun 1, 332 ( Pt 2), 525 - 31 Characterization of sphingosine kinase (SK) activity in Saccharomyces cerevisiae and isolation of SK-deficient mutants; Lanterman MM et al.; Sphingosine kinase (SK) catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, which is a second messenger involved in the proliferative responses of mammalian cells . Although the yeast Saccharomyces cerevisiae has similar phosphorylated sphingoid bases which appear to be involved in growth regulation and the response to stress, SK activity had not been previously demonstrated in yeast . In this study, an in vitro system was set up to characterize yeast SK activity . Activity was detected in the cytosol at neutral pH and 37 degreesC . Yeast SK phosphorylated the sphingoid bases sphingosine, dihydrosphingosine and phytosphingosine . (d,l)-threo-dihydrosphingosine, an inhibitor of mammalian SK, did not inhibit the yeast enzyme . Unique properties of yeast SK were an optimal temperature of 43 degreesC, and in vivo activation during nutrient deprivation . Spontaneous mutants with diminished SK activity were isolated utilizing a screen for resistance to sphingosine in a sphingosine-phosphate-lyase deletion background . Abnormal growth and heat sensitivity were observed in these mutants . These findings suggest that SK may function as a stress-response protein in yeast. Yeast, 1998 May, 14(7), 633 - 46 The Saccharomyces cerevisiae early secretion mutant tip20 is synthetic lethal with mutants in yeast coatomer and the SNARE proteins Sec22p and Ufe1p; Frigerio G; Tip20p is an 80 kDa cytoplasmic protein bound to the cytoplasmic surface of the endoplasmic reticulum (ER) by interaction with the type II integral membrane protein Sec20p . Both proteins are required for vesicular transport between the ER and Golgi complex . Recently, sec20-1 was found to be defective in retrograde transport . A collection of temperature-sensitive tip20 mutants are shown to be lethal in combination with ufe1-1, a target SNARE of the ER and ret2-1, yeast delta-COP . A subset of tip20 mutants was found to be lethal in combination with sec20-1, sec21-1, sec22-3 and sec27-1 . Since all pairwise combinations of a tip20 mutant, sec20-1, and ufe1-1 are lethal, Tip20p and Sec20p might be part of the docking complex for Golgi-derived retrograde transport vesicles . Since carboxy-terminal tip20 truncations are lethal in combination with mutants in three coatomer subunits, Tip20p might be involved in binding or uncoating of COPI coated retrograde transport vesicles. Yeast, 1998 May, 14(7), 623 - 31 Bicarbonate-mediated social communication stimulates meiosis and sporulation of Saccharomyces cerevisiae; Ohkuni K et al.; Meiosis and sporulation in the yeast Saccharomyces cerevisiae requires social communication, mediated by an extracellular factor which is secreted from cells during sporulation and accumulates in a cell density-dependent manner . We show here genetic and biochemical analyses supporting our conclusion that the extracellular factor is bicarbonate acting as an alkali to elevate extracellular pH . Sporulation defects of mdh1 (mitochondrial malate dehydrogenase) mutants and of wild-type cells at low density were rescued extracellularly by addition of bicarbonate or other alkaline solutions to raise medium pH . Addition of bicarbonate (or alkalization of medium) raised steady-state levels of mRNA in respiration-deficient mdh1 mutants and inhibited proliferation of wild-type cells at low density . These results indicate that the two conditions (respiration competency and high cell density), required for meiosis and sporulation, are essential for extracellular accumulation of bicarbonate and resulting alkalization of medium. Yeast, 1998 May, 14(7), 601 - 16 Physical mapping of chromosomes VII and XV of Saccharomyces cerevisiae at 3.5 kb average resolution to allow their complete sequencing; Tettelin H et al.; The high resolution complete physical maps of chromosomes VII and XV were constructed to form the basis for sequencing these chromosomes as part of the European systematic sequencing programme of the yeast genome, using a unique cosmid library from strain FY1679, and an original top-down mapping strategy involving I-Sce I chromosome fragmentation . A total of 138 and 196 cosmid clones were used to construct the maps for VII and XV, respectively, forming two unique contigs that cover the entirety of chromosomes (1091 kb each), except the telomeric repeats . Colinearity of the cosmid inserts with yeast DNA was verified, and the physical maps were eventually compared with the independently generated genetic maps. Mol Cells, 1998 Apr 30, 8(2), 130 - 7 Saccharomyces cerevisiae STE11 may contribute to the stabilities of a scaffold protein, STE5, in the pheromone signaling pathway; Kim SH et al.; Saccharomyces cerevisiae STE5 is an essential component of the pheromone-mediated-mitogen activated protein kinase (MAPK) pathway . The STE5 protein recruits MAPK module kinases (STE11, STE7, and FUS3) to give a specificity for the pheromone pathway . The STE5 protein contains a putative PEST motif for ubiquitin-dependent protein degradation, and its level may be important for regulation of pheromone signal transduction . In this article, we studied the roles of proteins associated with the STE5 protein for its stabilization by analyzing ste deletion mutants . Here, we found that the STE11 kinase performed the most important role in stabilization of the STE5 protein . The level of STE5 protein was significantly low in the absence of STE11 kinase, suggesting essential roles of STE11 in stabilization of the STE5 protein . Immunodetection and Northern blot analyses showed that the low level of the STE5 protein in the ste11 delta mutant is not due to the level of gene expression but to that of protein stability . Measurement of relative binding affinities showed that the STE11 protein tightly interacts with the STE5 protein for its stabilization. Mol Cell Biol, 1998 Jul, 18(7), 4368 - 76 Positive and negative autoregulation of REB1 transcription in Saccharomyces cerevisiae; Wang KL et al.; Reb1p is a DNA binding protein of Saccharomyces cerevisiae that has been implicated in the activation of transcription by polymerase (Pol) II, in the termination of transcription by Pol I, and in the organization of nucleosomes . Studies of the transcriptional control of the REB1 gene have led us to identify three Reb1p binding sites in the 5' region of the its gene, termed A, B, and C, at positions -110, -80, and +30 with respect to transcription initiation . In vitro, Reb1p binds to the three sites with the relative affinity of A >/= C > B . Kinetic parameters suggest that when both A and C sites are present on the same DNA molecule, the C site may recruit Reb1p for the A site . In vivo the A and B sites each contribute to the transcription activity of REB1 in roughly additive fashion . Mutation of both A and B sites abolishes transcription . On the other hand, the C site is a negative element, reducing transcription by 40% . In cells overexpressing Reb1p, the C site reduces transcription by more than 80% . This effect can be transposed to another transcription unit, demonstrating that the effect of Reb1p binding at the C site does not depend on interaction with upstream Reb1p molecules . Relocation of the C site to a position 105 bp downstream of the transcription initiation site abolishes its effect, suggesting that it does not act as a conventional attenuator of transcription . We conclude that binding of Reb1p at the C site hinders formation of the initiation complex . This arrangement of Reb1p binding sites provides a positive and negative mechanism to autoregulate the expression of REB1 . Such an arrangement could serve to dampen the inevitable fluctuation in Rep1p levels caused by the intermittent presence of its mRNA within an individual cell. Mol Cell Biol, 1998 Jul, 18(7), 4043 - 52 Prohibitin family members interact genetically with mitochondrial inheritance components in Saccharomyces cerevisiae; Berger KH et al.; Phb2p, a homolog of the tumor suppressor protein prohibitin, was identified in a genetic screen for suppressors of the loss of Mdm12p, a mitochondrial outer membrane protein required for normal mitochondrial morphology and inheritance in Saccharomyces cerevisiae . Phb2p and its homolog, prohibitin (Phb1p), were localized to the mitochondrial inner membrane and characterized as integral membrane proteins which depend on each other for their stability . In otherwise wild-type genetic backgrounds, null mutations in PHB1 and PHB2 did not confer any obvious phenotypes . However, loss of function of either PHB1 or PHB2 in cells with mitochondrial DNA deleted led to altered mitochondrial morphology, and phb1 or phb2 mutations were synthetically lethal when combined with a mutation in any of three mitochondrial inheritance components of the mitochondrial outer membrane, Mdm12p, Mdm10p, and Mmm1p . These results provide the first evidence of a role for prohibitin in mitochondrial inheritance and in the regulation of mitochondrial morphology. Mol Cell Biol, 1998 Jul, 18(7), 3752 - 61 Interaction of TATA-binding protein with upstream activation factor is required for activated transcription of ribosomal DNA by RNA polymerase I in Saccharomyces cerevisiae in vivo; Steffan JS et al.; Previous in vitro studies have shown that initiation of transcription of ribosomal DNA (rDNA) in the yeast Saccharomyces cerevisiae involves an interaction of upstream activation factor (UAF) with the upstream element of the promoter, forming a stable UAF-template complex; together with TATA-binding protein (TBP), UAF then recruits an essential factor, core factor (CF), to the promoter, forming a stable preinitiation complex . TBP interacts with both UAF and CF in vitro . In addition, a subunit of UAF, Rrn9p, interacts with TBP in vitro and in the two-hybrid system, suggesting the possible importance of this interaction for UAF function . Using the yeast two-hybrid system, we have identified three mutations in RRN9 that abolish the interaction of Rrn9p with TBP without affecting its interaction with Rrn10p, another subunit of UAF . Yeast cells containing any one of these individual mutations, L110S, L269P, or L274Q, did not show any growth defects . However, cells containing a combination of L110S with one of the other two mutations showed a temperature-sensitive phenotype, and