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 conformationa