Yeast, 1999 Oct, 15(14), 1503 - 10 Allelism of Saccharomyces cerevisiae genes PSO6, involved in survival after 3-CPs+UVA induced damage, and ERG3, encoding the enzyme sterol C-5 desaturase; Schmidt CL et al.; Sequencing of the yeast gene that complemented the sensitivity to the photoactivated monofunctional 3-carbethoxypsoralen of the pso6-1 mutant strain revealed that the ERG3 locus, encoding sterol C-5 desaturase involved in biosynthesis of ergosterol, is allelic to PSO6 . Disruption of the ERG3 gene yielded an erg3Delta mutant viable in ergosterol-containing YEPD media with the same pleiotropic mutant phenotype known for pso6-1 and erg3 mutants, including sensitivity to hydrogen peroxide and paraquat . Thus, the erg3/pso6 yeast mutant seems to be more sensitive than the WT to 3-CPs+UVA because of the oxidative damage contributed by this treatment and not because of an impaired repair of the furocoumarin-thymine monoadducts formed in the DNA . We found a significant increase of petites amongst erg3Delta and pso6-1 yeast mutant strains grown in conditions where respiration was mandatory . Mutant pso6-1, with its lowered content of ergosterol, exhibited enhanced synthesis of chitin that was maldistributed and not confined to the bud scars . Chitin overproduction in pso6/erg3 mutants resulted in hypersensitivity to Calcofluor White . Yeast, 1999 Oct, 15(14), 1471 - 84 STRE- and cAMP-independent transcriptional induction of Saccharomyces cerevisiae GSY2 encoding glycogen synthase during diauxic growth on glucose; Parrou JL et al.; It has been shown that the so-called stationary phase GSY2 gene encoding glycogen synthase was induced as the cells left the exponential phase of growth, while glucose and all other nutrients were still plentiful in the medium (Parrou et al., 1999) . Since this effect was essentially controlled at the transcriptional level, we looked for the cis- and trans-acting elements required for this specific growth-related genetic event . We demonstrated that mutations of the HAP2/3/4 binding site and of the two STress-Responsive cis-Elements (STRE) did not abolish the early induction of GSY2, although the latter mutation led to a 20-fold drop in the transcriptional activity of the promoter, as determined from lacZ gene fusions . Insertion of a DNA fragment (from -390 to -167 bp, relative to the ATG) of the promoter lacking the two STREs, upstream to the TATA box of a CYC1-lacZ fusion gene, allowed this reporter gene to be induced with a kinetic similar to that of GSY2-lacZ . Mutations in BCY1, which results in a hyperactive protein kinase A, did not alleviate the early induction, while causing a five- to 10-fold reduction in the transcriptional activity of GSY2 . In addition, the repressive effect of protein kinase A was quantitatively conserved when both STREs were mutated in GSY2 promoter, indicating that the negative control of gene expression by the RAS-cAMP signalling pathway does not act solely through STREs . Taken together, these results are indicative of an active process that couples growth control to dynamic glucose consumption . Yeast, 1999 Oct, 15(14), 1449 - 57 Role of the sulphate assimilation pathway in utilization of glutathione as a sulphur source by Saccharomyces cerevisiae; Miyake T et al.; Mutants unable to grow on medium containing glutathione as a sole source of sulphur (GSH medium) were isolated from Saccharomyces cerevisiae strains carrying met17(deficiency of O-acetylserine and O-acetylhomoserine sulphydrylase) . They were defective in the high-affinity glutathione transport system, GSH-P1 . Newly acquired mutations belonged to the same complementation group, gsh11 . However, it became apparent that gsh11 conferred the mutant phenotype not by itself but in collaboration with met17 . Moreover, mutations conferring the defect in sulphate assimilation made the cell unable to grow on GSH medium in collaboration with gsh11 . From this finding, we propose that the sulphate assimilation pathway acts as a sulphur-recycling system and that this function is especially vital to the cell when the supply of glutathione is limited . J Biol Chem, 1999 Oct 15, 274(42), 30310 - 4 Stimulation of DNA replication in Saccharomyces cerevisiae by a glutamine- and proline-rich transcriptional activation domain; Li R; Glutamine-rich Sp1 and proline-rich CTF1, two extensively studied mammalian transcription factors, bind to origins of replication in DNA tumor viruses and stimulate viral DNA replication in mammalian cells . Here it is shown that, when tethered to a plasmid-borne cellular origin of replication, the activation domains of both proteins can enhance origin function in Saccharomyces cerevisiae . Hydrophobic patches in Sp1 and CTF1 that mediate transcriptional activation in higher eukaryotes are also important for activation of replication in yeast . However, only the activation domain of CTF1 can enhance initiation of replication from a chromosomally embedded origin . This correlates with the ability of CTF1 to alter the local chromatin structure around the chromosomal origin of replication . The CTF1-induced chromatin remodeling occurs at multiple stages of the cell cycle . These findings strongly suggest a high degree of conservation in the mechanisms used by various types of transcription factors to stimulate viral and cellular DNA replication in eukaryotes. J Biol Chem, 1999 Oct 15, 274(42), 30052 - 8 Phospholipase C binds to the receptor-like GPR1 protein and controls pseudohyphal differentiation in Saccharomyces cerevisiae; Ansari K et al.; The hormone receptor-like protein Gpr1p physically interacts with phosphatidylinositol-specific phospholipase C (Plc1p) and with the Galpha protein Gpa2p, as shown by two-hybrid assays and co-immune precipitation of epitope-tagged proteins . Plc1p binds to Gpr1p in either the presence or absence of Gpa2, whereas the Gpr1p/Gpa2p association depends on the presence of Plc1p . Genetic interactions between the null mutations plc1Delta, gpr1Delta, gpa2Delta, and ras2Delta suggest that Plc1p acts together with Gpr1p and Gpa2p in a growth control pathway operating in parallel to the Ras2p function . Diploid cells lacking Gpr1p, Plc1p, or Gpa2p fail to form pseudohyphae upon nitrogen depletion, and the filamentation defect of gpr1Delta and plc1Delta strains is rescued by activating a mitogen-activated protein kinase pathway via STE11-4 or by activating a cAMP pathway via overexpressed Tpk2p . Plc1p is also required for efficient expression of the FG(TyA)::lacZ reporter gene under nitrogen depletion . In conclusion, we have identified two physically interacting proteins, Gpr1p and Plc1p, as novel components of a nitrogen signaling pathway controlling the developmental switch from yeast-like to pseudohyphal growth . Our data suggest that phospholipase C modulates the interaction of the putative nutrient sensor Gpr1p with the Galpha protein Gpa2p as a downstream effector of filamentation control. Mol Biol Cell, 1999 Oct, 10(10), 3521 - 38 The Saccharomyces cerevisiae homologue of human Wiskott-Aldrich syndrome protein Las17p interacts with the Arp2/3 complex; Madania A et al.; Yeast Las17 protein is homologous to the Wiskott-Aldrich Syndrome protein, which is implicated in severe immunodeficiency . Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization . Here we show that Las17p interacts with the Arp2/3 complex . LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells . Six of seven ARP2 ts mutants and at least one ARP3 ts mutant are synthetically lethal with las17Delta ts confirming functional interaction with the Arp2/3 complex . Further characterization of las17Delta cells showed that receptor-mediated internalization of alpha factor by the Ste2 receptor is severely defective . The polarity of normal bipolar bud site selection is lost . Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin . Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex . Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton. Mol Biol Cell, 1999 Oct, 10(10), 3125 - 36 Modulation of life-span by histone deacetylase genes in Saccharomyces cerevisiae; Kim S et al.; The yeast Saccharomyces cerevisiae has a limited life-span, which is measured by the number of divisions that individual cells complete . Among the many changes that occur as yeasts age are alterations in chromatin-dependent transcriptional silencing . We have genetically manipulated histone deacetylases to modify chromatin, and we have examined the effect on yeast longevity . Deletion of the histone deacetylase gene RPD3 extended life-span . Its effects on chromatin functional state were evidenced by enhanced silencing at the three known heterochromatic regions of the genome, the silent mating type (HM), subtelomeric, and rDNA loci, which occurred even in the absence of SIR3 . Similarly, the effect of the rpd3Delta on life-span did not depend on an intact Sir silencing complex . In fact, deletion of SIR3 itself had little effect on life-span, although it markedly accelerated the increase in cell generation time that is observed during yeast aging . Deletion of HDA1, another histone deacetylase gene, did not result in life-span extension, unless it was combined with deletion of SIR3 . The hda1Delta sir3Delta resulted in an increase in silencing, but only at the rDNA locus . Deletion of RPD3 suppressed the loss of silencing in rDNA in a sir2 mutant; however, the silencing did not reach the level found in the rpd3Delta single mutant, and RPD3 deletion did not overcome the life-span shortening seen in the sir2 mutant . Deletion of both RPD3 and HDA1 caused a decrease in life-span, which resulted from a substantial increase in initial mortality of the population . The expression of both of these genes declines with age, providing one possible explanation for the increase in mortality during the life-span . Our results are consistent with the loss of rDNA silencing leading to aging in yeast . The functions of RPD3 and HDA1 do not overlap entirely . RPD3 exerts its effect on chromatin at additional sites in the genome, raising the possibility that events at loci other than rDNA play a role in the aging process. J Mol Biol, 1999 Oct 15, 293(1), 19 - 28 Site-specific in vivo cleavages by DNA topoisomerase I in the regulatory regions of the 35 S rRNA in Saccharomyces cerevisiae are transcription independent; Vogelauer M et al.; Eukaryotic type I DNA topoisomerase controls DNA topology by transiently breaking and resealing one strand of DNA at a time . During transcription and replication its action reduces the torsional stress derived from these activities . The association of DNA topoisomerase I with the nucleolus has been reported and this enzyme was shown to be involved in yeast rDNA metabolism . Here, we have investigated the in vivo presence of DNA topoisomerase I cleavage sites in the non-transcribed spacer of the rDNA cluster . We show a specific profile of highly localized cleavage in relevant areas of this region . The sites are detected in the promoter and in the enhancer regions of the 35 S gene . The analysis of mutants in which transcription is prevented and/or reduced, namely a strain lacking the 43 kDa subunit of RNA polymerase I, a second one that does note transcribe, lacking a subunit of the core factor and another member of the RNA polymerase I transcription factors lacking one of the UAF component which transcribes at very low level, show that DNA topoisomerase I cleavage sites are not related to transcription by RNA polymerase I . These findings point to a role for DNA topoisomerase I that is additional to the commonly recognized function in removing the transcription-induced topological stress . Genetics, 1999 Oct, 153(2), 607 - 20 Saccharomyces cerevisiae checkpoint genes MEC1, RAD17 and RAD24 are required for normal meiotic recombination partner choice; Grushcow JM et al.; Checkpoint gene function prevents meiotic progression when recombination is blocked by mutations in the recA homologue DMC1 . Bypass of dmc1 arrest by mutation of the DNA damage checkpoint genes MEC1, RAD17, or RAD24 results in a dramatic loss of spore viability, suggesting that these genes play an important role in monitoring the progression of recombination . We show here that the role of mitotic checkpoint genes in meiosis is not limited to maintaining arrest in abnormal meioses; mec1-1, rad24, and rad17 single mutants have additional meiotic defects . All three mutants display Zip1 polycomplexes in two- to threefold more nuclei than observed in wild-type controls, suggesting that synapsis may be aberrant . Additionally, all three mutants exhibit elevated levels of ectopic recombination in a novel physical assay . rad17 mutants also alter the fraction of recombination events that are accompanied by an exchange of flanking markers . Crossovers are associated with up to 90% of recombination events for one pair of alleles in rad17, as compared with 65% in wild type . Meiotic progression is not required to allow ectopic recombination in rad17 mutants, as it still occurs at elevated levels in ndt80 mutants that arrest in prophase regardless of checkpoint signaling . These observations support the suggestion that MEC1, RAD17, and RAD24, in addition to their proposed monitoring function, act to promote normal meiotic recombination. Genetics, 1999 Oct, 153(2), 595 - 605 A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants; Merrill BJ et al.; To examine the role of the RAD52 recombinational repair pathway in compensating for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability . We isolated 10 mec1 mutations that display synthetic lethality with rad52 . These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p . Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting that Mec1p is required for the normal progression of DNA synthesis . sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress the checkpoint defect in mec1-srf mutants . Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p . By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-stranded DNA breaks that are not rapidly repaired in rad52 mutants . Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants. Genetics, 1999 Oct, 153(2), 585 - 94 Mks1p is a regulator of nitrogen catabolism upstream of Ure2p in Saccharomyces cerevisiae; Edskes HK et al.; The supply of nitrogen regulates yeast genes affecting nitrogen catabolism, pseudohyphal growth, and meiotic sporulation . Ure2p of Saccharomyces cerevisiae is a negative regulator of nitrogen catabolism that inhibits Gln3p, a positive regulator of DAL5, and other genes of nitrogen assimilation . Dal5p, the allantoate permease, allows ureidosuccinate uptake (Usa(+)) when cells grow on a poor nitrogen source such as proline . We find that overproduction of Mks1p allows uptake of ureidosuccinate on ammonia and lack of Mks1p prevents uptake of ureidosuccinate or Dal5p expression on proline . Overexpression of Mks1p does not affect cellular levels of Ure2p . An mks1 ure2 double mutant can take up ureidosuccinate on either ammonia or proline . Moreover, overexpression of Ure2p suppresses the ability of Mks1p overexpression to allow ureidosuccinate uptake on ammonia . These results suggest that Mks1p is involved in nitrogen control upstream of Ure2p as follows: NH(3) dash, vertical Mks1p dash, vertical Ure2p dash, vertical Gln3p --> DAL5 . Either overproduction of Mks1p or deletion of MKS1 interferes with pseudohyphal growth. Genetics, 1999 Oct, 153(2), 573 - 83 The conversion gradient at HIS4 of Saccharomyces cerevisiae . II . A role for mismatch repair directed by biased resolution of the recombinational intermediate; Foss HM et al.; Salient features of recombination at ARG4 of Saccharomyces provoke a variation of the double-strand-break repair (DSBR) model that has the following features: (1) Holliday junction cutting is biased in favor of strands upon which DNA synthesis occurred during formation of the joint molecule (this bias ensures that cutting both junctions of the joint-molecule intermediate arising during DSBR usually leads to crossing over); (2) cutting only one junction gives noncrossovers; and (3) repair of mismatches that are semirefractory to mismatch repair and/or far from the DSB site is directed primarily by junction resolution . The bias in junction resolution favors restoration of 4:4 segregation when such mismatches and the directing junction are on the same side of the DSB site . Studies at HIS4 confirmed the predicted influence of the bias in junction resolution on the conversion gradient, type of mismatch repair, and frequency of aberrant 5:3 segregation, as well as the predicted relationship between mismatch repair and crossing over. Genetics, 1999 Oct, 153(2), 555 - 72 The conversion gradient at HIS4 of Saccharomyces cerevisiae . I . Heteroduplex rejection and restoration of Mendelian segregation; Hillers KJ et al.; In Saccharomyces cerevisiae, some gene loci manifest gradients in the frequency of aberrant segregation in meiosis, with the high end of each gradient corresponding to a hotspot for DNA double-strand breaks (DSBs) . The slope of a gradient is reduced when mismatch repair functions fail to act upon heteroduplex DNA-aberrant segregation frequencies at the low end of the gradient are higher in the absence of mismatch repair . Two models for the role of mismatch repair functions in the generation of meiotic "conversion gradients" have been proposed . The heteroduplex rejection model suggests that recognition of mismatches by mismatch repair enzymes limits hybrid DNA flanking the site of a DSB . The restoration-conversion model proposes that mismatch repair does not affect the length of hybrid DNA, but instead increasingly favors restoration of Mendelian segregation over full conversion with increasing distance from the DSB site . In our experiment designed to distinguish between these two models, data for one subset of well repairable mismatches in the HIS4 gene failed to show restoration-type repair but did indicate reduction in the length of hybrid DNA, supporting the heteroduplex rejection model . However, another subset of data manifested restoration-type repair, indicating a relationship between Holliday junction resolution and mismatch repair . We also present evidence for the infrequent formation of symmetric hybrid DNA during meiotic DSB repair. Yeast, 1999 Sep 30, 15(13), 1411 - 7 Disruption of six ORFs on Saccharomyces cerevisiae chromosome X: the YJL069c gene of unknown function is essential to cell viability; Vandenbol M et al.; The short flanking homology PCR strategy (Wach et al., 1994) was used to disrupt six open reading frames (ORFs) on chromosome X of diploid strains (FY1679 and W303) of the yeast Saccharomyces cerevisiae . Two of the six ORFs analysed (YJL069c and YJL066c) display no similarity to known sequences . Three others (YJL065c, YJL068c, and YJL070c) are similar to those respectively encoding the DNA polymerase epsilon subunit c, human esterase D and rat AMP deaminase 1 . YJL071w has recently been identified as the ARG2 gene coding for acetylglutamate synthase . Inactivation of the YJL069c gene proved lethal and the yjl071w haploid disruptants were auxotrophic for arginine . For the four other gene inactivations, neither the heterozygous deletion diploids nor the corresponding haploid deletion mutants displayed any special phenotype when grown on rich glycerol or glucose medium or on synthetic minimal medium at three different temperatures, or on media containing compounds interfering with nucleic acid or protein synthesis . Mating and sporulation efficiencies were the same for the viable disruptants as for wild-type cells . The six kanMX4 disruption cassettes were cloned into the pUG7 vector and each of the cognate wild-type genes was inserted into the pRS416 centromeric plasmid . All strains and plasmids have been deposited in the EUROFAN collection (EUROSCARF, K . -D . Entian, Frankfurt, Germany) . Yeast, 1999 Sep 30, 15(13), 1365 - 75 Cysteine biosynthesis in Saccharomyces cerevisiae: a new outlook on pathway and regulation; Ono BI et al.; Using a Saccharomyces cerevisiae strain having the activities of serine O-acetyl-transferase (SATase), O-acetylserine/O-acetylhomoserine sulphydrylase (OAS/OAH SHLase), cystathionine beta-synthase (beta-CTSase) and cystathionine gamma-lyase (gamma-CTLase), we individually disrupted CYS3(coding for gamma-CTLase) and CYS4 (coding for beta-CTSase) . The obtained gene disruptants were cysteine-dependent and incorporated the radioactivity of (35)S-sulphate into homocysteine but not into cysteine or glutathione . We concluded, therefore, that SATase and OAS/OAH SHLase do not constitute a cysteine biosynthetic pathway and that cysteine is synthesized exclusively through the pathway constituted with beta-CTSase and gamma-CTLase; note that OAS/OAH SHLase supplies homocysteine to this pathway by acting as OAH SHLase . From further investigation upon the cys3-disruptant, we obtained results consistent with our earlier suggestion that cysteine and OAS play central roles in the regulation of sulphate assimilation . In addition, we found that sulphate transport activity was not induced at all in the cys4-disruptant, suggesting that CYS4 plays a role in the regulation of sulphate assimilation . Yeast, 1999 Sep 30, 15(13), 1331 - 45 Mutations that cause threonine sensitivity identify catalytic and regulatory regions of the aspartate kinase of Saccharomyces cerevisiae; Arevalo-Rodriguez M et al.; The HOM3 gene of Saccharomyces cerevisiae encodes aspartate kinase, which catalyses the first step in the branched pathway leading to the synthesis of threonine and methionine from aspartate . Regulation of the carbon flow into this pathway takes place mainly by feedback inhibition of this enzyme by threonine . We have isolated and characterized three HOM3 mutants that show growth inhibition by threonine due to a severe, threonine-induced reduction of the carbon flow into the aspartate pathway, leading to methionine limitation . One of the mutants has an aspartate kinase which is 30-fold more strongly inhibited by threonine than the wild-type enzyme . The predicted amino acid substitution in this mutant, A406T, is located in a region associated with the modulation of the enzymatic activity . The other two mutants carry an aspartate kinase with reduced affinity for its substrates, aspartate and ATP . The corresponding amino acid substitutions, K26I and G25D, affect residues located in the vicinity of a highly conserved lysine-phenylalanine-glycine-glycine (KFGG) stretch present in the N-terminal part of the aspartate kinase, to which no function has so far been assigned . We suggest that this region is involved in substrate binding . Mutagenesis of a HOM3 region centred in the KFGG-coding triplets generated alleles that determine threonine sensitivity or auxotrophy for threonine and methionine, but not a phenotype associated with a feedback-resistant aspartate kinase, indicating that this region is not involved in the allosteric response of the enzyme . EMBO J, 1999 Oct 1, 18(19), 5411 - 22 The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif; Dunckley T et al.; The major pathway of mRNA degradation in yeast occurs through deadenylation, decapping and subsequent 5' to 3' exonucleolytic decay of the transcript body . To identify proteins that control the activity of the decapping enzyme, which is encoded by the DCP1 gene, we isolated a high-copy suppressor of the temperature-sensitive dcp1-2 allele, termed DCP2 . Overexpression of Dcp2p partially suppressed the dcp1-2 decapping defect . Moreover, the Dcp2 protein was required for the decapping of both normal mRNAs and aberrant transcripts that are degraded by the mRNA surveillance pathway . The Dcp2 protein contains a MutT motif, which is found in a class of pyrophosphatases . Mutational analyses indicated that the region of Dcp2p containing the MutT motif is necessary and sufficient for Dcp2p's function in mRNA decapping . The Dcp2p also coimmunoprecipitates with the DCP1 decapping enzyme and is required for the production of enzymatically active decapping enzyme . These results suggest that direct or indirect interaction of Dcp1p with Dcp2p is required for the production of active decapping enzyme, perhaps in a process requiring the hydrolysis of a pyrophosphate bond. Appl Environ Microbiol, 1999 Oct, 65(10), 4685 - 7 Bacterial production of D-erythroascorbic acid and L-ascorbic acid through functional expression of Saccharomyces cerevisiae D-arabinono-1,4-lactone oxidase in Escherichia coli; Lee BH et al.; D-Arabinono-1,4-lactone oxidase, which catalyzes the terminal step in the biosynthesis of D-erythroascorbic acid in Saccharomyces cerevisiae, was functionally expressed in Escherichia coli inherently lacking the enzyme . The recombinant E . coli strain expressing the enzyme could overproduce D-erythroascorbic acid and L-ascorbic acid when supplied with D-arabinono-1,4-lactone and L-galactono-1,4-lactone, respectively. J Biol Chem, 1999 Oct 8, 274(41), 29211 - 9 Structure-function analysis of the protein-binding domains of Mac1p, a copper-dependent transcriptional activator of copper uptake in Saccharomyces cerevisiae; Serpe M et al.; The Mac1 protein in Saccharomyces cerevisiae is essential for the expression of yeast high affinity copper uptake . A positive transcription factor, Mac1p binds via its N-terminal domain to GCTC elements in the promoters of CTR1 and FRE1, encoding a copper permease and metal reductase, respectively . Mac1p-dependent transcriptional activation is negatively regulated by copper . We have mapped the domains in Mac1p responsible for its nuclear localization and for the protein-protein interactions that underlie its transcriptional activity . Immunofluorescence studies indicate that Mac1p contains two nuclear localization signals, one each in the N- and C-terminal halves of the protein . Yeast one-hybrid analysis demonstrates that the copper-dependent transcriptional activity in Mac1p resides primarily in a cysteine-rich element encompassing residues 264-279 . Two-hybrid analysis indicates that a copper-independent Mac1p-Mac1p interaction linked to DNA binding is due primarily to a predicted helix in the C-terminal region of the protein encompassing residues 388-406 . Point mutations within this putative helix abrogate the Mac1-Mac1 interaction in vivo and formation of a ternary (Mac1p)(2).DNA complex in vitro . When produced in normal abundance, Mac1pI396D and Mac1pF400D helix mutants do not support transcriptional activation in vivo consistent with an essential Mac1p dimerization in transcriptional activation . Lastly, the one- and two-hybrid data indicate that an intramolecular interaction between the DNA-binding and transactivation domains negatively modulates Mac1p activity. Genetika, 1999 Jun, 35(6), 744 - 50 {Cloning and determination of the structure of a new human gene, homologous to Saccharomyces cerevisiae rad proteins}; Loseva EF et al.; Cloning of a novel Homo sapiens gene from cDNA libraries