this phenotype was suppressed by fusing the mutant genes to SPT15, which encodes TBP . In addition, another mutation (F186S), which disrupts both Rrn9p-TBP and Rrn9p-Rrn10p interactions in the two-hybrid system, abolished UAF function in vivo, and this mutational defect was suppressed by fusion of the mutant gene to SPT15 combined with overexpression of Rrn10p . These experiments demonstrate that the interaction of UAF with TBP, which is presumably achieved by the interaction of Rrn9p with TBP, is indeed important for high-level transcription of rDNA by RNA polymerase I in vivo. Mutat Res, 1998 Mar, 407(2), 177 - 87 Requirement for DNA mismatch repair proteins in the transcription-coupled repair of thymine glycols in Saccharomyces cerevisiae; Leadon SA et al.; Defects in DNA mismatch repair have been shown to lead to increased genomic instability and mutability . We recently found that human cells defective in the DNA mismatch repair gene, hMSH2, were deficient in the transcription-coupled repair (TCR) of both oxidative DNA damage, including thymine glycols, and UV-induced DNA damage . However, in a hMLH1 mutant, only a reduction in the TCR of UV damage was observed . In this study, we examined whether TCR of thymine glycols in Saccharomyces cerecisiae also requires the genes involved in DNA mismatch repair . We found that yeast cells containing mutations in MSH2 were deficient in the removal of thymine glycols from the transcribed strand of the RPB2 gene, while cells with mutations in either MLH1 or PMS1 alone showed near normal levels of TCR of thymine glycols . Interestingly, double mutants in the MLH1 and PMS1 genes were deficient in TCR of thymine glycols . Taken together, these results suggest that these two MutL homologues can act independently of each other, but that they have overlapping roles in TCR . Overall levels of thymine glycol removal were not reduced in the mismatch repair mutants . In contrast to the results with thymine glycols, no defects in TCR of pyrimidine dimers were found in cells with mutations in MSH2, MLH1, PMS1, and MLH1/PMS1. Mutat Res, 1998 Mar, 407(2), 135 - 45 Genetic interactions between mutants of the 'error-prone' repair group of Saccharomyces cerevisiae and their effect on recombination and mutagenesis; Liefshitz B et al.; We have created an isogenic series of yeast strains that carry genetic systems to monitor different types of recombination and mutation {B . Liefshitz, A . Parket, R . Maya, M . Kupiec, The role of DNA repair genes in recombination between repeated sequences in yeast, Genetics 140 (1995) 1199-1211.} . In the present study we characterize the effect of mutations in genes of the 'error-prone' or postreplicative repair group on recombination and mutation . We show that rad5 and rad18 strains have elevated levels of spontaneous recombination, both of ectopic gene conversion and of recombination between direct repeats . The increase in recombination levels is similar in both mutants and in the rad5 rad18 double mutant, suggesting that the RAD5 and RAD18 gene products act together with respect to spontaneous recombination . In contrast, RAD5 and RAD18 play alternative roles in mutagenic repair: mutations in each of these genes elevate spontaneous forward mutation at the CAN1 locus, but when both genes are deleted, a low level of spontaneous mutagenesis is seen . The RAD5/RAD18 pathway of mutagenic repair is dependent on the REV3-encoded translesion polymerase . We analyze the interactions between the RAD5 and RAD18 gene products and other repair genes . The high recombination levels seen in rad5 and rad18 mutants is dependent on the RAD1, RAD51, RAD52, and RAD57 genes . The Srs2 helicase plays an important role in creating the recombinogenic substrate(s) processed by the RAD5 and RAD18 gene products. Biochemistry, 1998 Jun 16, 37(24), 8705 - 13 Insulin signaling in the yeast Saccharomyces cerevisiae . 3 . Induction of protein phosphorylation by human insulin; Muller G et al.; A low affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae has been identified recently (Muller, G., Rouveyre, N., Upshon, C., Gross, E., and Bandlow, W., preceding paper in this issue) . Since the mammalian insulin receptor functions as a tyrosine kinase with autophosphorylation capacity, kinase studies were performed with the partially purified insulin-binding protein preparation . Incubation with {gamma-32P}ATP in vitro led to phosphorylation of the 53-kDa insulin-binding protein on serine but not on tyrosine residues . In addition, a 70-kDa polypeptide, copurified with the insulin-binding protein preparation, was tyrosine-phosphorylated under the same conditions . Phosphorylation of both proteins was enhanced by human insulin . These results obtained by immunoprecipitation and immunoblotting using specific anti-phosphoserine/threonine/tyrosine antibodies were confirmed by phosphoamino acid analysis of the individual immunoprecipitated and gel-purified 32P-labeled phosphoproteins . During gel filtration, the 53-kDa protein coeluted as a 300-kDa complex together with the 70-kDa phosphotyrosine-containing protein and was coimmunoprecipitated with the latter using an anti-phosphotyrosine antibody, strongly arguing for complex formation between the two proteins . The data presented raise the possibility that stimulation of glycogen synthesis by insulin in yeast is mediated by a 53-kDa insulin-binding protein and a 70-kDa phosphotyrosine-containing protein which are organized in a large plasma membrane-bound signaling complex . Elucidation of the function and molecular mode of interaction of these components in yeast may help to understand metabolic insulin signaling in mammalian cells. Biochemistry, 1998 Jun 16, 37(24), 8696 - 704 Insulin signaling in the yeast Saccharomyces cerevisiae . 2 . Interaction of human insulin with a putative binding protein; Muller G et al.; A putative insulin-binding protein (Kd = 0.5 +/- 0.2 microM for human insulin) was partially purified from solubilized plasma membranes of Saccharomyces cerevisiaeby wheat germ agglutinin and insulin affinity chromatographies . The binding affinities of various mutant insulin analogues correlated well with their capacities to activate glycogen synthase and SNF1 kinase in glucose-induced yeast spheroplasts, the ranking of their relative efficacies in yeast and in isolated rat adipocytes being similar . Using a bifunctional cross-linker and two different experimental protocols, a 53-kDa polypeptide contained in the insulin-binding protein preparation was specifically affinity cross-linked to {125I}monoiodo{B26}insulin . The relative rankings of the insulin analogues with respect to inhibition of cross-linking and binding to the partially purified insulin-binding protein were identical . Incubation of intact yeast spheroplasts with {125I}monoiodo{AI4}insulin led to specific and time-dependent association of the radiolabeled insulin with the cell surface followed by its internalization and degradation . These processes were considerably delayed by low temperature and energy depletion of the spheroplasts, suggesting involvement of the ATP-dependent endosomal apparatus . These data provide evidence for the existence of a low-affinity insulin-binding protein in the plasma membrane of Saccharomyces cerevisiae. Biochemistry, 1998 Jun 16, 37(24), 8683 - 95 Insulin signaling in the yeast Saccharomyces cerevisiae . 1 . Stimulation of glucose metabolism and Snf1 kinase by human insulin; Muller G et al.; Effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae were studied in this report . Under two conditions of growth limitation (glucose-grown cells during transition to stationary phase or spheroplasts during incubation in synthetic glucose medium), human insulin (10 and 1 microM, respectively) enhanced glycogen accumulation and glycogen synthase activity by 40-60% compared to control cells . Glycogen phosphorylase activity was also increased under the same conditions, but this stimulation was diminished by 35-45% in insulin-treated compared to control cells . Thus, under growth limitation, insulin causes glycogen phosphorylase and glycogen synthase to become more sensitive to inactivation and activation, respectively . In glucose-induced spheroplasts, insulin (1 microM), in addition to glycogen accumulation, led to about 2-fold increases of the rates of ethanol production and glucose oxidation compared to control cells, and the maximal concentration of hexose 6-phosphate was increased by 30-40% . In contrast, glucose transport as well as the levels of the allosteric regulators, fructose 2,6-bisphosphate and cAMP, were not altered at all . Snf1 kinase is assumed to be involved in the regulation of glycogen metabolism in yeast, although it does not seem to be modulated directly by the glucose concentration . Snf1 kinase activity was elevated 5-10-fold in response to insulin both during glucose induction of yeast spheroplasts and during transition to stationary phase of glucose-grown cells . We conclude that Saccharomyces cerevisiae and insulin-sensitive mammalian cells share some parts of the signaling cascades regulating oxidative and nonoxidative glucose metabolism in response to glucose and insulin. Biochem Mol Biol Int, 1998 Jun, 45(1), 191 - 203 Luminol luminescence induced by oxidants in antioxidant-deficient yeasts Saccharomyces cerevisiae; Jakubowski W et al.; Luminol chemiluminescence induced in the presence of yeast cells and yeast cell homogenates was significantly induced by exogenous oxidants (hydrogen peroxide and menadione) . tert-Butyl hydroperoxide did not stimulate chemiluminescence by itself but augmented menadione-induced chemiluminescence . Comparison of yeast strains deficient in catalase, superoxide dismutase or glutathione showed that only glutathione-deficient strains showed elevated chemiluminescence in this system . These results support the idea that more reactive species than hydrogen peroxide and superoxide are critical in the induction of luminol chemiluminescence. Biochem Mol Biol Int, 1998 Jun, 45(1), 125 - 31 Soluble guanylate cyclase in Saccharomyces cerevisiae; Kuo WN et al.; After removing nonspecific immunoreactivities from crude extract by immunoaffinity chromatography, an immunoreactive-band at 40kDa of soluble guanylate cyclase (SGC) from Saccharomyces cerevisiae was detected by Western blot using rabbit anti-beta 1 subunit of SGC . Cyclic GMP level