Mol Cell Biol, 1995 Jun, 15(6), 3227 - 37 Suppression of mutations in two Saccharomyces cerevisiae genes by the adenovirus E1A protein; Zieler HA et al.; The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107 . To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth . The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid . Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth . A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well . The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly") . Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable . The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned . The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum . The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases . Deletion of either WEB1 or WEB2 is lethal . Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence. Mol Cell Biol, 1995 Jun, 15(6), 3187 - 96 Nutrient availability and the RAS/cyclic AMP pathway both induce expression of ribosomal protein genes in Saccharomyces cerevisiae but by different mechanisms; Neuman-Silberberg FS et al.; By differential hybridization, we identified a number of genes in Saccharomyces cerevisiae that are activated by addition of cyclic AMP (cAMP) to cAMP-depleted cells . A majority, but not all, of these genes encode ribosomal proteins . While expression of these genes is also induced by addition of the appropriate nutrient to cells starved for a nitrogen source or for a sulfur source, the pathway for nutrient activation of ribosomal protein gene transcription is distinct from that of cAMP activation: (i) cAMP-mediated transcriptional activation was blocked by prior addition of an inhibitor of protein synthesis whereas nutrient-mediated activation was not, and (ii) cAMP-mediated induction of expression occurred through transcriptional activation whereas nutrient-mediated induction was predominantly a posttranscriptional response . Transcriptional activation of the ribosomal protein gene RPL16A by cAMP is mediated through a upstream activation sequence element consisting of a pair of RAP1 binding sites and sequences between them, suggesting that RAP1 participates in the cAMP activation process . Since RAP1 protein decays during starvation for cAMP, regulation of ribosomal protein genes under these conditions may directly relate to RAP1 protein availability . These results define additional critical targets of the cAMP-dependent protein kinase, suggest a mechanism to couple ribosome production to the metabolic activity of the cell, and emphasize that nutrient regulation is independent of the RAS/cAMP pathway. Cancer Res, 1995 Jun 1, 55(11), 2299 - 303 DNA topoisomerase II is the molecular target of bisdioxopiperazine derivatives ICRF-159 and ICRF-193 in Saccharomyces cerevisiae; Ishida R et al.; Bisdioxopiperazines such as ICRF-159 and ICRF-193 have been shown to inhibit DNA topoisomerase II . To determine the molecular target of these compounds in vivo, we utilized a yeast genetic system in which the topoisomerase II activity can be modulated . To reduce topoisomerase II activity, we used top2-1 mutant yeast cells that have normal DNA topoisomerase II activity at 25 degrees C but greatly reduced enzyme activity at 30 degrees C, a temperature that is semipermissive for growth . At 25 degrees C top2-1 cells are as sensitive to the ICRF compounds as the wild-type strain; at 30 degrees C the cells became hypersensitive to these agents . In contrast, top2-1 strains become very resistant to the class of topoisomerase II inhibitors such as amsacrine and etoposide, which stabilize the covalent enzyme-DNA intermediate of the enzyme reaction . Overexpression of topoisomerase II from a plasmid-born TOP2 gene results in lower susceptibility to ICRF compounds and higher susceptibility to amsacrine than the parental strain exhibits . These results support the hypothesis that the main cellular target of ICRF compounds is DNA topoisomerase II, and that these compounds, unlike amsacrine and etoposide, inhibit topoisomerase II activity without stabilizing an enzyme-DNA covalent complex. C R Acad Sci III, 1995 Jun, 318(6), 665 - 9 The glyceraldehyde-3-phosphate dehydrogenase binds in vitro to the SH3 