of human testes using immunoscreening procedure with affinity-purified polyclonal antibodies against E . coli RecA protein was carried out . The nucleotide sequence and chromosomal localization of this gene were determined . Computer modeling of its primary transcript was conducted . The gene investigated is localized on the P arm of chromosome 19 within the 19p13.1 cluster and consists of seven exons and six introns . It has a leader, 3' untranslated, and translated regions . The translated sequence encodes a 33-kD protein that displays 30-40% homology to the Saccharomyces cerevisae Rad proteins . TATA- and CAAT-like sequences are situated at positions -30 and -40 bp as well as -102 and -148, respectively . The absence of CCGCCC- or GGGCGG-like sequences may indicate that this gene is tissue-specific. Chin J Biotechnol, 1998, 14(3), 195 - 200 Construction of engineering host by non-random method in Saccharomyces cerevisiae; Gong Y et al.; A yeast engineering host, GJ30, was constructed by a non-random method . GJ30 derivated from wild type yeast have leu2- and ura3- deficients which can be used as selection markers . The PCR analysis showed that the homologous recombinations occurred in the leu2 and ura3 locus of GJ30, respectively . The biological properties (cellular growth density, stability and efficiency for gene expression) of GJ30 were compared with other several yeast stains . The results showed that GJ30 is a suitable host strain for expression of foreign genes. Curr Genet, 1999 Sep, 36(3), 130 - 6 New insights into the pyrimidine salvage pathway of Saccharomyces cerevisiae: requirement of six genes for cytidine metabolism; Kurtz JE et al.; Cytidine metabolism in the yeast Saccharomyces cerevisiae was analyzed by genetic and biochemical approaches . Disruption of a unique ORF (Genbank accession No . U 20865) bearing homology with eucaryotic or bacterial cytidine deaminases abolished cytidine deaminase activity and resulted in 5-fluorocytidine resistance . The gene encoding cytidine deaminase will be referred to as CDD1 (Genbank accession number AF080089) . The ability to isolate mutants resistant to 5-fluorocytidine which mapped to five other loci demonstrated the existence of a complex cytidine metabolic network . Deciphering this network revealed several original features:(1) cytidine entry is mediated by the purine-cytosine transporter (Fcy2p),(2) cytidine is cleaved into cytosine by the uridine nucleosidase (Urh1p),(3) cytidine is phosphorylated into CMP by the uridine kinase (Urk1p),(4) a block in cytosine deaminase (Fcy1p), but not in cytidine deaminase (Cdd1p), constitutes a limiting step in cytidine utilisation as a UMP precursor. Curr Genet, 1999 Sep, 36(3), 124 - 9 Mutant allele pso7-1, that sensitizes Saccharomyces cerevisiae to photoactivated psoralen, is allelic with COX11, encoding a protein indispensable for a functional cytochrome c oxidase; Pungartnik C et al.; The yeast gene PSO7 was cloned from a genomic library by complementation of the pso7-1 mutant's sensitivity phenotype to 4-nitroquinoline-1-oxide (4NQO) . Sequence analysis revealed that PSO7 is allelic to the 1.1-kb ORF of the yeast gene COX11 which is located on chromosome XVI and encodes a protein of 28-kDa localized in the inner mitochondrial membrane . Allelism of PSO7/COX11 was verified by non-complementation of 4NQO-sensitivity in diploids homo- and hetero-allelic for the pso7-1 and cox11::TRP1 mutant alleles . Sensitivity to 4NQO was the same in exponentially growing cells of the pso7-1 mutant and the cox11::TRP1 disruptant . Allelism of COX11 and PSO7 indicates that the pso7 mutant's sensitivity to photoactivated 3-carbethoxypsoralen and to 4NQO is not caused by defective DNA repair, but rather is due to an altered metabolism of the pro-mutagen 4NQO in the absence of cytochrome oxidase (Cox) in pso7-1/cox11::TRP1 mutants/disruptants . Lack of Cox might also lead to a higher reactivity of the active oxygen species produced by photoactivated 3-carbethoxypsoralen . The metabolic state of the cells is important for their sensitivity phenotype since the largest enhancement of sensitivity to 4NQO between wild-type (WT) and the pso7 mutant occurs in exponentially growing cells, while cells in stationary phase or growing cells in phosphate buffer have the same 4NQO resistance, irrespective of their WT/mutant status . Strains containing the pso7-1 or cox11::TRP1 mutant allele were also sensitive to the oxidative stress-generating agents H(2)O(2) and paraquat . Mutant pso7-1, as well as disruptant cox11::TRP1, harboured mitochondria that in comparison to WT contained less than 5% and no detectable Cox activity, respectively. Biosci Biotechnol Biochem, 1999 Aug, 63(8), 1414 - 9 Isolation and characterization of the gene conferring thiamine-inducible expression from Saccharomyces cerevisiae; Shiba Y et al.; The production level of CPY in Saccharomyces cerevisiae KS58-2D/pCY303 was drastically decreased when thiamine was not added to the culture medium . We isolated and characterized the mutants that could produce CPY even though thiamine was absent from the medium . Using complementation screening in the mutants obtained, we isolated a gene that was involved in the thiamine-inducible expression, TIE1, which corresponded to the YDR325w ORF on chromosome IV . The predicted protein sequence of TIE1 did not have significant homology to proteins from public databases . The disruption of the TIE1 gene caused two phenotypes, increase of expression level in thiamine-free medium and ethanol sensitivity . This increase in thiamine-free medium was also observed in the expression under the control of ENO1 or ADH1 promoter in addition to the GAL10 promoter, suggesting that the TIE1 protein is associated with a similar kind of transcriptional mechanism regulated by thiamine. Appl Microbiol Biotechnol, 1999 Aug, 52(2), 215 - 20 Viability and thermal stability of a strain of Saccharomyces cerevisiae freeze-dried in different sugar and polymer matrices; Lodato P et al.; The viability and thermal stability of a freeze-dried yeast strain were studied in relation to some physical properties of the matrices in which the cells were freeze-dried . Samples of inoculum with solutions of the matrix components {polyvinylpyrrolidone (PVP), maltose, trehalose, maltodextrins, or mixtures of maltodextrin and trehalose} and controls without matrices were freeze-dried and then equilibrated at several relative humidities . Viability was determined before and after freeze-drying and after heat treatment (100 min at 70 degrees C) . Freeze-drying with trehalose, PVP, maltose or 1.8-kDa maltodextrin, and mixtures of maltodextrin/trehalose increased viability in comparison with controls . The 3.6-kDa maltodextrin was ineffective at protecting the cells during freeze-drying . The glass transition temperature (Tg), which depends on moisture content, was indicated as a possible factor to determine the stability of labile materials . Protective effects of the excipients during thermal treatment were analysed in relation to the physical changes (collapse or structural shrinkage) which were dependent on the Tg of the systems . The presence of a certain amount of amorphous disaccharides during freeze-drying and heating was found to be a critical factor for ensuring cell viability, which was protected even in rubbery (above Tg) matrices. J Biol Chem, 1999 Oct 1, 274(40), 28459 - 65 Thioredoxin deficiency causes the constitutive activation of Yap1, an AP-1-like transcription factor in Saccharomyces cerevisiae; Izawa S et al.; Yap1 is a transcription factor that responds to oxidative stress in Saccharomyces cerevisiae . The activity of Yap1 is regulated at the level of its intracellular localization, and a cysteine-rich domain at the C terminus of Yap1 is involved in this regulation . We investigated the effects of redox-regulatory proteins, thioredoxin and glutaredoxin, on the regulation of Yap1, using the deficient mutants of these thiol-disulfide oxidoreductases . In the thioredoxin-deficient mutant (trx1Delta/trx2Delta), Yap1 was constitutively concentrated in the nucleus and the level of expression of the Yap1 target genes was high under normal conditions, while this was not the case for the glutaredoxin-deficient mutant (grx1Delta/grx2Delta) . No distinct difference was observed in the levels of Yap1 protein between the wild type and trx1Delta/trx2Delta . The constitutive activation of Yap1 in trxDelta/trx2Delta was observed under aerobic conditions but not under anaerobic conditions . These findings suggest that thioredoxin has negative effects on this regulation via the redox states . We also show the synthetic lethality between yap1Delta and trx1Delta/trx2Delta mutation, but the yap1Delta/grx1Delta/grx2Delta triple mutant was viable, suggesting a difference of the functions between thioredoxin and glutaredoxin and a genetic interaction between Yap1 and thioredoxin in vivo. J Biol Chem, 1999 Oct 1, 274(40), 28121 - 7 Characterization and function in vivo of two novel phospholipases B/lysophospholipases from Saccharomyces cerevisiae; Merkel O et al.; The yeast genome contains two genes, designated as PLB2 and PLB3, that are 67% and 62% identical, respectively, to PLB1, which codes for a phospholipase B/lysophospholipase in yeast (Lee, S . K., Patton, J . L., Fido, M., Hines, L . K., Kohlwein, S . D., Paltauf, F., Henry, S . A., and Levin, D . E . (1994) J . Biol . Chem . 269, 19725-19730) . Deletion and overexpression studies and in vivo and in vitro activity measurements suggest that both genes indeed code for phospholipases B/lysophospholipases . In cell free extracts of a plb1 plb2 plb3 triple mutant, no phospholipase B activity was detectable . Upon overexpression of PLB2 in a plb1 plb3 mutant background, phospholipase B activity was detectable in the plasma membrane, periplasmic space extracts and the culture supernatant . Similar to Plb1p, Plb2p appears to accept all major phospholipid classes, with a preference for acidic phospholipids including phosphatidylinositol 3',4'-bisphosphate and phosphatidic acid . Consistent with a function as an extracellular lysophospholipase, PLB2 overexpression conferred resistance to lyso-phosphatidylcholine . Deletion of Plb2p function had no effect on glycerophosphoinositol or glycerophosphocholine release in vivo, in contrast to a deletion of Plb3p function, which resulted in a 50% reduction of phosphatidylinositol breakdown and glycerophosphoinositol release from the cells . In vitro, Plb3p hydrolyzes only phosphatidylinositol and phosphatidylserine and, to a lesser extent, their lyso-analogs . Plb3p activity in a plb1 plb2 mutant background was observed in periplasmic space extracts . Both Plb3p and Plb2p display transacylase activity in vitro, in the presence or absence, respectively, of detergent. J Biol Chem, 1999 Oct 1, 274(40), 28096 - 105 Transport of sulfonium compounds . Characterization of the s-adenosylmethionine and s-methylmethionine permeases from the yeast Saccharomyces cerevisiae; Rouillon A et al.; We report here the characterization and the molecular analysis of the two high affinity permeases that mediate the transport of S-adenosylmethionine (AdoMet) and S-methylmethionine (SMM) across the plasma membrane of yeast cells . Mutant cells unable to use AdoMet as a sulfur source were first isolated and demonstrated to lack high affinity AdoMet transport capacities . Functional complementation cloning allowed us to identify the corresponding gene (SAM3), which encodes an integral membrane protein comprising 12 putative membrane spanning regions and belonging to the amino acid permease family . Among amino acid permease members, the closest relative of Sam3p is encoded by the YLL061w open reading frame . Disruption of YLL061w was shown to specifically lead to cells unable to use SMM as a sulfur source . Accordingly, transport assays demonstrated that YLL061w disruption mutation impaired the high affinity SMM permease, and YLL061w was therefore renamed MMP1 . Further study of sam3Delta and mmp1Delta mutant cells showed that in addition to high affinity permeases, both sulfonium compounds are transported into yeast cells by low affinity transport systems that appear to be carrier-facilitated diffusion. Prikl Biokhim Mikrobiol, 1999 May-Jun, 35(3), 349 - 52 {Geothermal water as part of culture medium and morpho-physiological properties of Saccharomyces cerevisiae}; Abramov ShA et al.; Morphophysiological changes in Saccharomyces cerevisiae gamma-503 cells cultivated in nutrient media containing geothermal water as a source of mineral substances were studied . The optimal mineralization of the medium was found to be 4.0 g/l, supplemented with 2.6 g/l (NH4)2HPO4 . These conditions provided active growth and development of the culture with high yields of the biomass and the maximal enzymatic activity . Differences in cellular structures at certain stages of metabolism were demonstrated. Electrophoresis, 1999 Aug, 20(11), 2280 - 98 Two-dimensional gel protein database of Saccharomyces cerevisiae (update 1999); Perrot M et al.; By proving the opportunity to visualize several hundred proteins at a time, two-dimensional (2-D) gel electrophoresis is an important tool for proteome research . In order to take advantage of the full potential of this technique for yeast studies, we have undertaken a systematic identification of yeast proteins resolved by this technique . We report here the identification of 92 novel protein spots on the yeast 2-D protein