and SGC activity was measured by ELISA . Immunoprecipitated yeast SGC was activated by sodium nitroprusside, whereas inhibited by 1H-(1,2,4)oxadiazolo(4,3-A)quinoxalin-1-one . Increased cyclic GMP level was also noted when intact yeast cells were incubated with s-nitrosoglutathione, a NO donor . The result implies that NO can be utilized intracellularly and extracellularly . Moreover, the presence of SGC suggests the significance of NO/cyclic GMP signaling in unicellular eukaryotes. J Biol Chem, 1998 Jun 19, 273(25), 15787 - 93 MSS4, a phosphatidylinositol-4-phosphate 5-kinase required for organization of the actin cytoskeleton in Saccharomyces cerevisiae; Desrivieres S et al.; The Saccharomyces cerevisiae protein MSS4 is essential and homologous to mammalian phosphatidylinositol-4-phosphate (PI(4)P) 5-kinases . Here, we demonstrate that MSS4 is a lipid kinase . MSS4 has dual substrate specificity in vitro, converting PI(4)P to PI(4, 5)P2 and to a lesser extent PI(3)P to PI(3,4)P2; no activity was detected with PI or PI(5)P as a substrate . Cells overexpressing MSS4 contain an elevated level specifically of PI(4,5)P2, whereas mss4 mutant cells have only approximately 10% of the normal amount of this phosphorylated phosphoinositide . Furthermore, cells lacking MSS4 are unable to form actin cables and to properly localize their actin cytoskeleton during polarized cell growth . Overexpression of RHO2, encoding a Rho-type GTPase involved in regulation of the actin cytoskeleton, restores growth and polarized distribution of actin in an mss4 mutant . These results suggest that MSS4 is the major PI(4)P 5-kinase in yeast and provide a link between phosphoinositide metabolism and organization of the actin cytoskeleton in vivo. J Biol Chem, 1998 Jun 19, 273(25), 15352 - 7 Catabolite inactivation of wild-type and mutant maltose transport proteins in Saccharomyces cerevisiae; Brondijk TH et al.; The maltose transporter of Saccharomyces cerevisiae is subject to rapid, irreversible inactivation in the presence of glucose . Loss of transport function was paralleled by a decrease in amount of transporter protein and most likely involves endocytosis and degradation of the protein in the vacuole . This (catabolite) inactivation of Mal61p was triggered not only by glucose but also by 2-deoxy-D-glucose, which cannot be metabolized beyond 2-deoxy-D-glucose phosphate . The signal that targets membrane proteins specifically for catabolite inactivation is unknown . To investigate whether or not specific modification of Mal61p triggers the inactivation, putative protein kinase A and C phosphorylation sites were removed, and the transport activities and levels of the mutant proteins upon addition of glucose were followed in time . Three Mal61p mutants, i.e . S295A, T363A, and S487A, exhibited significantly reduced rates of inactivation in the presence of glucose . Likewise, in wild-type Mal61p the rate of inactivation and degradation of the protein paralleled each other in the case of T363A . On the contrary, for the S295A and S487A mutants the rates of protein degradation were slowed down more profoundly than was the loss of transport activity . These observations indicate that (i) some form of modification (e.g . phosphorylation) of the protein precedes breakdown, (ii) the modification inactivates Mal61p, and (iii) the inactivation of Mal61p is not necessarily followed by proteolytic degradation. Lett Appl Microbiol, 1998 Apr, 26(4), 279 - 82 Density of lectin-like receptors in the FLO1 phenotype of Saccharomyces cerevisiae; Patelakis SJ et al.; An improved lectin assay was recently developed to determine the density of lectin sites present on the surface of a haploid strain 1209 of Saccharomyces cerevisiae exhibiting the Flo1 phenotype . The assay is based on the use of FITC-avidin which contains a mannose side chain which functions as a binding site to the cell wall lectins termed zymolectins . An examination of the effect of culture time on the zymolectin density indicated no significant effect (P > 0.05) confirming other findings that Flo1 acts in a constitutive manner . Zymolectin densities for this strain averaged 5.4 x 10(6) sites cell-1. J Biol Chem, 1998 Jun 26, 273(26), 15954 - 60 Nucleotide-dependent tetramerization of CTP synthetase from Saccharomyces cerevisiae; Pappas A et al.; The nucleotide-dependent tetramerization of purified native URA7-encoded CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) from the yeast Saccharomyces cerevisiae was characterized . CTP synthetase existed as a dimer in the absence of ATP and UTP . In the presence of saturating concentrations of ATP and UTP, the CTP synthetase protein existed as a tetramer . Increasing concentrations of ATP and UTP caused a dose-dependent conversion of the dimeric species to a tetramer . The kinetics of enzyme tetramerization correlates with the kinetics of enzyme activity . The tetramerization of CTP synthetase was dependent on UTP and Mg2+ ions . ATP facilitated the UTP-dependent tetramerization of CTP synthetase by a mechanism that involved the ATP-dependent phosphorylation of UTP catalyzed by the enzyme . The glutaminase reaction that is catalyzed by the enzyme was not required for enzyme tetramerization . CTP, a potent inhibitor of CTP synthetase activity, did not inhibit the ATP/UTP-dependent tetramerization of the enzyme . Phosphorylation of the purified native CTP synthetase with protein kinase A and protein kinase C facilitated the nucleotide-dependent tetramerization . Dephosphorylation of native CTP synthetase with alkaline phosphatase prevented the nucleotide-dependent tetramerization of the enzyme . This correlated with the inactivation of CTP synthetase activity . Rephosphorylation of the dephosphorylated enzyme with protein kinase A and protein kinase C resulted in a partial restoration of the nucleotide-dependent tetramerization of the enzyme . This tetramerization correlated with the partial restoration of CTP synthetase activity . Taken together, these results indicated that enzyme tetramerization was required for CTP synthetase activity and that enzyme phosphorylation played an important role in the tetramerization and regulation of the enzyme. Mol Cell Biol, 1998 Jun, 18(6), 3431 - 44 Functional mapping of the U3 small nucleolar RNA from the yeast Saccharomyces cerevisiae; Samarsky DA et al.; The U3 small nucleolar RNA participates in early events of eukaryotic pre-rRNA cleavage and is essential for formation of 18S rRNA . Using an in vivo system, we have developed a functional map of the U3 small nucleolar RNA from Saccharomyces cerevisiae . The test strain features a galactose-dependent U3 gene in the chromosome and a plasmid-encoded allele with a unique hybridization tag . Effects of mutations on U3 production were analyzed by evaluating RNA levels in cells grown on galactose medium, and effects on U3 function were assessed by growing cells on glucose medium . The major findings are as follows: (i) boxes C' and D and flanking helices are critical for U3 accumulation; (ii) boxes B and C are not essential for U3 production but are important for function, most likely due to binding of a trans-acting factor(s); (iii) the 5' portion of U3 is required for function but not stability; and, (iv) strikingly, the nonconserved hairpins 2, 3, and 4, which account for 50% of the molecule, are not required for accumulation or function. Mol Cell Biol, 1998 Jun, 18(6), 3376 - 83 Processing of the intron-encoded U18 small nucleolar RNA in the yeast Saccharomyces cerevisiae relies on both exo- and endonucleolytic activities; Villa T et al.; Many small nucleolar RNAs (snoRNAs) are encoded within introns of protein-encoding genes and are released by processing of their host pre-mRNA . We have investigated the mechanism of processing of the yeast U18 snoRNA, which is found in the intron of the gene coding for translational elongation factor EF-1beta . We have focused our analysis on the relationship between splicing of the EF-1beta pre-mRNA and production of the mature snoRNA . Mutations inhibiting splicing of the EF-1beta pre-mRNA have been shown to produce normal U18 snoRNA levels together with the accumulation of intermediates deriving from the pre-mRNA, thus indicating that the precursor is an efficient processing substrate . Inhibition of 5'-->3' exonucleases obtained by insertion of G cassettes or by the use of a rat1-1 xrn1Delta mutant strain does not impair U18 release . In the Exo- strain, 3' cutoff products, diagnostic of an endonuclease-mediated processing pathway, were detected . Our data indicate that biosynthesis of the yeast U18 snoRNA relies on two different pathways, depending on both exonucleolytic and endonucleolytic activities: a major processing pathway based on conversion of the debranched intron and a minor one acting by endonucleolytic cleavage of the pre-mRNA. Mol Cell Biol, 1998 Jun, 18(6), 3149 - 62 The regulatory particle of the Saccharomyces cerevisiae proteasome; Glickman MH et al.; The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin . The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm . The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle . We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles . To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis . Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6) . Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine . In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex . Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts . One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes . Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution. EMBO J, 1998 May 1, 17(9), 2566 - 73 Glucose sensing and signaling by two glucose receptors in the yeast Saccharomyces cerevisiae; Ozcan S et al.; How eukaryotic cells sense availability of glucose, their preferred carbon and energy source, is an important, unsolved problem . Bakers' yeast (Saccharomyces cerevisiae) uses two glucose transporter homologs, Snf3 and Rgt2, as glucose sensors that generate a signal for induction of expression of genes encoding hexose transporters (HXT genes) . We present evidence that these proteins generate an intracellular glucose signal without transporting glucose . The Snf3 and Rgt2 glucose sensors contain unusually long C-terminal tails that are predicted to be in the cytoplasm . These tails appear to be the signaling domains of Snf3 and Rgt2 because they are necessary for glucose signaling by Snf3 and Rgt2, and transplantation of the C-terminal tail of Snf3 onto the Hxt1 and Hxt2 glucose transporters converts them into glucose sensors that can generate a signal for glucose-induced HXT gene expression . These results support the idea that yeast senses glucose using two modified glucose transporters that serve as glucose receptors. EMBO J, 1998 May 1, 17(9), 2543 - 53 The Ssn6-Tup1 repressor complex of Saccharomyces cerevisiae is involved in the osmotic induction of HOG-dependent and -independent genes; Marquez JA et al.; The response of yeast to osmotic stress has been proposed to rely on the HOG-MAP kinase signalling pathway and on transcriptional activation mediated by STRE promoter elements . However, the osmotic induction of HAL1, an important determinant of salt tolerance, is HOG independent and occurs through the release of transcriptional repression . We have identified an upstream repressing sequence in HAL1 promoter (URSHAL1) located between -231 and -156 . This promoter region was able to repress transcription from a heterologous promoter and to bind proteins in non-stressed cells, but not in salt-treated cells . The repression conferred by URSHAL1 is mediated through the Ssn6-Tup1 protein complex and is abolished in the presence of osmotic stress . The Ssn6-Tup1 co-repressor is also involved in the regulation of HOG-dependent genes such as GPD1, CTT1, ALD2, ENA1 and SIP18, and its deletion can suppress the osmotic sensitivity of hog1 mutants . We propose that the Ssn6-Tup1 repressor complex might be a general component in the regulation of osmostress responses at the transcriptional level of both HOG-dependent and -independent genes. J Theor Biol, 1998 Apr 21, 191(4), 397 - 414 A structured, minimal parameter model of the central nitrogen metabolism in Saccharomyces cerevisiae: the prediction of the behavior of mutants; van Riel NA et al.; In order to enable future pathway engineering of a complex system, such as the nitrogen metabolism in yeast, mathematical modelling tools have to be developed . The stoichiometric and biochemical characteristics of the glutamate and glutamine nodes (the Central Nitrogen Metabolism, CNM) are qualitatively known . Quantitative knowledge about the dynamics of the network lacks and needs to be developed for metabolic reprogramming . A model-based-experiment approach is proposed in which the development of a model initiates new experiments of which the results then improve the model . As a first step in this iterative system identification cycle, recent experimental data, both qualitative and quantitative, obtained from defined studies on the CNM of the yeast Saccharomyces cerevisiae have been translated into an initial mathematical model . The model approach is based on a combination of Flux Analysis and simple enzyme kinetics . The model is constructed using nonlinear Ordinary Differential Equations and regulation of the synthesis and activity of key enzymes of the CNM is included . The parameters of the model are estimated with a constrained Least Squares algorithm using the steady-state and dynamic pulse data of a glutamine limited continuous culture . The resulting model describes a continuous culture of a wild-type strain correctly and in general the trends of the dynamic behaviour after both glutamine and ammonia pulses to this culture are good . Inclusion of countercurrent reactions and compartmentation in the model is essential for the descriptive quality of the model under dynamic conditions . It is clear that more experimental work is needed . The model indicates that the GOGAT/Glutamine Synthetase (GS) pathway plays a more important physiological stabilizing role in yeast than is generally assumed . New, model-based, experiments have to investigate the function of GOGAT, especially under dynamic conditions . Also redox cofactors and ATP have to be measured . The resulting model is validated with data of similar experiments with a GS-mutant . The quality of the prediction of the behaviour of the mutant is comparable to the descriptive property, which is a very promising result, taking into account the limited dataset compared to the system complexity. Protein Expr Purif, 1998 Jun, 13(1), 111 - 9 The human D1A dopamine receptor: heterologous expression in Saccharomyces cerevisiae and purification of the functional receptor; Andersen B et al.; Functional human D1A dopamine receptor has been expressed in Saccharomyces cerevisiae . The primary sequence of the receptor was modified to include two affinity tags at the C-terminus of the protein, a FLAG tag (DYKDDDDK), and a His6 tag (HHHHHH) . These modifications allowed for purification to near homogeneity using immobilized metal affinity chromatography and immunoaffinity chromatography . Radioligand binding demonstrated that the purified and reconstituted receptor binds the antagonist {3H}SCH23390 with an affinity (KD = 8.0 +/- 3.2 nM) comparable to that of the native receptor. Protein Expr Purif, 1998 Jun, 13(1), 104 - 10 Expression and purification of correctly processed, active human TACE catalytic domain in Saccharomyces cerevisiae; Clarke HR et al.; Human tumor necrosis factor-alpha (TNF alpha) converting enzyme (TACE) releases soluble TNF alpha from cells . It is a member of the adamalysin family of metalloproteases . A truncated form of TACE cDNA was expressed in Saccharomyces cerevisiae and purified to homogeneity in order to study TACE structure and function . Recombinant TACE was expressed as a preproprotein including the pro- and catalytic (PROCAT) domains fused to the yeast alpha-factor leader . A C-terminal immunoreactive FLAG peptide was added for Western blot detection and anti-FLAG antibody column purification . We constructed two glycosylation mutant PROCAT TACE isoforms to facilitate purification . A PROCAT isoform, mutated to eliminate two N-linked glycosylation sites, was buffer exchanged and purified to homogeneity by ion exchange chromatography and an anti-FLAG antibody affinity step . N-terminal sequence analysis showed that the mutant preproprotein was processed in yeast at the furin protease cleavage site and yielded an active catalytic domain which has TNF alpha peptide-specific protease activity . Mass spectrometry of the purified catalytic domain showed that removal of both N-linked sites results in a homogeneous sized polypeptide lacking further posttranslational modifications. Biochim Biophys Acta, 1998 Jun 15, 1392(2-3), 233 - 44 Human lamin B receptor exhibits sterol C14-reductase activity in Saccharomyces cerevisiae; Silve S et al.; Lamin B receptor (LBR), a nuclear protein of avian and mammalian cells, contains an hydrophobic domain that shares extensive structural similarities with the members of the sterol reductase family . To test if the sterol-reductase-like domain of LBR could be enzymatically competent, several sterol reductase-defective strains of Saccharomyces cerevisiae were transformed with a human-LBR expressing vector . LBR production did not change the ergosterol biosynthesis defect in an erg4 mutant impaired in sterol C24(28) reductase . In contrast, the sterol C14 reduction step and ergosterol prototrophy were restored in LBR-producing erg24 transformants which lack endogenous sterol C14 reductase . To test the effects of C14 reductase inhibitors on LBR activity, we constructed EMY54, an ergosterol-requiring strain that is devoid of both sterol C8-C7 isomerase and sterol C14 reductase activities . EMY54 cells recovered the capability of synthesizing ergost-8-en-3beta-ol upon transformation with a vector that expressed either yeast sterol C14 reductase or hLBR . In addition, growth in sterol-free medium was restored in these transformants . Sterol biosynthesis and proliferation of LBR-producing cells were found to be highly susceptible to fenpropimorph and tridemorph, but only moderately susceptible to SR 31747 . Our results strongly suggest that hLBR is a sterol C14 reductase. Gene, 1998 Jun 15, 213(1-2), 159 - 67 Functional genetic tests of DNA mismatch repair protein activity in Saccharomyces cerevisiae; Polaczek P et al.; Hereditary non-polyposis colorectal cancer (HNPCC) is associated with mutations in four different genes encoding proteins involved in DNA mismatch repair (DMR) . As many as 30% of the observed sequence variations in human DMR genes predict minor alterations in the encoded protein, such as amino acid (aa) replacements or small in-frame deletions/insertions . For such sequence variants, a functional genetic test will be required to discriminate mutations from polymorphisms . We have constructed a series of isogenic yeast strains in which individual genes involved in DMR are disrupted, and have standardized an assay which measures GT tract stability (Strand et al., 1993) to characterize these gene products . Disruptions of the yeast MSH2, MLH1, and PMS1 genes result in, respectively, a 290-, 450- and 390-fold increased tract instability over the wild type (wt) strain under optimized assay conditions . Expression of the wt MSH2 and PMS1 gene from plasmids results in complementation of the corresponding chromosomal gene disruption . Two different aa replacements which correspond to previously observed sequence variants of the human MSH2 gene, and implicated in HNPCC, were created in the conserved aa of the yeast MSH2 gene by site directed mutagenesis . Conversion of the Pro640 in the yeast protein to Leu resulted in a complete loss of protein function . In contrast, a yeast MSH2p protein in which the His658 is changed to Tyr retains full function in this in vivo assay . These results indicate that the Pro-->Leu and His-->Tyr variants observed in humans constitute, respectively, a mutation and a polymorphism . The system described here may be used for further structure/function analysis of yeast DMR proteins . Such studies may provide insight into the effects that specific sequence variations observed in human DMR proteins have on their function . J Biochem (Tokyo), 1998 May, 123(5), 912 - 7 Functional expression of the human UDP-galactose transporters in the yeast Saccharomyces cerevisiae; Sun-Wada GH et al.; We describe the functional expression of the putative human Golgi UDP-galactose transporters (hUGT1 and hUGT2) in the yeast Saccharomyces cerevisiae . Both hUGT1 and hUGT2 were expressed under the control of the yeast constitutive GAPDH