domain of Saccharomyces cerevisiae Cdc25p; Buu A et al.; In order to isolate yeast proteins able to bind to the SH3 domain of the Cdc25p exchange factor, a biochemical approach was used . The SH3 (src homolog type 3) domain of yeast Cdc25p, fused both to a tail of 6 histidine (His) and to glutathione-S-transferase (GST), was purified and then, using His affinity for Ni2+ ions, bound to a Ni-NTA column . This column was used for isolating yeast proteins which have affinity for the yeast SH3-Cdc25p domain . The major protein thus isolated, was sequenced and identified as a yeast glyceraldehyde-3-phosphate dehydrogenase (GAP3DH). Microbiology, 1995 Jun, 141 ( Pt 6), 1289 - 99 Actin cortical cytoskeleton and cell wall synthesis in regenerating protoplasts of the Saccharomyces cerevisiae actin mutant DBY 1693; Kopecka M et al.; The relationship between the actin cytoskeleton and cell wall synthesis was studied by light and electron microscopy in protoplasts of Saccharomyces cerevisiae DBY 1693 containing the act1-1 allele . Since protoplasting also disturbs the actin cytoskeleton, these mutant protoplasts had a double error in their actin cytoskeletons . In the period between the onset of wall synthesis and completion of the wall, protoplasts grown at the permissive temperature showed an even distribution of actin patches all over the surface on which a new cell wall was being synthesized . After wall completion, actin patches partially disappeared, but then re-appeared, accumulated in growth regions at the start of polarized growth . This was compared with the pattern of actin patches observed in intact temperature-sensitive actin mutant cells cultivated at the permissive temperature . Electron microscopy of freeze-etched replicas revealed finger-like invaginations of the plasma membrane in both the actin mutant cells and their protoplasts . These structures showed a very similar distribution to the actin patches detected by rhodamine phalloidin staining in the fluorescence microscope . A hypothesis is presented, explaining the role of actin patches/finger-like invaginations of the plasma membrane in the synthesis of beta-(1-->3)-D-glucan wall microfibrils in yeast cells. J Ind Microbiol, 1995 Jun, 14(6), 461 - 6 Flocculation of industrial and laboratory strains of Saccharomyces cerevisiae; Sieiro C et al.; A comparative study has been made of different laboratory and industrial wild-type strains of Saccharomyces cerevisiae in relation to their flocculation behavior . All strains were inhibited by mannose and only one by maltose . In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains . This was to such an extent that it did not allow their inclusion in the Flo1 or NewFlo phenotypes . Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic to FLO1 found in genetic strains. J Bacteriol, 1995 Jun, 177(12), 3534 - 9 Oxidative cell wall damage mediated by bleomycin-Fe(II) in Saccharomyces cerevisiae; Lim ST et al.; Bleomycin mediates cell wall damage in the yeast Saccharomyces cerevisiae . Bleomycin treatments in the presence of Fe(II) increased the rate of spheroplast formation by lytic enzymes by 5- to 40-fold . Neither Fe(III) nor other tested ions caused significant cell wall damage in the presence of bleomycin . The effect of bleomycin-Fe(II) on the cell wall mimicked the characteristics of bleomycin-Fe(II)-mediated DNA damage in dependence on aeration, inhibition by ascorbate, and potentiation by submillimolar concentrations of sodium phosphate . Bleomycin-mediated cell wall damage was time and dose dependent, with incubations as short as 20 min and drug concentrations as low as 3.3 x 10(-7)M causing measurable cell wall damage in strain CM1069-40 . These times and concentrations are within the range of effectiveness for bleomycin-mediated DNA damage and for the cytotoxicity of the drug . Although Fe(III) was inactive with bleomycin and O2, the bleomycin-Fe(III) complex damaged walls and lysed cells in the presence of H2O2 . H2O2 causes similar activation of bleomycin-Fe(III) in assays of DNA scission . These results suggest that an activated bleomycin-Fe-O2 complex disrupts essential cell wall polymers in a manner analogous to bleomycin-mediated cleavage of