map . These identifications extend the number of protein spots identified on our yeast reference map to 401 . These spots correspond to the products of 279 different genes . They have been essentially identified by three methods: gene overexpression, amino acid composition and mass spectrometry . These data can be accessed on the Yeast Protein Map server (htpp://www.ibgc.u-bordeaux2.fr/YPM). Biochimie, 1999 Jul, 81(7), 751 - 6 RNA:pseudouridine synthetase Pus1 from Saccharomyces cerevisiae: oligomerization property and stoichiometry of the complex with yeast tRNA(Phe); Arluison V et al.; Yeast RNA:pseudouridine synthetase Pus1 catalyzes the formation of pseudouridines in tRNAs . We report here the quaternary structure of purified recombinant Pus1 in solution . At low concentration, in the absence of tRNA, Pus1 oligomerizes while at high concentration it precipitates . This oligomerization/aggregation can be prevented by addition of dodecyl-beta-D-maltoside or of yeast tRNA(Phe) . The detergent does not significantly interfere with substrate binding or with activity of Pus1 . The stoichiometry of the Pus1/tRNA(Phe) complex is 1/1 . We conclude that the detergent covers an hydrophobic region of the RNA binding pocket responsible for Pus1 aggregation. Mol Cell Biol, 1999 Oct, 19(10), 7041 - 9 NORF5/HUG1 is a component of the MEC1-mediated checkpoint response to DNA damage and replication arrest in Saccharomyces cerevisiae; Basrai MA et al.; Analysis of global gene expression in Saccharomyces cerevisiae by the serial analysis of gene expression technique has permitted the identification of at least 302 previously unidentified transcripts from nonannotated open reading frames (NORFs) . Transcription of one of these, NORF5/HUG1 (hydroxyurea and UV and gamma radiation induced), is induced by DNA damage, and this induction requires MEC1, a homolog of the ataxia telangiectasia mutated (ATM) gene . DNA damage-specific induction of HUG1, which is independent of the cell cycle stage, is due to the alleviation of repression by the Crt1p-Ssn6p-Tup1p complex . Overexpression of HUG1 is lethal in combination with a mec1 mutation in the presence of DNA damage or replication arrest, whereas a deletion of HUG1 rescues the lethality due to a mec1 null allele . HUG1 is the first example of a NORF with important biological functional properties and defines a novel component of the MEC1 checkpoint pathway. Mol Cell Biol, 1999 Oct, 19(10), 6972 - 9 Rsp5 ubiquitin-protein ligase mediates DNA damage-induced degradation of the large subunit of RNA polymerase II in Saccharomyces cerevisiae; Beaudenon SL et al.; Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins . We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro . We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect . The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1 . These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage . A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells. Mol Cell Biol, 1999 Oct, 19(10), 6929 - 39 The morphogenesis checkpoint in Saccharomyces cerevisiae: cell cycle control of Swe1p degradation by Hsl1p and Hsl7p; McMillan JN et al.; In Saccharomyces cerevisiae, the Wee1 family kinase Swe1p is normally stable during G(1) and S phases but is unstable during G(2) and M phases due to ubiquitination and subsequent degradation . However, perturbations of the actin cytoskeleton lead to a stabilization and accumulation of Swe1p . This response constitutes part of a morphogenesis checkpoint that couples cell cycle progression to proper bud formation, but the basis for the regulation of Swe1p degradation by the morphogenesis checkpoint remains unknown . Previous studies have identified a protein kinase, Hsl1p, and a phylogenetically conserved protein of unknown function, Hsl7p, as putative negative regulators of Swe1p . We report here that Hsl1p and Hsl7p act in concert to target Swe1p for degradation . Both proteins are required for Swe1p degradation during the unperturbed cell cycle, and excess Hsl1p accelerates Swe1p degradation in the G(2)-M phase . Hsl1p accumulates periodically during the cell cycle and promotes the periodic phosphorylation of Hsl7p . Hsl7p can be detected in a complex with Swe1p in cell lysates, and the overexpression of Hsl7p or Hsl1p produces an effective override of the G(2) arrest imposed by the morphogenesis checkpoint . These findings suggest that Hsl1p and Hsl7p interact directly with Swe1p to promote its recognition by the ubiquitination complex, leading ultimately to its destruction. Mol Cell Biol, 1999 Oct, 19(10), 6775 - 87 Erf2, a novel gene product that affects the localization and palmitoylation of Ras2 in Saccharomyces cerevisiae; Bartels DJ et al.; Plasma membrane localization of Ras requires posttranslational addition of farnesyl and palmitoyl lipid moieties to a C-terminal CaaX motif (C is cysteine, a is any aliphatic residue, X is the carboxy terminal residue) . To better understand the relationship between posttranslational processing and the subcellular localization of Ras, a yeast genetic screen was undertaken based on the loss of function of a palmitoylation-dependent RAS2 allele . Mutations were identified in an uncharacterized open reading frame (YLR246w) that we have designated ERF2 and a previously described suppressor of hyperactive Ras, SHR5 . ERF2 encodes a 41-kDa protein with four predicted transmembrane (TM) segments and a motif consisting of the amino acids Asp-His-His-Cys (DHHC) within a cysteine-rich domain (CRD), called DHHC-CRD . Mutations within the DHHC-CRD abolish Erf2 function . Subcellular fractionation and immunolocalization experiments reveal that Erf2 tagged with a triply iterated hemagglutinin epitope is an integral membrane protein that colocalizes with the yeast endoplasmic reticulum marker Kar2 . Strains lacking ERF2 are viable, but they have a synthetic growth defect in the absence of RAS2 and partially suppress the heat shock sensitivity resulting from expression of the hyperactive RAS2(V19) allele . Ras2 proteins expressed in an erf2Delta strain have a reduced level of palmitoylation and are partially mislocalized to the vacuole . Based on these observations, we propose that Erf2 is a component of a previously uncharacterized Ras subcellular localization pathway . Putative members of an Erf2 family of proteins have been uncovered in yeast, plant, worm, insect, and mammalian genome databases, suggesting that Erf2 plays a role in Ras localization in all eucaryotes. Mol Cell Biol, 1999 Oct, 19(10), 6608 - 20 DOT4 links silencing and cell growth in Saccharomyces cerevisiae; Kahana A et al.; Transcriptional silencing in Saccharomyces cerevisiae occurs at specific loci and is mediated by a multiprotein complex that includes Rap1p and the Sir proteins . We studied the function of a recently identified gene, DOT4, that disrupts silencing when overexpressed . DOT4 encodes an ubiquitin processing protease (hydrolase) that is primarily located in the nucleus . By two-hybrid analysis, the amino-terminal third of Dot4p interacts with the silencing protein Sir4p . Cells lacking DOT4 exhibited reduced silencing and a corresponding decrease in the level of Sir4p . Together, these findings suggest that Dot4p regulates silencing by acting on Sir4p . In strains with several auxotrophic markers, loss of DOT4 ubiquitin hydrolase activity also results in a slow-growth defect . The defect can be partially suppressed by mutations in a subunit of the 26S proteasome, suggesting that Dot4p has the ability to prevent ubiquitin-mediated degradation . Furthermore, wild-type SIR2, SIR3, and SIR4 are required for full manifestation of the growth defect in a dot4 strain, indicating that the growth defect is caused in part by a silencing-related mechanism . We propose that Dot4p helps to restrict the location of silencing proteins to a limited set of genomic loci. Nature, 1999 Sep 9, 401(6749), 177 - 80 Saccharomyces cerevisiae telomerase is an Sm small nuclear ribonucleoprotein particle; Seto AG et al.; Activation of the chromosome end-replicating enzyme telomerase can greatly extend the lifespan of normal human cells and is associated with most human cancers . In all eukaryotes examined, telomerase has an RNA subunit, a conserved reverse transcriptase subunit and additional proteins, but little is known about the assembly of these components . Here we show that the Saccharomyces cerevisiae telomerase RNA has a 5'-2,2,7-trimethylguanosine (TMG) cap and a binding site for the Sm proteins, both hallmarks of small nuclear ribonucleoprotein particles (snRNPs) that are involved in nuclear messenger RNA splicing . Immunoprecipitation of telomerase from yeast extracts shows that Sm proteins are assembled on the RNA and that most or all of the telomerase activity is associated with the Sm-containing complex . These data support a model in which telomerase RNA is transcribed by RNA polymerase II and 7-methylguanosine-capped, binds the seven Sm proteins, becomes TMG-capped and picks up the other protein subunits . We conclude that the functions of snRNPs assembled by this pathway are not restricted to RNA processing, but also include chromosome telomere replication. Acta Crystallogr D Biol Crystallogr, 1999 Sep, 55 ( Pt 9), 1586 - 8 Crystallization and preliminary X-ray analysis of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (tyrosine inhibitable) from Saccharomyces cerevisiae; Schneider TR et al.; 3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (E.C . 4.1.2.15) catalyses the first step in the biosynthesis of aromatic amino acids: the condensation of phophoenolpyruvate and erythrose 4-phosphate to 3-deoxy-D-arabino-heptulosonate-7-phosphate . Diffraction-quality crystals of the tyrosine-inhibitable form of the enzyme from Saccharomyces cerevisiae have been obtained by the hanging-drop vapour-diffusion method in the presence of polyethylene glycol . The crystals belong to the triclinic space group P1, with unit-cell parameters a = 81.5, b = 94.0, c = 104.6 A, alpha = 65.5, beta = 85.2, gamma = 75.0 degrees, and can be flash-cooled using glycerol as a cryoprotectant . A data set to 2.3 A has been collected at 120 K. J Biol Chem, 1999 Sep 24, 274(39), 28026 - 34 Overlapping positive and negative GATA factor binding sites mediate inducible DAL7 gene expression in Saccharomyces cerevisiae; Rai R et al.; Allantoin pathway gene expression in Saccharomyces cerevisiae responds to two different environmental stimuli . The expression of these genes is induced in the presence of allantoin or its degradative metabolites and repressed when a good nitrogen source (e . g . asparagine or glutamine) is provided . Three types of cis-acting sites and trans-acting factors are required for allantoin pathway gene transcription as follows: (i) UAS(NTR) element associated with the transcriptional activators Gln3p and Gat1p, (ii) URS(GATA) element associated with the repressor Dal80p, and (iii) UIS(ALL) element associated with the Dal82 and Dal81 proteins required for inducer-dependent transcription . Most of the work leading to the above conclusions has employed inducer-independent allantoin pathway genes (e.g . DAL5 and DAL3) . The purpose of this work is to extend our understanding of these elements and their roles to inducible allantoin pathway genes using the DAL7 (encoding malate synthase) as a model . We show that eight distinct cis-acting sites participate in the process as follows: a newly identified GC-rich element, two UAS(NTR), two UIS(ALL), and three URS(GATA) elements . The two GATA-containing UAS(NTR) elements are coincident with two of the three GATA sequences that make up the URS(GATA) elements . The remaining URS(GATA) GATA sequence, however, is not a UAS(NTR) element but appears to function only in repression . The data provide insights into how these cis- and trans-acting factors function together to accomplish the regulated expression of the DAL7 gene that is observed in vivo. J Biol Chem, 1999 Sep 24, 274(39), 27590 - 6 Oxidative stress and iron are implicated in fragmenting vacuoles of Saccharomyces cerevisiae lacking Cu,Zn-superoxide dismutase; Corson LB et al.; The absence of the antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) is shown here to cause vacuolar fragmentation in Saccharomyces cerevisiae . Wild-type yeast have 1-3 large vacuoles whereas the sod1Delta yeast have as many as 50 smaller vacuoles . Evidence that this fragmentation is oxygen-mediated includes the findings that aerobically (but not anaerobically) grown sod1Delta yeast exhibit aberrant vacuoles and genetic suppressors of other oxygen-dependent sod1 null phenotypes rescue the vacuole defect . Surprisingly, iron also is implicated in the fragmentation process as iron addition exacerbates the sod1Delta vacuole defect while iron starvation ameliorates it . Because the vacuole is reported to be a site of iron storage and iron reacts avidly with reactive oxygen species to generate toxic side products, we propose that vacuole damage in sod1Delta cells arises from an elevation of iron-mediated oxidation within the vacuole or from elevated pools of "free" iron that may bind nonproductively to vacuolar ligands . Furthermore, additional pleiotropic phenotypes of sod1Delta cells (including increased sensitivity to pH, nutrient deprivation, and metals) may be secondary to vacuolar compromise . Our findings support the hypothesis that oxidative stress alters cellular iron homeostasis which in turn increases oxidative damage . Thus, our findings may have