promoter . The expression level of hUGT1 seemed to be considerably lower than that of hUGT2, although hUGT1 has an amino acid sequence identical to that of hUGT2 except for 5 amino acid residues at the C-terminus . The hUGT product was expressed in the membranes of Golgi and other organellar compartments . The membrane vesicles prepared from the hUGT1- or the hUGT2-expressing yeast cells exhibited UDP-galactose specific transport activity . The apparent Km values of the yeast-expressed hUGT1 and hUGT2 for UDP-galactose were 1.2 and 2 microM, respectively, which were comparable with the Km obtained with mammalian Golgi vesicles . Transport was dependent on temperature and integrity of vesicles, and was inhibited by UMP, as observed with mammalian Golgi vesicles . Our results demonstrate that the previously described hUGT1 and hUGT2 encode the UDP-galactose transporters, rather than regulatory proteins . The development of a convenient yeast expression system should facilitate analysis of the structure-function relationships of the UDP-galactose transporters. Arch Microbiol, 1998 Jun, 169(6), 517 - 24 The two 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition; Schnappauf G et al.; Activity of the tyrosine-inhibitable 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Saccharomyces cerevisiae that was encoded by the ARO4 gene cloned on a high-copy-number plasmid was enhanced 64-fold as compared to the wild-type . The enzyme was purified to apparent homogeneity from the strain that harbored this recombinant plasmid . The estimated molecular weight of 42,000 of the enzyme corresponded to the calculated molecular mass of 40 kDa deduced from the DNA sequence . The enzyme could be inactivated by EDTA in a reaction that was reversed by several bivalent metal ions; presumably a metal cofactor is required for enzymatic catalysis . The Michaelis constant of the enzyme was 125 microM for phosphoenolpyruvate and 500 microM for erythrose 4-phosphate . The rate constant was calculated as 6 s-1, and kinetic data indicated a sequential mechanism of the enzymatic reaction . Tyrosine was a competitive inhibitor with phosphoenolpyruvate as substrate of the enzyme (Ki of 0.9 microM) and a noncompetitive inhibitor with erythrose 4-phosphate as substrate . This is in contrast to the ARO3-encoded isoenzyme, where phenylalanine is a competitive inhibitor with erythrose 4-phosphate as a substrate of the enzyme and a noncompetitive inhibitor with phosphoenolpyruvate as substrate. Nucleic Acids Res, 1998 Jul 1, 26(13), 3286 - 92 A mutation in NPS1/STH1, an essential gene encoding a component of a novel chromatin-remodeling complex RSC, alters the chromatin structure of Saccharomyces cerevisiae centromeres; Tsuchiya E et al.; The NPS1/STH1 gene encodes a nuclear protein essential for the progression of G2/M phase in Saccharomyces cerevisiae . Nps1p shares homology to Snf2/Swi2p, a subunit of a protein complex known as the SNF/SWI complex . Recently, Nps1p was found to be a component of a protein complex termed RSC (3) essential for mitotic growth, whereas its function is unknown . We isolated a temperature-sensitive mutant allele of NPS1 , nps1-105, and found that the mutation increases the sensitivity to thiabendazole (TBZ) . At the restrictive temperature, nps1-105 arrested at the G2/M phase in MAD1-dependent manner and missegregated the mini-chromosome with higher frequency than the wild type cells . The nuclease digestion of the chromatin of the mutant cells revealed that the mutation causes the alteration of the chromatin structure around centromeres at the restrictive temperature . The results suggested that, in the nps1-105 mutant, impaired chromatin structure surrounding centromeres may lead to an impairment of kinetochore function and the cells arrest at G2/M phase through the spindle-assembly checkpoint system. Nucleic Acids Res, 1998 Jul 1, 26(13), 3154 - 8 Meiotic role of SWI6 in Saccharomyces cerevisiae; Leem SH et al.; The transcript levels of DNA replication genes and some recombination genes in Saccharomyces cerevisiae fluctuate and peak at the G1/S boundary in the mitotic cell cycle . This fluctuation is regulated by MCB (Mlu I cell cycle box) elements which are bound by the DSC1/MBF1 complex consisting of Swi6 and Mbp1 . It is also known that some of the MCB-regulated genes are induced by treatment with DNA damaging agents and in meiosis . In this report, the function of SWI6 in meiosis was investigated . Delta swi6 cells underwent sporulation as did wild-type cells . However, the deletion mutant cells showed reduced spore viability and lower frequency of recombination . The transcript levels of the recombination genes RAD51 and RAD54 , which have MCB elements, were reduced in Delta swi6 cells . The transcript levels of SWI6 itself were also induced and declined in meiosis . Furthermore, an increased dosage of SWI6 enhanced the transcript level of the RAD51 gene and also the recombination frequency in meiosis . These results suggest that SWI6 enhances the expression level of the recombination genes in meiosis in a dosage-dependent manner, which results in an effect on the frequency of meiotic recombination. EMBO J, 1998 Jun 15, 17(12), 3326 - 41 Involvement of distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae; Colombo S et al.; Adenylate cyclase activity in Saccharomyces cerevisiae is dependent on Ras proteins . Both addition of glucose to glucose-deprived (derepressed) cells and intracellular acidification trigger an increase in the cAMP level in vivo . We show that intracellular acidification, but not glucose, causes an increase in the GTP/GDP ratio on the Ras proteins independent of Cdc25 and Sdc25 . Deletion of the GTPase-activating proteins Ira1 and Ira2, or expression of the RAS2(val19) allele, causes an enhanced GTP/GDP basal ratio and abolishes the intracellular acidification-induced increase . In the ira1Delta ira2Delta strain, intracellular acidification still triggers a cAMP increase . Glucose also did not cause an increase in the GTP/GDP ratio in a strain with reduced feedback inhibition of cAMP synthesis . Further investigation indicated that feedback inhibition by cAPK on cAMP synthesis acts independently of changes in the GTP/GDP ratio on Ras . Stimulation by glucose was dependent on the Galpha-protein Gpa2, whose deletion confers the typical phenotype associated with a reduced cAMP level: higher heat resistance, a higher level of trehalose and glycogen and elevated expression of STRE-controlled genes . However, the typical fluctuation in these characteristics during diauxic growth on glucose was still present . Overexpression of Ras2(val19) inhibited both the acidification- and glucose-induced cAMP increase even in a protein kinase A-attenuated strain . Our results suggest that intracellular acidification stimulates cAMP synthesis in vivo at least through activation of the Ras proteins, while glucose acts through the Gpa2 protein . Interaction of Ras2(val19) with adenylate cyclase apparently prevents its activation by both agonists. FEMS Microbiol Lett, 1998 May 15, 162(2), 249 - 55 Green fluorescent protein-cell wall fusion proteins are covalently incorporated into the cell wall of Saccharomyces cerevisiae; Ram AF et al.; Tagging of two cell wall mannoproteins, Cwp1p and Cwp2p, in Saccharomyces cerevisiae with the green fluorescent protein from Aequorea victoria resulted in incorporation of fluorescent fusion proteins into the cell wall . Both living cells and isolated cell walls became brightly labeled . Intriguingly, the incorporation patterns of both fusion proteins differed . Western analysis of enzymatically released fusion proteins showed that they were covalently linked to the beta-1,6-glucan part of the cell wall . Removal of the glycosylphosphatidyl-inositol anchor signal sequence of the green fluorescent protein-cell wall protein fusion proteins resulted in secretion of the proteins into the culture medium . These results indicate that green fluorescent protein-cell wall protein fusion proteins can be used as a convenient fluorescent marker to study the incorporation of specific cell wall proteins and the control mechanisms involved. Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6876 - 80 A human homolog of the Saccharomyces cerevisiae REV3 gene, which encodes the catalytic subunit of DNA polymerase zeta; Gibbs PE et al.; To get a better understanding of mutagenic mechanisms in humans, we have cloned and sequenced the human homolog of the Saccharomyces cerevisiae REV3 gene . The yeast gene encodes the catalytic subunit of DNA polymerase zeta, a nonessential enzyme that is thought to carry out translesion replication and is responsible for virtually all DNA damage-induced mutagenesis and the majority of spontaneous mutagenesis . The human gene encodes an expected protein of 3,130 residues, about twice the size of the yeast protein (1,504 aa) . The two proteins are 29% identical in an amino-terminal region of approximately 340 residues, 39% identical in a carboxyl-terminal region of approximately 850 residues, and 29% identical in a 55-residue region in the middle of the two genes . The sequence of the expected protein strongly predicts that it is the catalytic subunit of a DNA polymerase of the pol zeta type; the carboxyl-terminal domain possesses, in the right order, the six motifs characteristic of eukaryotic DNA polymerases, most closely resembles yeast pol zeta among all polymerases in the GenBank database, and is different from the human alpha, delta, and epsilon enzymes . Human cells expressing high levels of an hsREV3 antisense RNA fragment grow normally, but show little or no UV-induced mutagenesis and are slightly more sensitive to killing by UV . The human gene therefore appears to carry out a function similar to that of its yeast counterpart. Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6779 - 84 The human homologue of Saccharomyces cerevisiae Gle1p is required for poly(A)+ RNA export; Watkins JL et al.; The mechanism of mRNA export is a complex issue central to cellular physiology . We characterized previously yeast Gle1p, a protein with a leucine-rich (LR) nuclear export sequence (NES) that is essential for poly(A)+ RNA export in Saccharomyces cerevisiae . To characterize elements of the vertebrate mRNA export pathway, we identified a human homologue of yeast Gle1p and analyzed its function in mammalian cells . hGLE1 encodes a predicted 75-kDa polypeptide with high sequence homology to yeast Gle1p, but hGle1p does not contain a sequence motif matching any of the previously characterized NESs . hGLE1 can complement a yeast gle1 temperature-sensitive export mutant only if a LR-NES is inserted into it . To determine whether hGle1p played a role in nuclear export, anti-hGle1p antibodies were microinjected into HeLa cells . In situ hybridization of injected cells showed that poly(A)+ RNA export was inhibited . In contrast, there was no effect on the nuclear import of a glucocorticoid receptor reporter . We conclude that hGle1p functions in poly(A)+ RNA export, and that human cells facilitate such export with a factor similar to yeast but without a recognizable LR-NES . With hGle1p localized at the nuclear pore complexes, hGle1p is positioned to act at a terminal step in the export of mature RNA messages to the cytoplasm. Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6716 - 21 Rpp2, an essential protein subunit of nuclear RNase P, is required for processing of precursor tRNAs and 35S precursor rRNA in Saccharomyces cerevisiae; Stolc V et al.; RPP2, an essential gene that encodes a 15.8-kDa protein subunit of nuclear RNase P, has been identified in the genome of Saccharomyces cerevisiae . Rpp2 was detected by sequence similarity with a human protein, Rpp20, which copurifies with human RNase P . Epitope-tagged Rpp2 can be found in association with both RNase P and RNase mitochondrial RNA processing in immunoprecipitates from crude extracts of cells . Depletion of Rpp2 protein in vivo causes accumulation of precursor tRNAs with unprocessed introns and 5' and 3' termini, and leads to defects in the processing of the 35S precursor rRNA . Rpp2-depleted cells are defective in processing of the 5.8S rRNA . Rpp2 immunoprecipitates cleave both yeast precursor tRNAs and precursor rRNAs accurately at the expected sites and contain the Rpp1 protein orthologue of the human scleroderma autoimmune antigen, Rpp30 . These results demonstrate that Rpp2 is a protein subunit of nuclear RNase P that is functionally conserved in eukaryotes from yeast to humans. Mol Biol Cell, 1998 Jun, 9(6), 1565 - 76 Chs6p-dependent anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces cerevisiae; Ziman M et al.; Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomyces cerevisiae, may be transported to the cell surface in a regulated manner . Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the cell surface . We find that chs6Delta strains have morphological abnormalities similar to those of chs3 mutants . Subcellular fractionation and indirect immunofluorescence indicate that Chs3p distribution is altered in chs6 mutant cells . Order-of-function experiments using end4-1 (endocytosis-defective) and chs6 mutants indicate that Chs6p is required for anterograde transport of Chs3p from an internal endosome-like membrane compartment, the chitosome, to the plasma membrane . As a result, chs6 strains accumulate Chs3p in chitosomes . Chs1p, a distinct chitin synthase that acts during or after cell separation, is transported normally in chs6 mutants, suggesting that Chs1p and Chs3p are independently packaged during protein transport through the late secretory pathway. Mol Biol Cell, 1998 Jun, 9(6), 1351 - 65 A role for the lumenal domain in Golgi localization of the Saccharomyces cerevisiae guanosine diphosphatase; Vowels JJ et al.; Integral membrane proteins (IMPs) contain localization signals necessary for targeting to their resident subcellular compartments . To define signals that mediate localization to the Golgi complex, we have analyzed a resident IMP of the Saccharomyces cerevisiae Golgi complex, guanosine diphosphatase (GDPase) . GDPase, which is necessary for Golgi-specific glycosylation reactions, is a type II IMP with a short amino-terminal cytoplasmic domain, a single transmembrane domain (TMD), and a large catalytic lumenal domain . Regions specifying Golgi localization were identified by analyzing recombinant proteins either lacking GDPase domains or containing corresponding domains from type II vacuolar IMPs . Neither deletion nor substitution of the GDPase cytoplasmic domain perturbed Golgi localization . Exchanging the GDPase TMD with vacuolar protein TMDs only marginally affected Golgi localization . Replacement of the lumenal domain resulted in mislocalization of the chimeric protein from the Golgi to the vacuole, but a similar substitution leaving 34 amino acids of the GDPase lumenal domain intact was properly localized . These results identify a major Golgi localization determinant in the membrane-adjacent lumenal region (stem) of GDPase . Although necessary, the stem domain is not sufficient to mediate localization; in addition, a membrane-anchoring domain and either the cytoplasmic or full-length lumenal domain must be present to maintain Golgi residence . The importance of lumenal domain sequences in GDPase Golgi localization and the requirement for multiple hydrophilic protein domains support a model for Golgi localization invoking protein-protein interactions rather than interactions between the TMD and the lipid bilayer. Mol Biol Cell, 1998 Jun, 9(6), 1253 - 63 Nitrogen-regulated ubiquitination of the Gap1 permease of Saccharomyces cerevisiae; Springael JY et al.; Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces rapid inactivation and degradation of the general amino acid permease Gap1 through a process requiring the Npi1/Rsp5 ubiquitin (Ub) ligase . In this study, we show that NH4+ induces endocytosis of Gap1, which is then delivered into the vacuole where it is degraded . This down-regulation is accompanied by increased conversion of Gap1 to ubiquitinated forms . Ubiquitination and subsequent degradation of Gap1 are impaired in the npi1 strain . In this mutant, the amount of Npi1/Rsp5 Ub ligase is reduced >10-fold compared with wild-type cells . The C-terminal tail of Gap1 contains sequences, including a di-leucine motif, which are required for NH4+-induced internalization and degradation of the permease . We show here that mutant Gap1 permeases affected in these sequences still bind Ub . Furthermore, we provide evidence that only a small fraction of Gap1 is modified by Ub after addition of NH4+ to mutants defective in endocytosis. J Biol Chem, 1998 Jun 12, 273(24), 14838 - 44 Ectopic potassium uptake in trk1 trk2 mutants of Saccharomyces cerevisiae correlates with a highly hyperpolarized membrane potential; Madrid R et al.; Null trk1 trk2 mutants of Saccharomyces cerevisiae exhibit a low-affinity uptake of K+ and Rb+ . We show that this low-affinity Rb+ uptake is mediated by several independent transporters, and that trk1Delta cells and especially trk1Delta trk2Delta cells are highly hyperpolarized . Differences in the membrane potentials were assessed for sensitivity to hygromycin B and by flow cytometric analyses of cellular DiOC6(3) fluorescence . On the basis of the latter analyses, it is proposed that Trk1p and Trk2p are involved in the control of the membrane potential, preventing excessive hyperpolarizations . K+ starvation and nitrogen starvation hyperpolarize both TRK1 TRK2 and trk1Delta trk2Delta cells, thus suggesting that other proteins, in addition to Trk1p and Trk2p, participate in the control of the membrane potential . The HAK1 K+ transporter from Schwanniomyces occidentalis suppresses the K+-defective transport of trk1Delta trk2Delta cells but not the high hyperpolarization, and the HKT1 K+ transporter from wheat suppresses both defects, in the presence of Na+ . We discuss the mechanism involved in the control of the membrane potential by Trk1p and Trk2p and the causal relationship between the high membrane potential (negative inside) of trk1Delta trk2Delta cells and its ectopic transport of alkali cations. J Cell Biol, 1998 May 4, 141(3), 567 - 84 Rvs161p interacts with Fus2p to promote cell fusion in Saccharomyces cerevisiae; Brizzio V et al.; FUS7 was previously identified by a mutation that causes a defect in cell fusion in a screen for bilateral mating defects . Here we show that FUS7 is allelic to RVS161/END6, a gene implicated in a variety of processes including viability after starvation, endocytosis, and actin cytoskeletal organization . Two lines of evidence indicate that RVS161/END6's endocytic function is not required for cell fusion . First, several other endocytic mutants showed no cell fusion defects . Second, we isolated five function-specific alleles of RVS161/FUS7 that were defective for endocytosis, but not mating, and three alleles that were defective for cell fusion but not endocytosis . The organization of the actin cytoskeleton was normal in the cell fusion mutants, indicating that Rvs161p's function in cell fusion is independent of actin organization . The three to fourfold induction of RVS161 by mating pheromone and the localization of Rvs161p-GFP to the cell fusion zone suggested that Rvs161p plays a direct role in cell fusion . The phenotypes of double mutants, the coprecipitation of Rvs161p and Fus2p, and the fact that the stability of Fus2p was strongly dependent on Rvs161p's mating function lead to the conclusion that Rvs161p is required to interact with Fus2p for efficient cell fusion. J Bacteriol, 1998 Jun, 180(12), 3131 - 6 Altered Na+ and Li+ homeostasis in Saccharomyces cerevisiae cells expressing the bacterial cation antiporter NhaA; Ros R et al.; The bacterial Na+ (Li+)/H+ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae . NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance . In cells containing the yeast Ena1-4 (Na+, Li+) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H+ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations . In yeast mutants without (Na+, Li+) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H+ ATPase and correlated with a decrease of intracellular lithium . NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase . These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane . The phenotypes conferred by its expression depend on the functionally of plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase. Genetics, 1998 Jun, 149(2), 893 - 901 The transcriptional activator Imp2p maintains ion homeostasis in Saccharomyces cerevisiae; Masson JY et al.; Yeast cells deficient in the transcriptional activator Imp2p are viable, but display marked hypersensitivity to a variety of