DNA. Mol Cell Biol, 1995 Jun, 15(6), 3291 - 300 Analysis of the Saccharomyces cerevisiae mitochondrial COX3 mRNA 5' untranslated leader: translational activation and mRNA processing; Wiesenberger G et al.; We used transformation of yeast mitochondria and homologous gene replacement to study features of the 613-base COX3 mRNA 5' untranslated leader (5'-UTL) required for translational activation by the protein products of the nuclear genes PET54, PET122, and PET494 in vivo . Elimination of the single AUG triplet in the 5'-UTL had no detectable effect on expression, indicating that activator proteins do not work by allowing ribosomes to bypass that AUG . Deletion of the entire 5'-UTL completely prevented translation, suggesting that the activator proteins do not function by antagonizing any other negative element in the 5'-UTL . Removal of the 15 terminal bases from the 5' end of the 5'-UTL did not block activator-dependent translation . The largest internal deletion that did not interfere with translation removed 125 bases from the upstream portion of the leader . However, two large deletions that blocked translation could be reverted to activator-dependent expression by secondary changes in the remaining 5'-UTL sequences, indicating that the original deletions had not removed the translational activator target but only deformed it . Taken together, the deletion mutations and revertants define a region of 151 bases (between positions -480 and -330 relative to the start codon) containing sequences that are sufficient for translational activation when modified slightly . Suppression of the respiratory phenotypes of two 5'-UTL mutations by overexpression of PET54, PET122, and PET494 indicated functional interactions between the leader and the three activator proteins . The mature COX3 mRNA is cleaved from a precursor immediately downstream of the preceding tRNAVal in a fashion resembling mRNA processing in vertebrate mitochondria . Our results indicate that the site of this cleavage in Saccharomyces cerevisiae is determined solely by the position of the tRNA. Genetics, 1995 Jun, 140(2), 443 - 56 Specificities of the Saccharomyces cerevisiae rad6, rad18, and rad52 mutators exhibit different degrees of dependence on the REV3 gene product, a putative nonessential DNA polymerase; Roche H et al.; The Saccharomyces cerevisiae rad6, rad18, and rad52 mutants exhibit DNA repair deficiencies and distinct mutator phenotypes . DNA replication past unrepaired spontaneous damage might contribute to the specificities of these mutators . Because REV3 is thought to encode a DNA polymerase that specializes in translesion synthesis, we determined the REV3 dependence of the rad mutator specificities . Spontaneous mutagenesis at a plasmid-borne SUP4-o locus was examined in isogenic strains having combinations of normal or mutant REV3 and RAD6, RAD18, or RAD52 alleles . For the rad6 and rad18 mutators, the mutation rate increase relied largely, but not exclusively, on REV3 whereas the rad52 mutator was entirely REV3 dependent . The influence of REV3 on the specificity of the rad6 mutator differed markedly depending on the mutational class examined . However, the requirement of rev3 for the production of G.C-->T.A transversions by the rad18 mutator, which induces only these substitutions, was similar to that for rad6-mediated G.C-->T.A transversion . This supports a role for the Rad6-Rad18 protein complex in the control of spontaneous mutagenesis . The available data imply that the putative Rev3 polymerase can process a variety of spontaneous DNA lesions that normally are substrates for error-free repair. FEBS Lett, 1995 May 29, 365(2-3), 198 - 202 PTF1 encodes an essential protein in Saccharomyces cerevisiae, which shows strong homology with a new putative family of PPIases; Hani J et al.; Complementation of a temperature sensitive mutant of the yeast Saccharomyces cerevisiae resulted in the isolation of PTF1 (processing/termination factor 1), an essential gene encoding a putative 3'-end processing or transcription termination factor of pre-mRNAs . Ptf1p shows significant homology to a newly discovered family of PPIases . This family is characterized by its insensitivity to immunosuppressive drugs and the lack of homology with cyclophilins and FK-506 binding proteins {Rahfeld et al . (1994) FEBS Lett . 