medical relevance as both oxidative stress and alterations in iron homeostasis have been implicated in diverse human disease processes . Our findings suggest that strategies to decrease intracellular iron may significantly reduce oxidatively induced cellular damage. J Biol Chem, 1999 Sep 24, 274(39), 27567 - 72 The Saccharomyces cerevisiae HCR1 gene encoding a homologue of the p35 subunit of human translation initiation factor 3 (eIF3) is a high copy suppressor of a temperature-sensitive mutation in the Rpg1p subunit of yeast eIF3; Valasek L et al.; The complex eukaryotic initiation factor 3 (eIF3) was shown to promote the formation of the 43 S preinitiation complex by dissociating 40 S and 60 S ribosomal subunits, stabilizing the ternary complex, and aiding mRNA binding to 40 S ribosomal subunits . Recently, we described the identification of RPG1 (TIF32), the p110 subunit of the eIF3 core complex in yeast . In a screen for Saccharomyces cerevisiae multicopy suppressors of the rpg1-1 temperature-sensitive mutant, an unknown gene corresponding to the open reading frame YLR192C was identified . When overexpressed, the 30-kDa gene product, named Hcr1p, was able to support, under restrictive conditions, growth of the rpg1-1 temperature-sensitive mutant, but not of a Rpg1p-depleted mutant . An hcr1 null mutant was viable, but showed slight reduction of growth when compared with the wild-type strain . Physical interaction between the Hcr1 and Rpg1 proteins was shown by co-immunoprecipitation analysis . The combination of Deltahcr1 and rpg1-1 mutations resulted in a synthetic enhancement of the slow growth phenotype at a semipermissive temperature . In a computer search, a significant homology to the human p35 subunit of the eIF3 complex was found . We assume that the yeast Hcr1 protein participates in translation initiation likely as a protein associated with the eIF3 complex. Yeast, 1999 Sep 15, 15(12), 1275 - 85 Deletion of six open reading frames from the left arm of chromosome IV of Saccharomyces cerevisiae; Tuller G et al.; The construction of six deletion mutants of Saccharomyces cerevisiae and their basic phenotypic characterization are described . Open reading frames YDL148c, YDL109c, YDL021w, YDL019c, YDL018c and YDL015c from the left arm of chromosome IV were deleted using a polymerase chain reaction (PCR)-based disruption technique, introducing the kanMX4 resistance marker into the respective genes . Gene replacement cassettes (pYORCs) for use in other strain backgrounds were cloned by PCR using DNA templates from haploid or diploid deletion mutants, and inserted into episomal plasmids . Cognate clones of all six ORFs were obtained by gap repair . Deletions were carried out in diploid cells and, after sporulation, yielded four viable spores for clones disrupted in YDL109c, YDL021w, YDL019c and YDL018c . Spores harbouring disruptions in ORFs YDL148c and YDL015c germinated but underwent only a few divisions before ceasing growth, suggesting that the respective genes are essential for vegetative growth on YPD complete media . The other deletion mutants grew like wild-type at different temperatures and on different carbon sources . A brief computational analysis of the six ORFs studied in this work is presented . Yeast, 1999 Sep 15, 15(12), 1183 - 97 Molecular mechanism of the multiple regulation of the Saccharomyces cerevisiae ATF1 gene encoding alcohol acetyltransferase; Fujiwara D et al.; The ATF1 gene encodes an alcohol acetyl transferase (AATase), that catalyses the synthesis of acetate esters from acetyl CoA and several kinds of alcohols . ATF1 transcription is negatively regulated by unsaturated fatty acids and oxygen . A series of analyses of the ATF1 promoter identified an 18 bp element essential for transcriptional activation . Ligation of the 18 bp element into a plasmid carrying the CYC1 promoter deleted UAS-activated transcription and conferred transcriptional repression by unsaturated fatty acids . The 18 bp element contains a binding sequence for Rap1p, which is a transcriptional repressor and activator . In vitro binding studies showed that Rap1p binds to the 18 bp element essential for transcriptional activation . The results of internal deletion studies of the promoter region suggested that there was also a region responsible for ATF1 oxygen regulation . This region contained the consensus binding sequence for the hypoxic repressor Rox1p . In vitro binding studies showed that Rox1p binds to the region responsible for oxygen regulation . To investigate the effect of the hypoxic repressor complex on transcription, ATF1 expression was measured in rox1, tup1 and ssn6 disruptant strains . It was found that rox1, tup1 and ssn6 disruption caused elevated expression of ATF1 under aerobic conditions . Thus, the activation of ATF1 transcription is dependent on Rap1p, and the Rox1p-Tup1p-Ssn6p hypoxic repressor complex is responsible for repression by oxygen . Furthermore, a study of ATF1 expression in a sch9 null mutant suggested that the Sch9p protein kinase is involved in ATF1 trancriptional activation . Bioinformatics, 1999 Jul-Aug, 15(7-8), 607 - 11 SCPD: a promoter database of the yeast Saccharomyces cerevisiae; Zhu J et al.; MOTIVATION: In order to facilitate a systematic study of the promoters and transcriptionally regulatory cis-elements of the yeast Saccharomyces cerevisiae on a genomic scale, we have developed a comprehensive yeast-specific promoter database, SCPD . RESULTS: Currently SCPD contains 580 experimentally mapped transcription factor (TF) binding sites and 425 transcriptional start sites (TSS) as its primary data entries . It also contains relevant binding affinity and expression data where available . In addition to mechanisms for promoter information (including sequence) retrieval and a data submission form, SCPD also provides some simple but useful tools for promoter sequence analysis . AVAILABILITY: SCPD can be accessed from the URL The database is continually updated. Biochem Biophys Res Commun, 1999 Sep 16, 263(1), 47 - 51 Heterologous expression of Mucor rouxii delta(12)-desaturase gene in Saccharomyces cerevisiae; Passorn S et al.; In this study we present the cloning and functional characterization of a gene whose product is responsible for Delta(12)-desaturase activity and is involved in the metabolic pathway of gamma-linolenic acid (GLA) synthesis of Mucor rouxii . A cDNA encoding for Delta(12)-desaturase of M . rouxii was obtained using the combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification cDNA ends (RACE) techniques . The 1188 {corrected} bp code for an open reading frame of 396 amino acid residues . The deduced amino acid sequence of the cloned cDNA comprises three conserved histidine regions and two hydrophobic domains and showed similarity with microsomal omega-3 and omega-6 desaturases of plants . Expression of this open reading frame in Saccharomyces cerevisiae resulted in the accumulation of linoleic acid (C18:2), suggesting that this gene encodes for a membrane-bound desaturase, Delta(12)-desaturase, of M . rouxii that is functional in yeast . Mol Gen Genet, 1999 Jul, 261(6), 967 - 76 Genetic interactions between a null allele of the RIT1 gene encoding an initiator tRNA-specific modification enzyme and genes encoding translation factors in Saccharomyces cerevisiae; Astrom SU et al.; The Saccharomyces cerevisiae gene RIT1 encodes a phospho-ribosyl transferase that exclusively modifies the initiator tRNA (tRNAMet(i)) by the addition of a 2'-O-ribosyl phosphate group to Adenosine 64 . As a result, tRNAMet(i) is prevented from participating in the elongation steps of protein synthesis . We previously showed that the modification is not essential for the function of tRNAMet(i) in the initiation of translation, since rit1 null strains are viable and show no obvious growth defects . Here, we demonstrate that yeast strains in which a rit1 null allele is combined with mutations in any of the genes for the three subunits of eukaryotic initiation factor-2 (eIF-2), or with disruption alleles of two of the four initiator methionine tRNA (IMT) genes, show synergistic growth defects . A multicopy plasmid carrying an IMT gene can alleviate these defects . On the other hand, introduction of a high-copy-number plasmid carrying the TEF2 gene, which encodes the eukaryotic elongation factor 1alpha (eEF-1alpha), into rit1 null strains with two intact IMT genes had the opposite effect, indicating that increased levels of eEF-1alpha are deleterious to these strains, presumably due to sequestration of the unmodified met-tRNAMet(i) for elongation . Thus, under conditions in which the components of the ternary met-tRNAMet(i):GTP:eIF-2 complex become limiting or are functionally impaired, the presence of the 2'-O-ribosyl phosphate modification in tRNAMet(i) is important for the provision of adequate amounts of tRNAMet(i) for formation of this ternary complex. Mol Gen Genet, 1999 Jul, 261(6), 917 - 32 Dominant negative mutations in the alpha-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae; Leavitt LM et al.; The alpha-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals . We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor . Two dominant negative alleles have been recovered in this manner . In addition, we find that previously isolated loss-of-function mutations in the alpha-factor receptor exhibit dominant negative effects . Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors . Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles . Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein alpha subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein . Expression of increased levels of G protein subunits partially reverses the dominant negative effects . The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling . The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation . This interaction appears to be mediated by the SST2 gene product. J Biotechnol, 1999 May 28, 71(1-3), 267 - 74 Cybernetic modeling of spontaneous oscillations in continuous cultures of Saccharomyces cerevisiae; Kompala DS; We have developed a 'cybernetic' model to simulate the dynamic competition between all the available metabolic pathways of yeast Saccharomyces cerevisiae . This computer model predicts all the key experimentally observed aspects of the sustained oscillations in all the measured concentrations in continuous cultures, such as the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitation rate are changed. FEBS Lett, 1999 Sep 17, 458(2), 215 - 8 Overexpression of L-glutamine:D-fructose-6-phosphate amidotransferase provides resistance to methylmercury in Saccharomyces cerevisiae; Miura N et al.; To identify novel genes that confer resistance to methylmercury (MeHg), a yeast genomic DNA library was transfected into Saccharomyces cerevisiae . Two functional plasmids were isolated from transfected yeast clones D1 and H5 that exhibited resistance to MeHg . The yeast transfected with plasmid isolated from clone H5 was several-fold more resistant than yeast transfected with plasmid from clone D1 . Functional characterization of the genomic DNA fragment obtained from clone H5 determined that the GFA1 gene conferred resistance to MeHg . GFA1 was reported to encode L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) which catalyzes the synthesis of glucosamine-6-phosphate from glutamine and fructose-6-phosphate . Accumulation of mercury in yeast clone W303B/pGFA1, which contains the transfected GFA1 gene, did not differ from that in control yeast clone W303B/pYES2 . The W303B/pGFA1 strain did not show resistance to mercuric chloride, zinc chloride, cadmium chloride or copper chloride, suggesting that the resistance acquired by GFA1 gene transfection might be specific to MeHg . This is the first report of a gene involved in MeHg resistance in eukaryotic cells identified by screening a DNA library. J Biol Chem, 1999 Sep 17, 274(38), 27002 - 9 Genetic analysis of glutathione peroxidase in oxidative stress response of Saccharomyces cerevisiae; Inoue Y et al.; Three glutathione peroxidase homologs (YKL026C, YBR244W, and YIR037W/HYR1) were found in the Saccharomyces Genome Database . We named them GPX1, GPX2, and GPX3, respectively, and we investigated the function of each gene product . The gpx3Delta mutant was hypersensitive to peroxides, whereas null mutants of the GPX1 and GPX2 did not show any obvious phenotypes . Glutathione peroxidase activity decreased approximately 57 and 93% in the gpx3Delta and gpx1Delta/gpx2Delta/gpx3Delta mutants, respectively, compared with that of wild type . Expression of the GPX3 gene was not induced by any stresses tested, whereas that of the GPX1 gene was induced by glucose starvation . The GPX2 gene expression was induced by oxidative stress, which was dependent upon the Yap1p . The TSA1 (thiol-specific antioxidant) gene encodes thioredoxin peroxidase that can reduce peroxides by using thioredoxin as a reducing power . Disruption of the TSA1 gene enhanced the basal expression level of the Yap1p target genes such as GSH1, GLR1, and GPX2 and that resulted in increases of total glutathione level and activities of glutathione reductase and glutathione peroxidase . However, expression of the TSA1 gene did not increase in the gpx1Delta/gpx2Delta/gpx3Delta mutant . Therefore, de novo synthesis and recycling of glutathione were increased in the tsa1Delta mutant to maintain the catalytic cycle of glutathione peroxidase reaction efficiently as a backup system for