oxidative agents . We now report that imp2 null mutants are also extremely sensitive to elevated levels of the monovalent ions, Na+ and Li+, as well as to the divalent ions Ca2+, Mn2+, Zn2+, and Cu2+, but not to Cd2+, Mg2+, Co2+, Ni2+, and Fe2+, as compared to the parent strain . We next searched for multicopy suppressor genes that would allow the imp2Delta mutant to grow under high salt conditions . Two genes that independently restored normal salt-resistance to the imp2Delta mutant, ENA1 and HAL3, were isolated . ENA1 encodes a P-type ion pump involved in monovalent ion efflux from the cell, while HAL3 encodes a protein required for activating the expression of Ena1p . Neither ENA1 nor HAL3 gene expression was positively regulated by Imp2p . Moreover, the imp2 ena1 double mutant was exquisitely sensitive to Na+/Li+ cations, as compared to either single mutant, implying that Imp2p mediates Na+/Li+ cation homeostasis independently of Ena1p. Genetics, 1998 Jun, 149(2), 879 - 92 Mot3, a Zn finger transcription factor that modulates gene expression and attenuates mating pheromone signaling in Saccharomyces cerevisiae; Grishin AV et al.; In the yeast Saccharomyces cerevisiae, mating pheromone response is initiated by activation of a G protein- and mitogen-activated protein (MAP) kinase-dependent signaling pathway and attenuated by several mechanisms that promote adaptation or desensitization . To identify genes whose products negatively regulate pheromone signaling, we screened for mutations that suppress the hyperadaptive phenotype of wild-type cells overexpressing signaling-defective G protein beta subunits . This identified recessive mutations in MOT3, which encodes a nuclear protein with two Cys2-His2 Zn fingers . MOT3 was found to be a dosage-dependent inhibitor of pheromone response and pheromone-induced gene expression and to require an intact signaling pathway to exert its effects . Several results suggested that Mot3 attenuates expression of pheromone-responsive genes by mechanisms distinct from those used by the negative transcriptional regulators Cdc36, Cdc39, and Mot2 . First, a Mot3-lexA fusion functions as a transcriptional activator . Second, Mot3 is a dose-dependent activator of several genes unrelated to pheromone response, including CYC1, SUC2, and LEU2 . Third, insertion of consensus Mot3 binding sites (C/A/T)AGG(T/C)A activates a promoter in a MOT3-dependent manner . These findings, and the fact that consensus binding sites are found in the 5' flanking regions of many yeast genes, suggest that Mot3 is a globally acting transcriptional regulator . We hypothesize that Mot3 regulates expression of factors that attenuate signaling by the pheromone response pathway. Genetics, 1998 Jun, 149(2), 865 - 78 Ion tolerance of Saccharomyces cerevisiae lacking the Ca2+/CaM-dependent phosphatase (calcineurin) is improved by mutations in URE2 or PMA1; Withee JL et al.; Calcineurin is a conserved, Ca2+/CaM-stimulated protein phosphatase required for Ca2+-dependent signaling in many cell types . In yeast, calcineurin is essential for growth in high concentrations of Na+, Li+, Mn2+, and OH-, and for maintaining viability during prolonged treatment with mating pheromone . In contrast, the growth of calcineurin-mutant yeast is better than that of wild-type cells in the presence of high concentrations of Ca2+ . We identified mutations that suppress multiple growth defects of calcineurin-deficient yeast (cnb1Delta or cna1Delta cna2Delta) . Mutations in URE2 suppress the sensitivity of calcineurin mutants to Na+, Li+, and Mn2+, and increase their survival during treatment with mating pheromone . ure2 mutations require both the transcription factor Gln3p and the Na+ ATPase Pmr2p to confer Na+ and Li+ tolerance . Mutations in PMA1, which encodes the yeast plasma membrane H+-ATPase, also suppress many growth defects of calcineurin mutants . pma1 mutants display growth phenotypes that are opposite to those of calcineurin mutants; they are resistant to Na+, Li+, and Mn2+, and sensitive to Ca2+ . We also show that calcineurin mutants are sensitive to aminoglycoside antibiotics such as hygromycin B while pma1 mutants are more resistant than wild type . Furthermore, pma1 and calcineurin mutations have antagonistic effects on intracellular {Na+} and {Ca2+} . Finally, we show that yeast expressing a constitutively active allele of calcineurin display pma1-like phenotypes, and that membranes from these yeast have decreased levels of Pma1p activity . These studies further characterize the roles that URE2 and PMA1 play in regulating intracellular ion homeostasis. Genetics, 1998 Jun, 149(2), 843 - 56 Involvement of protein N-glycosyl chain glucosylation and processing in the biosynthesis of cell wall beta-1,6-glucan of Saccharomyces cerevisiae; Shahinian S et al.; beta-1,6-Glucan plays a key structural role in the yeast cell wall . Of the genes involved in its biosynthesis, the activity of Cwh41p is known, i.e., the glucosidase I enzyme of protein N-chain glucose processing . We therefore examined the effects of N-chain glucosylation and processing mutants on beta-1,6-glucan biosynthesis and show that incomplete N-chain glucose processing results in a loss of beta-1,6-glucan, demonstrating a relationship between N-chain glucosylation/processing and beta-1,6-glucan biosynthesis . To explore the involvement of other N-chain-dependent events with beta-1,6-glucan synthesis, we investigated the Saccharomyces cerevisiae KRE5 and CNE1 genes, which encode homologs of the "quality control" components UDP-Glc:glycoprotein glucosyltransferase and calnexin, respectively . We show that the essential activity of Kre5p is separate from its possible role as a UDP-Glc:glycoprotein glucosyltransferase . We also observe a approximately 30% decrease in beta-1,6-glucan upon disruption of the CNE1 gene, a phenotype that is additive with other beta-1,6-glucan synthetic mutants . Analysis of the cell wall anchorage of the mannoprotein alpha-agglutinin suggests the existence of two beta-1,6-glucan biosynthetic pathways, one N-chain dependent, the other involving protein glycosylphosphatidylinositol modification. Genetics, 1998 Jun, 149(2), 817 - 32 Meiotic chromosome morphology and behavior in zip1 mutants of Saccharomyces cerevisiae; Tung KS et al.; The yeast Zip1 protein (Zip1p) is a component of the central region of the synaptonemal complex (SC) . Zip1p is predicted to form a dimer consisting of a coiled-coil domain flanked by globular domains . To analyze the organization of Zip1p within the SC, in-frame deletions of ZIP1 were constructed and analyzed . The results demonstrate that the C terminus but not the N terminus of Zip1p is required for its localization to chromosomes . Deletions in the carboxy half of the predicted coiled-coil region cause decreases in the width of the SC . Based on these results, a model for the organization of Zip1p within the SC is proposed . zip1 deletion mutations were also examined for their effects on sporulation, spore viability, crossing over, and crossover interference . The results demonstrate that the extent of synapsis is positively correlated with the levels of spore viability, crossing over, and crossover interference . In contrast, the role of Zip1p in synapsis is separable from its role in meiotic cell cycle progression . zip1 mutants display interval-specific effects on crossing over. Genetics, 1998 Jun, 149(2), 795 - 805 A nucleolar protein that affects mating efficiency in Saccharomyces cerevisiae by altering the morphological response to pheromone; Kim J et al.; SSF1 and SSF2 are redundant essential yeast genes that, when overexpressed, increase the mating efficiency of cells containing a defective Ste4p Gbeta subunit . To identify the precise function of these genes in mating, different responses to pheromone were assayed in cells that either lacked or overexpressed SSF gene products . Cells containing null alleles of both SSF1 and SSF2 displayed the normal transcriptional induction response to pheromone but were unable to form mating projections . Overexpression of SSF1 conferred the ability to form mating projections on cells containing a temperature-sensitive STE4 allele, but had only a small effect on transcriptional induction . SSF1 overexpression preferentially increased the mating efficiency of a strain containing a null allele of SPA2, a gene that functions specifically in cell morphology . To investigate whether Ssf1p plays a direct physical role in mating projection formation, its subcellular location was determined . An Ssf1p-GFP fusion was found to localize to the nucleolus, implying that the role of SSF gene products in projection formation is indirect . The region of Ssf1p-GFP localization in cells undergoing projection formation was larger and more diffuse, and was often present in a specific orientation with respect to the projection . Although the function of Ssf1p appears to originate in the nucleus, it is likely that it ultimately acts on one or more of the proteins that is directly involved in the morphological response to pheromone . Because many of the proteins required for projection formation during mating are also required for bud formation during vegetative growth, regulation of the activity or amount of one or more of these proteins by Ssf1p could explain its role in both mating and dividing cells. EMBO J, 1998 May 15, 17(10), 2759 - 66 Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae; Gilon T et al.; Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins . There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation . Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins . We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions . From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants . Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants . The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system . Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes. Plant Mol Biol, 1998 Jun, 37(3), 513 - 21 HSP 12 is a LEA-like protein in Saccharomyces cerevisiae; Mtwisha L et al.; LEA group I, II and III antibodies all recognised soluble proteins present in an extract of yeast (Saccharomyces cerevisiae) . The smaller protein of the two recognised by the group I antibody displayed identical migration on SDS-PAGE to the pea seed LEA group I protein against which the antibody was raised . However, the antibody failed to recognise the predominant protein present after heating the extract at 80 degrees C for 10 min . This predominant protein, which also displayed identical migration on SDS-PAGE, was purified from the supernatant of the extract heated at 80 degrees C for 10 min . Peptide sequencing after CNBr cleavage identified the isolated protein as the heat shock protein HSP 12 . Despite a previous report that HSP 12 is a heat shock protein, HSP 12 was found to increase in yeast grown at 37 degrees C compared with growth at 30 degrees C . However, increased amounts of HSP 12 were present in yeast after entry into stationary phase; this was enhanced by growth in the osmolytes NaCl and mannitol. J Biol Chem, 1998 May 1, 273(18), 11062 - 8 Syringomycin action gene SYR2 is essential for sphingolipid 4-hydroxylation in Saccharomyces cerevisiae; Grilley MM et al.; The Saccharomyces cerevisiae gene SYR2, necessary for growth inhibition by the cyclic lipodepsipeptide syringomycin E, is shown to be required for 4-hydroxylation of long chain bases in sphingolipid biosynthesis . Four lines of support for this conclusion are presented: (a) the predicted Syr2p shows sequence similarity to diiron-binding membrane enzymes involved in oxygen-dependent modifications of hydrocarbon substrates, (b) yeast strains carrying a disrupted SYR2 allele produced sphingoid long chain bases lacking the 4-hydroxyl group present in wild type strains, (c) 4-hydroxylase activity was increased in microsomes prepared from a SYR2 overexpression strain, and (d) the syringomycin E resistance phenotype of a syr2 mutant strain was suppressed when grown under conditions in which exogenous 4-hydroxysphingoid long chain bases were incorporated into sphingolipids . The syr2 strain produced wild type levels of sphingolipids, substantial levels of hydroxylated very long chain fatty acids, and the full complement of normal yeast sphingolipid head groups . These results show that the SYR2 gene is required for the 4-hydroxylation reaction of sphingolipid long chain bases, that this hydroxylation is not essential for growth, and that the 4-hydroxyl group of sphingolipids is necessary for syringomycin E action on yeast. J Biol Chem, 1998 May 1, 273(18), 10819 - 22 The peptidyl-prolyl isomerase domain of the CyP-40 cyclophilin homolog Cpr7 is not required to support growth or glucocorticoid receptor activity in Saccharomyces cerevisiae; Duina AA et al.; CyP-40 cyclophilins are found in association with molecular chaperone Hsp90.steroid receptor complexes . The amino-terminal portion of these cyclophilins harbors the characteristic peptidyl-prolyl isomerase (PPIase) domain, whereas three copies of the tetratricopeptide (TPR) motif, a structure shown to be involved in protein-protein interactions, and a putative calmodulin-binding domain are located in the carboxyl-terminal half of the protein . The TPR domains mediate binding to Hsp90, but a requirement for the PPIase domain has not been established . To address this, we have investigated the effects of mutations that alter the PPIase domain of the Saccharomyces cerevisiae CyP-40 homolog, Cpr7 . Because Cpr7 is required for rapid growth and full Hsp90 activity, a functional assessment of the PPIase domain could be performed in vivo . A mutation in the catalytic domain altering a conserved site predicted to be essential for isomerase activity did not compromise Cpr7 function . Furthermore, deletion of the entire PPIase domain did not significantly affect growth or Hsp90-mediated steroid receptor activity . These results indicate that the TPR-containing carboxyl terminus of Cpr7 is sufficient for fundamental Cpr7-dependent activity. Mol Gen Genet, 1998 Apr, 258(1-2), 104 - 16 The Cdc14 phosphatase is functionally associated with the Dbf2 protein kinase in Saccharomyces cerevisiae; Grandin N et al.; The Saccharomyces cerevisiae Cdc14 protein phosphatase and Dbf2 protein kinase have been implicated to act during late M phase, but their functions are not known . We report here that CDC14 is a low-copy suppressor of the dbf2-2 mutation at 37 degrees C . The kinase activity of Dbf2 accumulated at a high level, in vivo, during a cdc14 arrest and was also much higher in cdc14 mutant cells at the permissive temperature of growth, therefore in cycling mutant cells than in cycling wild-type cells . This correlated with the accumulation of the more slowly migrating form of Dbf2, previously shown to correspond to the hyperphosphorylated form of the protein . The finding that the dbf2-2 mutation could be rescued following overproduction of catalytically inactive forms of Cdc14 suggested that the control of Dbf2 activity by Cdc14 might be only indirect and independent of Cdc14 phosphatase activity . However, it was found that Cdc14 could form oligomers within the cell, thus leaving open the possibility that catalytically inactive Cdc14 might associate with wild-type Cdc14 and rescue dbf2-2 in a phosphatase-dependent manner . We confirmed that overexpression of CDC14 could rescue mutations in CDC15, which encodes another kinase also implicated to act in late M phase . Cells of a cdc15-2 dbf2-2 double mutant died at temperatures much lower than did either single mutant, whereas there was only a slight additive phenotype in the cdc14-1 dbf2-2 and cdc14-1 cdc15-2 double mutant cells . Finally, functional association between Cdc14 and Dbf2 (and also Cdc15) was confirmed by the finding that the cdc14, dbf2 and cdc15 mutations could be partially rescued by the addition of 1.2 M sorbitol to the culture medium . Our data are the first to demonstrate a functional link between Cdc14 and Dbf2 based on both biochemical and genetic information. Mol Gen Genet, 1998 Apr, 258(1-2), 53 - 9 Screening for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in Saccharomyces cerevisiae; Hamada K et al.; Open reading frames in the genome of Saccharomyces cerevisiae were screened for potential glycosylphosphatidylinositol (GPI)-attached proteins . The identification of putative GPI-attached proteins was based on three criteria: the presence of a GPI-attachment signal sequence, a signal sequence for secretion and a serine- or threonine-rich sequence . In all, 53 ORFs met these three criteria and 38 were further analyzed as follows . The sequence encoding the 40 C-terminal amino acids of each was fused with the structural gene for a reporter protein consisting of a secretion signal, alpha-galactosidase and a hemagglutinin (HA) epitope, and examined for the ability to become incorporated into the cell wall . On this basis, 14 of fusion proteins were classified as GPI-dependent cell wall proteins because cells expressing these fusion proteins: (i) had high levels of alpha-galactosidase activity on their surface; (ii) released significant amounts of the fusion proteins from the membrane on treatment with phosphatidylinositol-specific phospholipase C (PI-PLC); and (iii) released fusion proteins from the cell wall following treatment with laminarinase . Of the 14 identified putative GPI-dependent cell wall proteins, 12 had novel ORFs adjacent to their GPI-attachment signal sequence . Amino acid sequence alignment of the C-terminal sequences of the 12 ORFs, together with those of known cell wall proteins, reveals some sequence similarities among them. Mol Gen Genet, 1998 Apr, 258(1-2), 16 - 25 Effects of mutations in the Saccharomyces cerevisiae RNA14 gene on the abundance and polyadenylation of its transcripts; Mandart E; In the yeast Saccharomyces cerevisiae, the RNA14 and RNA15 gene products have been implicated in RNA cleavage and polyadenylation in vitro and in the choice of polyadenylation site of ACT1 mRNA in vivo . The RNA14 gene produces three transcripts that differ in their 3' end, suggesting the use of different polyadenylation sites . The appearance of the three RNA14 transcripts was examined in different rna14 and/or rna15 mutant strains . In the rna14-1 or rna15-2 mutant strains, only the large transcript is present at the non-permissive temperature, showing that the rna14-1 and rna15-2 mutations lead to the use of the most distal RNA14 polyadenylation site, which turns out to be the most efficient . The rna14-5 mutation, which does not primarily modify the choice of poly(A) site, increases the global amount of RNA14 transcript . Surprisingly, this RNA14 mRNA overproduction is also observed in the double rna14-1 rna15-2 mutant strain . Moreover, in the strains in which the RNA14 transcripts are overproduced, short heterogeneous polyadenylated antisense RNAs are detected in the 3' region of the RNA14 large transcript . Taken together these observations suggest that, in addition to poly(A) site choice, Rna14 protein has another function involved in the control of global RNA14 mRNA level. Microbiology, 1998 May, 144 ( Pt 5), 1451 - 62 Repression of nitrogen catabolic genes by ammonia and glutamine in nitrogen-limited continuous cultures of Saccharomyces cerevisiae; ter Schure EG et al.; Growth of Saccharomyces cerevisiae on ammonia and glutamine decreases the expression of many nitrogen catabolic genes to low levels . To discriminate between ammonia- and glutamine-driven repression of GAP1, PUT4, GDH1 and GLN1, a gln1-37 mutant was used . This mutant is not able to convert ammonia into glutamine . Glutamine-limited continuous cultures were used to completely derepress the expression of GAP1, PUT4, GDH1 and GLN1 . Following an ammonia pulse, the expression of GAP1, PUT4 and GDH1 decreased while the intracellular glutamine concentration remained constant, both in the cytoplasm and in the vacuole . Therefore, it was concluded that ammonia causes gene repression independent of the intracellular glutamine concentration . The expression of GLN1 was not decreased by an ammonia pulse but solely by a glutamine pulse . Analysis of the mRNA levels of ILV5 and HIS4 showed that the response of the two biosynthetic genes, GDH1 and GLN1, to ammonia and glutamine in the wild-type and gln1-37 was not due to changes in general transcription of biosynthetic genes . Ure2p has been shown to be an essential element for nitrogen-regulated gene expression . Deletion of URE2 in the gln1-37 background prevented repression of gene expression by ammonia, showing that the ammonia-induced repression is not caused by a general stress response but represents a specific signal for nitrogen catabolite regulation.
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