352, 180-184} . Should Ptf1p display PPIase activity, it would be the first characterized, eukaryotic member of this putative family, which is essential for growth. Ned Tijdschr Geneeskd, 1995 May 27, 139(21), 1093 - 5 {Vaginal infection caused by Saccharomyces cerevisiae}; van Doorn HC et al.; In a woman of 26, who suffered from a vulvovaginal infection and had previously been treated for Candida vaginitis, Saccharomyces cerevisiae was cultured and identified . At her work she sold baking yeast . Topical treatment with amphotericin B 100 mg suppositories was successful . Microscopic examination (1000 x) of the discharge in saline showed haloed yeast cells . For treatment, oral ketoconazole or topical administration of amphotericin B or clotrimazole, in relatively high doses, may be applied . This yeast might be the cause of 'chronic candidiasis' more often than suspected, notably in women working in a bakery or a brewery. Gene, 1995 May 26, 158(1), 113 - 7 Gene disruption with PCR products in Saccharomyces cerevisiae; Lorenz MC et al.; We describe here the generation of gene disruption constructs using PCR amplification of selectable markers with primers that provide homology to the target gene of interest . We find that regions of homology as short as 38 to 50 bp suffice to mediate homologous recombination in yeast . We describe applications of this technology to three specific yeast genes that would have been difficult to disrupt with current methods . By dispensing with the need to either clone the gene of interest or engineer a standard disruption construct, this method should facilitate analysis of sequenced genes of unknown function, which will soon include the entire yeast genome. J Biol Chem, 1995 May 26, 270(21), 12717 - 24 Casein kinase I gamma subfamily . Molecular cloning, expression, and characterization of three mammalian isoforms and complementation of defects in the Saccharomyces cerevisiae YCK genes; Zhai L et al.; Casein kinase I, one of the first protein kinases identified biochemically, is known to exist in multiple isoforms in mammals . Using a partial cDNA fragment corresponding to an isoform termed CK1 gamma, three full-length rat testis cDNAs were cloned that defined three separate members of this subfamily . The isoforms, designated CK1 gamma 1, CK1 gamma 2, and CK1 gamma 3, have predicted molecular masses of 43,000, 45,500, and 49,700 . CK1 gamma 3 may also exist in an alternatively spliced form . The proteins are more than 90% identical to each other within the protein kinase domain but only 51-59% identical to other casein kinase I isoforms within this region . Messages for CK1 gamma 1 (2 kilobases (kb)), CK1 gamma 2 (1.5 and 2.4 kb), and CK1 gamma 3 (2.8 kb) were detected by Northern hybridization of testis RNA . Message for CK1 gamma 3 was also observed in brain, heart, kidney, lung, liver, and muscle whereas CK1 gamma 1 and CK1 gamma 2 messages were restricted to testis . All three CK1 gamma isoforms were expressed as active enzymes in Escherichia coli and partially purified . The enzymes phosphorylated typical in vitro casein kinase I substrates such as casein, phosvitin, and a synthetic peptide, D4 . Phosphorylation of the D4 peptide was activated by heparin whereas phosphorylation of the protein substrates was inhibited . The known casein kinase I inhibitor CK1-7 also inhibited the CK1 gamma s although less effectively than the CK1 alpha or CK1 delta isoforms . All three CK1 gamma s underwent autophosphorylation when incubated with ATP and Mg2+ . The YCK1 and YCK2 genes in Saccharomyces cerevisiae encode casein kinase I homologs, defects in which lead to aberrant morphology and growth arrest . Expression of mammalian CK1 gamma 1 or CK1 gamma 3 restored growth and normal morphology to a yeast mutant carrying a disruption of YCK1 and a temperature-sensitive allele of YCK2, suggesting overlap of function between the yeast Yck proteins and these CK1 isoforms. Nucleic Acids Res, 1995 May 25, 23(10), 1679 - 85 Regulation of the Saccharomyces cerevisiae DNA repair gene RAD16; Bang DD et al.; The RAD16 gene product has been shown to be essential for the repair of the silenced mating type loci {Bang et al . (1992) Nucleic Acids Res . 