thioredoxin peroxidase. J Biol Chem, 1999 Sep 17, 274(38), 26720 - 6 Interaction of translation initiation factor eIF4G with eIF4A in the yeast Saccharomyces cerevisiae; Dominguez D et al.; Eukaryotic initiation factor (eIF) 4A is an essential protein that, in conjunction with eIF4B, catalyzes the ATP-dependent melting of RNA secondary structure in the 5'-untranslated region of mRNA during translation initiation . In higher eukaryotes, eIF4A is assumed to be recruited to the mRNA through its interaction with eIF4G . However, the failure to detect this interaction in yeast brought into question the generality of this model . The work presented here demonstrates that yeast eIF4G interacts with eIF4A both in vivo and in vitro . The eIF4A-binding site was mapped to amino acids 542-883 of yeast eIF4G1 . Expression in yeast cells of the eIF4G1 domain that binds eIF4A results in cell growth inhibition, and addition of this domain to an eIF4A-dependent in vitro system inhibits translation in a dose-dependent manner . Both in vitro translation and cell growth can be specifically restored by increasing the eIF4A concentration . These data demonstrate that yeast eIF4A and eIF4G interact and suggest that this interaction is required for translation and cell growth. J Biol Chem, 1999 Sep 17, 274(38), 26668 - 82 Biochemical characterization of the interaction between the Saccharomyces cerevisiae MSH2-MSH6 complex and mispaired bases in DNA; Marsischky GT et al.; The interaction of the Saccharomyces cerevisiae MSH2-MSH6 complex with mispaired bases was analyzed using gel mobility shift assays and surface plasmon resonance methods . Under equilibrium binding conditions, MSH2-MSH6 bound to homoduplex DNA with a K(d) of 3.9 nM and bound oligonucleotide duplexes containing T:G, +1, +2, +4, and +10 insertion/deletion loop (IDL) mispairs with K(d) values of 0.20, 0.25, 11, 3.2, and 0.55 nM, respectively . Competition binding experiments using 65 different substrates revealed a 10-fold range in mispair discrimination . In general, base-base mispairs and a +1 insertion/deletion mispair were recognized better than intermediate sized insertion/deletion mispairs of 2-8 bases . Larger IDL mispairs (>8 bases) were recognized almost as well as the +1 IDL mispair . Recognition of mispairs by MSH2-MSH6 was influenced by sequence context, with the 6-nucleotide region surrounding the mispair being primarily responsible for influencing mispair recognition . Effects of sequences as far away as 15 nucleotides were also observed . Differential effects of ATP on the stability of MSH2-MSH6-mispair complexes suggested that base-base mispairs and the smaller IDL mispairs were recognized by a different binding mode than larger IDL mispairs, consistent with genetic experiments indicating that MSH2-MSH6 functions primarily in the repair of base-base and small IDL mispairs. J Chromatogr A, 1999 Aug 6, 852(1), 161 - 73 Affinity chromatography of human estrogen receptor-alpha expressed in Saccharomyces cerevisiae . Combination of heparin- and 17beta-estradiol-affinity chromatography; Feng W et al.; Estrogen receptor-alpha is a member of the nuclear hormone receptor superfamily and is considered as a very important regulatory protein . Human estrogen receptor-alpha has been cloned into Saccharomyces cerevisiae as a fusion to ubiquitin and expression is controlled by a metallothionin promotor . Pilot scale quantities of receptor have been produced by a yeast strain transformed with expression plasmid YEpE13 {Graumann et al., J . Steroid Biochem . Mol . Biol . 57 (1996) 293} in a 14 l stirred tank reactor . The yeast extract contained 2-4 pmol of receptor protein per mg total protein . A purification scheme has been developed using heparin-affinity chromatography combined with affinity chromatography with immobilized 17beta-estradiol 17-hemisuccinate . Heparin-affinity chromatography was very efficient to remove host cell protein . Accompanying proteins that stabilize unoccupied receptor have not been dissociated during elution . The receptor could be purified 5-10-fold in ligand-free state . In contrast to previous reports, we did not find a difference of the binding affinity of liganded and unliganded receptor for heparin immobilized onto Sepharose . The unoccupied receptor could be further purified 100-fold with ligand-affinity chromatography using 17beta-estradiol 17-hemisuccinate-bovine serum albumin-Sepharose . The receptor could be kept in its native state, although saturated with 17beta-estradiol . The purification sequence allows an efficient production of receptor . Further improvement of productivity can be only accomplished by increasing the expression level. Lipids, 1999 Jul, 34(7), 681 - 8 19-Azasqualene-2,3-epoxide and its N-oxide: metabolic fate and inhibitory effect on sterol biosynthesis in Saccharomyces cerevisiae; Milla P et al.; 19-Azasqualene-2,3-epoxide was more inhibitory than the corresponding N-oxide against 2,3-oxidosqualene cyclase (OSC) solubilized from Saccharomyces cerevisiae (IC50 7+/-2 and 25+/-5 microM, respectively) . Both compounds showed a reversible, noncompetitive-type inhibition on solubilized OSC . Different inhibitory properties between the compounds were especially evident when measuring {14C}acetate incorporation into nonsaponifiable lipids extracted from treated cells . In cells treated with 19-azasqualene-2,3-epoxide at 30 microM, the radioactivity associated with the oxidosqualene fraction, which was negligible in the controls, rose to over 40% of the nonsaponifiable lipids, whereas it remained at a slightly appreciable level in cells treated with the N-oxide derivative under the same conditions . 19-Azasqualene-2,3-epoxide was also more effective than the N-oxide as a cell growth inhibitor (minimal concentration of compound needed to inhibit yeast growth: 45 and >100 microM, respectively) . The two inhibitors underwent different metabolic fates in the yeast: while 19-azasqualene-2,3-epoxide did not undergo any transformation, its N-oxide was actively reduced to the corresponding amine in whole and in "ultrasonically stimulated" cells . The N-oxide reductases responsible for this transformation appear to be largely confined within the microsomal fractions and require NADPH for their activity . A possible relationship between the inhibitory properties of the two compounds and their metabolic fates is discussed. J Cell Biol, 1999 Sep 6, 146(5), 1019 - 32 The role of actin in spindle orientation changes during the Saccharomyces cerevisiae cell cycle; Theesfeld CL et al.; In the budding yeast Saccharomyces cerevisiae, the mitotic spindle must align along the mother-bud axis to accurately partition the sister chromatids into daughter cells . Previous studies showed that spindle orientation required both astral microtubules and the actin cytoskeleton . We now report that maintenance of correct spindle orientation does not depend on F-actin during G2/M phase of the cell cycle . Depolymerization of F-actin using Latrunculin-A did not perturb spindle orientation after this stage . Even an early step in spindle orientation, the migration of the spindle pole body (SPB), became actin-independent if it was delayed until late in the cell cycle . Early in the cell cycle, both SPB migration and spindle orientation were very sensitive to perturbation of F-actin . Selective disruption of actin cables using a conditional tropomyosin double-mutant also led to defects in spindle orientation, even though cortical actin patches were still polarized . This suggests that actin cables are important for either guiding astral microtubules into the bud or anchoring them in the bud . In addition, F-actin was required early in the cell cycle for the development of the actin-independent spindle orientation capability later in the cell cycle . Finally, neither SPB migration nor the switch from actin-dependent to actin-independent spindle behavior required B-type cyclins. Bioorg Med Chem Lett, 1999 Aug 16, 9(16), 2413 - 8 Selective A1-adenosine receptor antagonists identified using yeast Saccharomyces cerevisiae functional assays; Campbell RM et al.; Evaluation of a biased "library" of pyrrolo{2,3-d}pyrimidines using yeast-based functional assays expressing human A1- and A2a-adenosine receptors, led to the A1 selective antagonist 4b . A direct correlation between yeast functional activity and binding data was established . Practical compounds with polar residues at C-4 of the pyrrolopyrimidine system required H-bond donor functionality for high potency. Plant J, 1999 Aug, 19(3), 237 - 47 Cloning and characterisation of PGA1 and PGA2: two G protein alpha-subunits from pea that promote growth in the yeast Saccharomyces cerevisiae; Marsh JF 3rd et al.; We report here on the cloning and characterization of two G protein alpha-subunits from pea: PGA1 and PGA2 . Based on DNA gel blot analysis, PGA1 and PGA2 are the only Galpha homologous sequences in pea . RT-PCR analysis reveals that PGA1 and PGA2 transcripts are present in a variety of adult pea tissues . However, PGA2 mRNA is consistently detected at a lower level than PGA1 and demonstrates some degree of tissue specificity relative to PGA1 . In the apical bud of pea seedlings, PGA1 and PGA2 transcripts decrease in response to 24 h of white light following growth for 6 days in darkness . The G protein mediated, yeast mating pathway was used to analyse the function of PGA1 and PGA2 in vivo . PGA1 downregulates the mating pathway, but through a mechanism that is independent of Gbetagamma sequestration . Unexpectedly, both PGA1 and PGA2 promote growth through a mating pathway independent mechanism. Mol Microbiol, 1999 Sep, 33(5), 904 - 18 Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae; Thevelein JM et al.; The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae plays a major role in the control of metabolism, stress resistance and proliferation, in particular in connection with the available nutrient conditions . Extensive information has been obtained on the core section of the pathway, i.e . Cdc25, Ras, adenylate cyclase, PKA, and on components interacting directly with this core section, such as the Ira proteins, Cap/Srv2 and the two cAMP phosphodiesterases . Recent work has now started to reveal upstream regulatory components and downstream targets of the pathway . A G-protein-coupled receptor system (Gpr1-Gpa2) acts upstream of adenylate cyclase and is required for glucose activation of cAMP synthesis in concert with a glucose phosphorylation-dependent mechanism . Although a genuine signalling role for the Ras proteins remains unclear, they appear to mediate at least part of the potent stimulation of cAMP synthesis by intracellular acidification . Recently, several new targets of the PKA pathway have been discovered . These include the Msn2 and Msn4 transcription factors mediating part of the induction of STRE-controlled genes by a variety of stress conditions, the Rim15 protein kinase involved in stationary phase induction of a similar set of genes and the Pde1 low-affinity cAMP phosphodiesterase, which specifically controls agonist-induced cAMP signalling . A major issue that remains to be resolved is the precise connection between the cAMP-PKA pathway and other nutrient-regulated components involved in the control of growth and of phenotypic characteristics correlated with growth, such as the Sch9 and Yak1 protein kinases . Cln3 appears to play a crucial role in the connection between the availability of certain nutrients and Cdc28 kinase activity, but it remains to be clarified which nutrient-controlled pathways control Cln3 levels. FEMS Microbiol Lett, 1999 Aug 15, 177(2), 199 - 204 Effect of Cu,Zn superoxide dismutase disruption mutation on replicative senescence in Saccharomyces cerevisiae; Barker MG et al.; The role of oxidative damage in determining the replicative lifespan of Saccharomyces cerevisiae was investigated using a wild-type haploid laboratory yeast and a Cu,Zn superoxide dismutase (sod1) mutant derivative on glucose, ethanol, glycerol and galactose media . SOD1 expression was necessary to ensure longevity on all carbon sources tested . Whilst carbon source and SOD1 gene expression do influence yeast lifespan, the relationship between the two factors is complex. J Mol Evol, 1999 Sep, 49(3), 352 - 7 The role of interelement selection in Saccharomyces cerevisiae Ty element evolution; Jordan IK et al.; Retrotransposons are mobile genetic elements that are ubiquitous components of eukaryotic genomes . The evolutionary success of retrotransposons is explained by their ability to replicate faster than the host genomes in which they reside . Elements with higher rates of genomic replication possess a selective advantage over less active elements . Retrotransposon populations, therefore, are shaped largely by selective forces acting at the genomic level between elements . To evaluate rigorously the effects of selective forces acting on retrotransposons, detailed information on the patterns of molecular variation within and between retrotransposon families is needed . The sequencing of the Saccharomyces cerevisiae genome, which includes the entire genomic complement of yeast retrotransposons, provides an unprecedented opportunity to access and analyze such data . In this study, we analyzed in detail the patterns of nucleotide variation within the open reading frames of two parental (Ty1 and Ty2) and one hybrid (Ty1/2) family of yeast retrotransposons . The pattern and distribution of nucleotide changes