20, 3925-3931} . More recently we demonstrated that the RAD16 and RAD7 proteins are also required for repair of non-transcribed strands of active genes in Saccharomyces cerevisiae {Waters et al . (1993) Mol . Gen . Genet . 239, 28-32} . We have studied the regulation of the RAD16 gene and found that the RAD16 transcript levels increased up to 7-fold upon UV irradiation . Heat shock at 42 degrees C also results in elevated levels of RAD16 mRNA . In sporulating MAT alpha/MATa diploid cells RAD16 mRNA is also induced . The basal level of the RAD16 transcript is constant during the mitotic cell cycle . G1-arrested cells show normal induction of RAD16 mRNA upon UV irradiation demonstrating that the induction is not a secondary consequence of G2 cell cycle arrest following UV irradiation . However, in cells arrested in G1 the induction of RAD16 mRNA after UV irradiation is not followed by a rapid decline as occurs in normal growing cells suggesting that the down regulation of RAD16 transcription is dependent on progression into the cell cycle. Mol Gen Genet, 1995 May 20, 247(4), 409 - 15 Genetics of a-agglutunin function in Saccharomyces cerevisiae; de Nobel H et al.; The Saccharomyces cerevisiae cell adhesion protein a-agglutinin is composed of an anchorage subunit (Aga1p) and an adhesion subunit (Aga2p) . Although functional a-agglutinin is expressed only by a cells, previous results indicated that AGA1 RNA is expressed in both a and alpha cells after pheromone induction . Expression of the Aga2p adhesion subunit in alpha cells allowed a-agglutinability, indicating that alpha cells express the a-agglutinin anchorage subunit, although no role for Aga1p in alpha cells has been identified . Most of the a-specific agglutination-defective mutants isolated previously were defective in AGA1; a single mutant (La199) was a candidate for an aga2 mutant . Expression of AGA2 under PGK control allowed secretion of active Aga2p from control strains but did not complement the La199 agglutination defect or allow secretion of Aga2p from La199, suggesting that the La199 mutation might identify a new gene required for a-agglutinin function . However, the La199 agglutination defect showed tight linkage to aga2::URA3 and did not complement aga2::URA3 in a/a diploids . The aga2 gene cloned from La199 was nonfunctional and contained an ochre mutation . The inability of pPGK-AGA2 to express functional Aga2p in La199 was shown to result from an additional mutation(s) that reduces expression of plasmid-borne genes . AGA2 was mapped to the left arm of chromosome VII approximately 28 cM from the centromere. FEMS Microbiol Lett, 1995 May 15, 128(3), 279 - 82 Lack of lactate-proton symport activity in pck1 mutants of Saccharomyces cerevisiae; Casal M et al.; Mutants of Saccharomyces cerevisiae without phosphoenolpyruvate carboxykinase activity showed no measurable lactate proton symport, while mutants without fructose-1,6-bisphosphatase had normal transport activity . Incubation of a pck1 mutant, under derepression conditions in the presence of glycerol, restored the activity of the lactate-proton symport, with identical kinetic characteristics to that in the wild-type . For efficient lactate-proton symport activity, not only is an external inducer such as lactic acid needed, but also a molecule derived from the acid metabolism may be necessary. J Biol Chem, 1995 May 12, 270(19), 11549 - 54 Isolation and characterization of the small nucleolar ribonucleoprotein particle snR30 from Saccharomyces cerevisiae; Lubben B et al.; The nucleolus of the yeast Saccharomyces cerevisiae contains the small nucleolar RNA snR30 (snoRNA), that is found associated with at least two proteins, NOP1 and GAR1 . All three of these molecules are essential for the cell's viability and have been implicated in pre-rRNA maturation . NOP1 and GAR1 are believed to be general rRNA-processing factors or, alternatively, integral protein components of the small nucleolar ribonucleoprotein particle snR30 (snoRNP) . In this paper, we describe procedures for the biochemical isolation of snR30 RNP, and we