on the phylogenetic reconstructions of the three families of Ty elements reveal evidence of negative selection on both internal and external branches of the Ty phylogenies . These results indicate that most, if not all, Ty elements examined represent active or recently active retrotransposon lineages . We discuss the relevance of these findings with respect to the coevolutionary dynamic operating between genomic element populations and the host organisms in which they reside. J Biol Chem, 1999 Sep 10, 274(37), 26550 - 6 Abortive initiation by Saccharomyces cerevisiae RNA polymerase III; Bhargava P et al.; Promoter escape can be rate-limiting for transcription by bacterial RNA polymerases and RNA polymerase II of higher eukaryotes . Formation of a productive elongation complex requires disengagement of RNA polymerase from promoter-bound eukaryotic transcription factors or bacterial sigma factors . RNA polymerase III (pol III) stably associates with the TFIIIB-DNA complex even in the absence of localized DNA unwinding associated with the open promoter complex . To explore the role that release of pol III from the TFIIIB-DNA complex plays in limiting the overall rate of transcription, we have examined the early steps of RNA synthesis . We find that, on average, only three rounds of abortive initiation precede the formation of each elongation complex and that nearly all pol III molecules escape the abortive initiation phase of transcription without significant pausing or arrest . However, when elongation is limited to 5 nucleotides, the intrinsic exoribonuclease activity of pol III cleaves 5-mer RNA at a rate considerably faster than product release or reinitiation . This cleavage also occurs in the normal process of forming a productive elongation complex . The possible role of nucleolytic retraction in disengaging pol III from TFIIIB is discussed. Eur J Cell Biol, 1999 Jul, 78(7), 435 - 40 The processing alpha1,2-mannosidase of Saccharomyces cerevisiae depends on Rer1p for its localization in the endoplasmic reticulum; Massaad MJ et al.; The yeast alpha1,2-mannosidase Mns1p is involved in N-linked oligosaccharide processing in Saccharomyces cerevisiae by converting Man9GlcNAc2 to a single isomer of Man8GlcNAc2 . alpha1,2-Mannosidase is a 63 kDa type II resident membrane protein of the endoplasmic reticulum that has none of the known endoplasmic reticulum localization signals (HDEL/KDEL, KKXX, or RRXX) . Using antibodies against recombinant alpha1,2-mannosidase, indirect immunofluorescence showed that alpha1,2-mannosidase localization is abnormal in rer1 cells and that the alpha1,2-mannosidase localizes in the vacuoles of rer1/deltapep4 cells whereas in wild-type and deltapep4 cells it is found in the endoplasmic reticulum . 35S-labeled cell extracts were subjected to double immunoprecipitation, first with antibodies to alpha1,2-mannosidase, then with either alpha1,2-mannosidase antibodies or antibodies to alpha1,6-mannose residues added in the Golgi . The labeled proteins were examined by autoradiography after sodium dodecyl sulfate polyacrylamide gel electrophoresis . A significant proportion of the labeled alpha1,2-mannosidase was immunoprecipitated by alpha1,6-mannose antibodies in wild-type, deltapep4 and rer1/deltapep4 cells with endogenous levels of alpha1,2-mannosidase, and in wild-type, deltapep4, rer1 and rer1/deltapep4 cells overexpressing alpha1,2-mannosidase . The alpha1,2-mannosidase of rer1/deltapep4 cells had a slower mobility on the gels than alpha1,2-mannosidase precipitated from wild-type or deltapep4 cells, indicating increased glycosylation due to transport through the Golgi to the vacuoles . It is concluded that the endoplasmic reticulum localization of alpha1,2-mannosidase in wild-type cells depends on Rer1p for retrieval from an early Golgi compartment. Genetics, 1999 Sep, 153(1), 81 - 94 Genetic study of interactions between the cytoskeletal assembly protein sla1 and prion-forming domain of the release factor Sup35 (eRF3) in Saccharomyces cerevisiae; Bailleul PA et al.; Striking similarities between cytoskeletal assembly and the "nucleated polymerization" model of prion propagation suggest that similar or overlapping sets of proteins may assist in both processes . We show that the C-terminal domain of the yeast cytoskeletal assembly protein Sla1 (Sla1C) specifically interacts with the N-terminal prion-forming domain (Sup35N) of the yeast release factor Sup35 (eRF3) in the two-hybrid system . Sla1C and several other Sup35N-interacting proteins also exhibit two-hybrid interactions with the poly-Gln-expanded N-proximal fragment of human huntingtin, which promotes Huntington disease-associated aggregation . The Sup35N-Sla1C interaction is inhibited by Sup35N alterations that make Sup35 unable to propagate the {PSI(+)} state and by the absence of the chaperone protein Hsp104, which is essential for {PSI} propagation . In a Sla1(-) background, {PSI} curing by dimethylsulfoxide or excess Hsp104 is increased, while translational readthrough and de novo {PSI} formation induced by excess Sup35 or Sup35N are decreased . These data show that, in agreement with the proposed function of Sla1 during cytoskeletal formation, Sla1 assists in {PSI} formation and propagation, but is not required for these processes . Sla1(-) strains are sensitive to some translational inhibitors, and some sup35 mutants, obtained in a Sla1(-) background, are sensitive to Sla1, suggesting that the interaction between Sla1 and Sup35 proteins may play a role in the normal function of the translational apparatus . We hypothesize that Sup35N is involved in regulatory interactions with intracellular structural networks, and {PSI} prion may be formed as a by-product of this process. Genetics, 1999 Sep, 153(1), 69 - 79 Pleiotropic defects caused by loss of the proteasome-interacting factors Rad23 and Rpn10 of Saccharomyces cerevisiae; Lambertson D et al.; Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains . We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome . Similarly, a small fraction of Rpn10 is a component of the proteasome . Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo . We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant . rad23Delta rpn10Delta displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10 . Although rad23Delta rpn10Delta displays similar sensitivity to DNA damage as a rad23Delta single mutant, deletion of RAD23 in rpn10Delta significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Delta single mutant . A mutant Rad23 that is unable to bind the proteasome ((DeltaUbL)rad23) does not suppress the canavanine or cold-sensitive defects of rad23Delta rpn10Delta, demonstrating that Rad23/proteasome interaction is related to these effects . Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Delta rpn10Delta suggest that proteasome function is altered. Genetics, 1999 Sep, 153(1), 35 - 47 Temperature-sensitive mutations in the Saccharomyces cerevisiae MRT4, GRC5, SLA2 and THS1 genes result in defects in mRNA turnover; Zuk D et al.; In a screen for factors involved in mRNA turnover, four temperature-sensitive yeast strains (ts1189, ts942, ts817, and ts1100) exhibited defects in the decay of several mRNAs . Complementation of the growth and mRNA decay defects, and genetic experiments, revealed that ts1189 is mutated in the previously unknown MRT4 gene, ts942 is mutated in GRC5 (encoding the L9 ribosomal protein), ts817 contains a mutation in SLA2 (encoding a membrane protein), and ts1100 contains a mutation in THS1 (encoding the threonyl-tRNA synthetase) . Three of the four mutants (mrt4, grc5, and sla2) were not defective in protein synthesis, suggesting that these strains contain mutations in factors that may play a specific role in mRNA decay . The mRNA stabilization observed in the ths1 strain, however, could be due to the significant drop in translation observed in this mutant at 37 degrees . While the three interesting mutants appear to encode novel mRNA decay factors, at least one could be linked to a previously characterized mRNA decay pathway . The growth and mRNA decay defects of ts942 (grc5) cells were suppressed by overexpression of the NMD3 gene, encoding a protein shown to participate in a two-hybrid interaction with the nonsense-mediated decay protein Upf1p. Genetics, 1999 Sep, 153(1), 25 - 33 SAS4 and SAS5 are locus-specific regulators of silencing in Saccharomyces cerevisiae; Xu EY et al.; Sir2p, Sir3p, Sir4p, and the core histones form a repressive chromatin structure that silences transcription in the regions near telomeres and at the HML and HMR cryptic mating-type loci in Saccharomyces cerevisiae . Null alleles of SAS4 and SAS5 suppress silencing defects at HMR; therefore, SAS4 and SAS5 are negative regulators of silencing at HMR . This study revealed that SAS4 and SAS5 contribute to silencing at HML and the telomeres, indicating that SAS4 and SAS5 are positive regulators of silencing at these loci . These paradoxical locus-specific phenotypes are shared with null alleles of SAS2 and are unique among phenotypes of mutations in other known regulators of silencing . This work also determined that these SAS genes play roles that are redundant with SIR1 at HML, yet distinct from SIR1 at HMR . Furthermore, these SAS genes are not redundant with each other in silencing HML . Collectively, these data suggest that SAS2, SAS4, and SAS5 constitute a novel class of regulators of silencing and reveal fundamental differences in the regulation of silencing at HML and HMR . We provide evidence for a model that accounts for the observation that these SAS genes are both positive and negative regulators of silencing. Genetics, 1999 Sep, 153(1), 13 - 23 Identification of SAS4 and SAS5, two genes that regulate silencing in Saccharomyces cerevisiae; Xu EY et al.; In Saccharomyces cerevisiae, chromatin-mediated silencing inactivates transcription of the genes at the HML and HMR cryptic mating-type loci and genes near telomeres . Mutations in the Rap1p and Abf1p binding sites of the HMR-E silencer (HMRa-e**) result in a loss of silencing at HMR . We characterized a collection of 15 mutations that restore the alpha-mating phenotype to MATalpha HMRa-e** strains . These mutations defined three complementation groups, two new groups and one group that corresponded to the previously identified SAS2 gene . We cloned the genes that complemented members of the new groups and identified two previously uncharacterized genes, which we named SAS4 and SAS5 . Neither SAS4 nor SAS5 was required for viability . Null alleles of SAS4 and SAS5 restored SIR4-dependent silencing at HMR, establishing that each is a regulator of silencing . Null alleles of SAS4 and SAS5 bypassed the role of the Abf1p binding site of the HMR-E silencer but not the role of the ACS or Rap1p binding site . Previous analysis indicated that SAS2 is homologous to a human gene that is a site of recurring translocations involved in acute myeloid leukemia . Similarly, SAS5 is a member of a gene family that included two human genes that are the sites of recurring translocations involved in acute myeloid leukemia. Biochem Biophys Res Commun, 1999 Sep 7, 262(3), 726 - 30 Functional expression of the tandem-repeated homodimer of the mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae; Hatanaka T et al.; The mitochondrial ADP/ATP carrier (AAC) is believed to function as a dimer . To characterize the oligomeric state of the yeast type 2 AAC (yAAC2), we tried to express its tandem-repeated homodimer, in which the C-terminus of the first repeat was fused to the N-terminus of the second repeat, in yeast mitochondria . The tandem dimer was expressed in the mitochondrial membrane at the same level as that of yAAC2, being inserted into the mitochondrial membrane as in yAAC2, and it showed very similar transport activity to that of yAAC2 . It was suggested that the two carrier molecules in a dimeric form are located in the membrane facing each other in the same orientation . Biochemistry, 1999 Aug 31, 38(35), 11340 - 8 Dimer dissociation and thermosensitivity kinetics of the Saccharomyces cerevisiae and human TATA binding proteins; Jackson-Fisher AJ et al.; A kinetic analysis of dimer dissociation, TATA DNA binding, and thermal inactivation of the yeast Saccharomyces cerevisiae and human TATA binding proteins (TBP) was conducted . We find that yeast TBP dimers, like human TBP dimers, are slow to dissociate in vitro (t(1/2) approximately 20 min) . Mild mutations in the crystallographic dimer interface accelerate the rate of dimer dissociation, whereas severe mutations prevent dimerization . In the presence of excess TATA DNA, which measures the entire active TBP population, dimer dissociation represents the rate-limiting step in DNA binding . These findings provide a biochemical extension to genetic studies demonstrating that TBP dimerization prevents unregulated gene expression in yeast {Jackson-Fisher, A . J., Chitikila, C., Mitra, M., and Pugh, B . F . (1999) Mol . Cell 3, 717-727} . In the presence of vast excesses of TBP over TATA DNA, which measures only a very small fraction of the total TBP, the monomer population in a monomer/dimer equilibrium binds DNA rapidly, which is consistent with a simultaneous binding and bending of the DNA . Under conditions where other studies failed to detect dimers, yeast TBP's DNA binding activity was extremely labile in the absence of TATA DNA, even at temperatures as low as 0 degrees C . Kinetic analyses of TBP instability in the absence of DNA at 30 degrees C revealed that even under fairly stabilizing solution conditions, TBP's DNA binding activity rapidly dissipated with t(1/2) values ranging from 6 to 26 min . TBP's stability appeared to vary with the square root of the TBP concentration, suggesting that TBP dimerization helps prevent TBP inactivation. Proc Natl Acad Sci U S A, 1999 Aug 31, 96(18), 10200 - 5 Altered dosage of the Saccharomyces cerevisiae spindle pole body duplication gene, NDC1, leads to aneuploidy and polyploidy; Chial HJ et al.; Saccharomyces cerevisiae cells are exquisitely sensitive to altered dosage of the spindle pole body duplication gene, NDC1 . We show that the NDC1 locus is haploinsufficient because diploid yeast cells cannot survive with a single chromosomal copy of the NDC1 gene . Diploid cells with a single copy of NDC1 can survive by gaining an extra copy of the NDC1-containing chromosome . NDC1 haploinsufficiency is a dominant loss-of-function phenotype that leads to aneuploidy . Furthermore, we report that overexpression of NDC1 leads to spindle pole body duplication defects indistinguishable from those observed in ndc1-1 mutant cells . Cells overexpressing NDC1 arrest with monopolar spindles and exhibit increase-in-ploidy phenotypes . Thus, both increased and decreased NDC1 dosage can lead to aneuploidy . The striking sensitivity of yeast cells to changes in NDC1 gene dosage suggests a model for the behavior of some tumor suppressor genes and oncogenes in which loss-of-function mutations and overexpression, respectively, lead to increased genetic instability. Proc Natl Acad Sci U S A, 1999 Aug 31, 96(18), 10206 - 11 Evidence for a conserved system for iron metabolism in the mitochondria of Saccharomyces cerevisiae; Schilke B et al.; nifU of nitrogen-fixing bacteria is involved in the synthesis of the Fe-S cluster of nitrogenase . In a synthetic lethal screen with the mitochondrial heat shock protein (HSP)70, SSQ1, we identified a gene of Saccharomyces cerevisiae, NFU1, which encodes a protein with sequence identity to the C-terminal domain of NifU . Two other yeast genes were found to encode proteins related to the N-terminal domain of bacterial NifU . They have been designated ISU1 and ISU2 . Isu1, Isu2, and Nfu1 are located in the mitochondrial matrix . ISU genes of yeast carry out an essential function, because a Deltaisu1Deltaisu2 strain is inviable . Growth of Deltanfu1Delta isu1 cells is significantly compromised, allowing assessment of the physiological roles of Nfu and Isu proteins . Mitochondria from Deltanfu1Deltaisu1 cells have decreased activity of several respiratory enzymes that contain Fe-S clusters . As a result, Deltanfu1Deltaisu1 cells grow poorly on carbon sources requiring respiration . Deltanfu1Deltaisu1 cells also accumulate abnormally high levels of iron in their mitochondria, similar to Deltassq1 cells, indicating a role for these proteins in iron metabolism . We suggest that NFU1 and ISU1 gene products play a role in iron homeostasis, perhaps in assembly, insertion, and/or repair of mitochondrial Fe-S clusters . The conservation of these protein domains in many organisms suggests that this role has been conserved throughout evolution. Cell Stress Chaperones, 1999 Mar, 4(1), 46 - 53 Direct evidence for the intracellular localization of Hsp104 in Saccharomyces cerevisiae by immunoelectron microscopy; Kawai R et al.; To reveal the intracellular localization of Hsp104 in the yeast Saccharomyces cerevisiae before and after heat-shock, we performed immunoelectron microscopy after immunogold labeling with anti-Hsp104 antibody . At normal temperature (25 degrees C), a small amount of Hsp104 was located in the cytoplasm and nucleus . On exposure to mild heat-shock at 40 degrees C, protein aggregates appeared in the cytoplasm and nucleus, and Hsp104 increased around the aggregates with increasing time of the mild heat-shock treatment . Moreover, at lethal heat-shock temperature (51 degrees C) for 20 min after mild heat treatment at 40 degrees C, the intracellular localization of Hsp104 and intracellular structures were similar to those of the mild heat-shocked cells . However, in the lethally heat-shocked cells, certain intracellular structures were destroyed, and Hsp104 was not expressed . In the hsp104 null mutant strain Deltahsp104 which was treated at 40 degrees C, Hsp104 was not localized around the aggregates . Additionally, in the Deltahsp104 strain, even mild heat-shocked cells at 37 degrees C or 40 degrees C, showed destruction of intracellular structure compared to the wild-type strain . Our data suggest the following: (1) Hsp104 is associated closely with protein aggregates during heat-shock treatment, (2) Hsp104 is important for maintenance of the intracellular structure under lethal heat-shock conditions, (3) acquisition of thermotolerance depends on the amount of Hsp104 produced during mild heat-shock treatment . Curr Genet, 1999 Jul, 35(6), 609 - 17 Cysteine uptake by Saccharomyces cerevisiae is accomplished by multiple permeases; During-Olsen L et al.; Uptake by Saccharomyces cerevisiae of the sulphur-containing amino acid L-cysteine was found to be non-saturable under various conditions, and uptake kinetics suggested the existence of two or more transport systems in addition to the general amino-acid permease, Gap1p . Overexpression studies identified BAP2, BAP3, AGP1 and GNP1 as genes encoding transporters of cysteine . Uptake studies with disruption mutants confirmed this, and identified two additional genes for transporters of cysteine, TAT1 and TAT2, both very homologous to BAP2, BAP3, AGP1 and GNP1 . While Gap1p and Agp1p appear to be the main cysteine transporters on the non-repressing nitrogen source proline, Bap2p, Bap3p, Tat1p, Tat2p, Agp1p and Gnp1p are all important for cysteine uptake on ammonium-based medium . Furthermore, whereas Bap2p, Bap3p, Tat1p and Tat2p seem most important under amino acid-rich conditions, Agp1p contributes significantly when only ammonium is present, and Gnp1p only contributes under the latter condition. J Bacteriol, 1999 Sep, 181(17), 5219 - 24 The dual-specificity protein phosphatase Yvh1p acts upstream of the protein kinase mck1p in promoting spore development in Saccharomyces cerevisiae; Beeser AE et al.; Diploid Saccharomyces cerevisiae cells induce YVH1 expression and enter the developmental pathway, leading to sporulation when starved for nitrogen . We show that yvh1 disruption causes a defect in spore maturation; overexpression of MCK1 or IME1 suppresses this yvh1 phenotype . While mck1 mutations are epistatic to those in yvh1 relative to spore maturation, overexpression of MCK1 does not suppress the yvh1 slow-vegetative-growth phenotype . We conclude that (i) Yvh1p functions earlier than Mck1p and Ime1p in the signal transduction cascade that regulates sporulation and is triggered by nitrogen starvation and (ii) the role of Yvh1p in gametogenesis can be genetically distinguished from its role in vegetative growth. Yeast, 1999 Aug, 15(11), 1141 - 9 Designer deletion and prototrophic strains derived from Saccharomyces cerevisiae strain W303-1a; Replogle K et al.; We report the construction of Saccharomyces cerevisiae strains isogenic to W303-1a that are designed to allow efficient genetic analysis . To facilitate the generation of null alleles of target genes by PCR-mediated gene disruption, we constructed designer deletion alleles of the ARG4, TRP1 and URA3 genes . In addition, a single pair of oligonucleotide primers were designed that can be used to amplify any of several marker genes for use in PCR-mediated gene disruption . A new version of the 'reusable' hisG-URA3-hisG cassette was constructed for use in PCR-mediated gene disruption . Finally, to facilitate the formation of isogenic diploids by selection, we constructed strains that contain combinations of wild-type alleles of ADE2, HIS3, LEU2, TRP1 and URA3 . Yeast, 1999 Aug, 15(11), 1111 - 24 Mutations in the Saccharomyces cerevisiae gene SAC1 cause multiple drug sensitivity; Hughes WE et al.; Wild-type yeast Saccharomyces cerevisiae are surprisingly resistant to a wide range of drugs and agents . We had previously isolated novobiocin-sensitive mutants to aid the study of the intracellular target for this drug . Characterization of one of these mutants, mds1, revealed that it was sensitive not only to novobiocin but also to a wide range of drugs . The nature of this multiple drug-sensitive phenotype was shown to be different from that of previously isolated multiple drug-sensitive mutants . We have shown that the multiple drug-sensitivity of mds1 is due to mutations within the gene SAC1 and have identified a variety of mutations within the gene from the Mds1 strain . SAC1 encodes a protein which has been previously implicated in the correct function of the actin cytoskeleton, in inositol metabolism, in ATP transport in the endoplasmic reticulum and in Sec14p (PI-TP) function . We have shown that multiple drug-sensitivity is a new phenotype seen in some, but not all, of the previously characterized sac1 mutants . Based on our findings, we propose a mechanism by which Sac1p could affect drug resistance and also mediate other effects on cell growth . J Biol Chem, 1999 Aug 27, 274(35), 24514 - 21 Alternatives to the isomerase-dependent pathway for the beta-oxidation of oleic acid are dispensable in Saccharomyces cerevisiae . Identification of YOR180c/DCI1 encoding peroxisomal delta(3,5)-delta(2,4)-dienoyl-CoA isomerase; Gurvitz A et al.; Fatty acids with double bonds at odd-numbered positions such as oleic acid can enter beta-oxidation via a pathway relying solely on the auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase, termed the isomerase-dependent pathway . Two novel alternative pathways have recently been postulated to exist in mammals, and these additionally depend on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (di-isomerase-dependent) or on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase (reductase-dependent) . We report the identification of the Saccharomyces cerevisiae oleic acid-inducible DCI1 (YOR180c) gene encoding peroxisomal di-isomerase . Enzyme assays conducted on soluble extracts derived from yeast cells overproducing Dci1p using 3,5,8,11,14-eicosapentenoyl-CoA as substrate demonstrated a specific di-isomerase activity of 6 nmol x min(-1) per mg of protein . Similarly enriched extracts from eci1Delta cells lacking peroxisomal 3,2-isomerase additionally contained an intrinsic 3,2-isomerase activity that could generate 3, 5,8,11,14-eicosapentenoyl-CoA from 2,5,8,11,14-eicosapentenoyl-CoA but not metabolize trans-3-hexenoyl-CoA . Amplification of this intrinsic activity replaced Eci1p since it restored growth of the eci1Delta strain on petroselinic acid for which di-isomerase is not required whereas Eci1p is . Heterologous expression in yeast of rat di-isomerase resulted in a peroxisomal protein that was enzymatically active but did not re-establish growth of the eci1Delta mutant on oleic acid . A strain devoid of Dci1p grew on oleic acid to wild-type levels, whereas one lacking both Eci1p and Dci1p grew as poorly as the eci1Delta mutant . Hence, we reasoned that yeast di-isomerase does not additionally represent a physiological 3,2-isomerase and that Dci1p and the postulated alternative pathways in which it is entrained are dispensable for degrading oleic acid. Nucleic Acids Res, 1999 Aug 15, 27(16), 3259 - 66 Excision repair of nitrogen mustard-DNA adducts in Saccharomyces cerevisiae; McHugh PJ et al.; The bifunctional alkylating anticancer drug nitrogen mustard forms a variety of DNA lesions, including monoadducts and intrastrand and interstrand crosslinks . Although it is known that nucleotide excision repair (NER) is important in processing these adducts, the role of the other principal excision repair pathway, base excision repair (BER) is less well defined . Using isogenic Saccharomyces cerevisiae strains disrupted for a variety of NER and BER genes we have examined the relative importance of the two pathways in the repair of nitrogen mustard adducts . As expected, NER defective cells (rad4 and rad14 strains) are extremely sensitive to the drug . One of the BER mutants, a 3-methyladenine glycosylase defective (mag1) strain also shows significant hypersensitivity . Using a rad4/mag1 double mutant it is shown that the two excision repair pathways are epistatic to each other for nitrogen mustard sensitivity . Furthermore, both rad14 and mag1 disruptants show elevated levels of nitrogen mustard-induced forward mutation . Measurements of repair rates of nitrogen mustard N-alkylpurine adducts in the highly transcribed RPB2 gene demonstrate defects in the processing of mono-adducts in rad4, rad14 and mag1 strains . However, there are differences in the kinetics of adduct removal in the NER mutants compared to the mag1 strain . In the mag1 strain significant repair occurs within 1 h with evidence of enhanced repair on the transcribed strand . Adducts however accumulate at later times in this strain . In contrast, in the NER mutants repair is only evident at times greater than 1 h . In a mag1/rad4 double mutant damage accumulates with no evidence of repair . Comparison of the rates of repair in this gene with those in a different genomic region indicate that the contributions of NER and BER to the repair of nitrogen mustard adducts may not be the same genome wide. Mol Cell Biol, 1999 Sep, 19(9), 6065 - 75 Interactions of TLC1 (which encodes the RNA subunit of telo