identify seven snR30 RNP proteins of molecular masses of 10, 23, 25, 38, 46, 48, and 65 kDa, including the previously reported GAR1 protein . Additional proteins, including NOP1, may also be components of snR30 RNP but are lost during our stringent isolation procedure . The 10-, 23-, and 25-kDa (GAR1) and 65-kDa proteins remain tightly associated with the snR30 RNA even after isopycnic sedimentation in cesium sulfate gradients . Electron microscopy of Mono Q-purified snR30 RNPs show a slightly elongated two-domain structure approximately 20 nm long and 14 nm wide. J Biol Chem, 1995 May 12, 270(19), 11415 - 23 The effect of the S14A mutation on the conformation and thermostability of Saccharomyces cerevisiae G-actin and its interaction with adenine nucleotides; Chen X et al.; The actin Ser14 hydroxyl is one of a number of ligands that binds to the gamma-phosphate of ATP thereby stabilizing the actin.ATP complex . In yeast actin, conversion of Ser14 to Ala (S14A), causes a temperature-sensitive phenotype in vivo and temperature-sensitive polymerization defects in vitro (Chen, X., and Rubenstein, P . A . (1995) J . Biol . Chem . 270, 11406-11414) . Here, using a new luciferase-based procedure, we show that the mutation results in a 40-60-fold decrease in actin's affinity for ATP . The mutation causes a decrease in the intrinsic ATPase activity of both Ca- and Mg-G-actin at 30 degrees C and alters the protease susceptibility of sites on subdomain 2 . Ca-S14A-actin but not Mg-S14A-actin binds etheno-ATP at 37 degrees C . Intrinsic tryptophan fluorescence measurements show that at 37 degrees C, Mg-S14A-actin but not the calcium form unfolds . CD measurements show the mutation causes a decrease in the apparent denaturation temperature for Ca-actin from 57 to 45 degrees C and for the magnesium form a decrease from 52 to 40 degrees C . Based on a re-examination of actin's crystal structure coordinates, we propose that the Ser14 hydroxyl forms a polar bridge between the ATP gamma-phosphate and the amide nitrogen of Gly74, thus conferring additional stability on the actin small domain. J Biol Chem, 1995 May 12, 270(19), 11406 - 14 A mutation in an ATP-binding loop of Saccharomyces cerevisiae actin (S14A) causes a temperature-sensitive phenotype in vivo and in vitro; Chen X et al.; The Ser14 hydroxyl group of actin is one of six groups that potentially form hydrogen bonds with the gamma-phosphate of the ATP bound in the cleft separating the two domains of the protein . To understand the importance of this group in actin function, we mutated Ser14 of Saccharomyces cerevisiae actin and studied the effects of these mutations in vivo and in vitro . Substitution of Cys of Gly resulted in cell death . Substitution of Thr for Ser resulted in an actin with wild-type properties in vivo and in vitro . Cells carrying the Ser14-->Ala (S14A) mutation were viable but displayed a temperature sensitive lethality at 37 degrees C preceded by delocalization of actin patches, the appearance of bar-like structures, and finally the disappearance of identifiable actin structures . The mutation caused no effect on the critical concentration of polymerization but resulted in an actin with an increased rate of polymerization, an altered protease susceptibility, and a decreased filament ATPase activity . At 37 degrees C, Mg-, but not Ca-S14A-actin irreversibly lost the ability to polymerize . These results demonstrate the importance of the ATP-Ser14 hydroxyl hydrogen bond in regulating actin function in vivo and in vitro and the magnification of the effects of the mutation when Mg2+ is substituted for Ca2+ in the protein. Nucleic Acids Res, 1995 May 11, 23(9), 1614 - 20 8-Methoxypsoralen photoinduced plasmid-chromosome recombination in Saccharomyces cerevisiae using a centromeric vector; Meira LB et al.; The characterization of a new system to study the induction of plasmid-chromosome recombination is described . Single-stranded and double-stranded centromeric vectors bearing 8-methoxypsoralen photoinduced lesions were used to transform a wild-type yeast strain bearing the leu2-3,112 marker . Using the SSCP methodology and DNA sequencing, it was demonstrated that repair of the lesions in plasmid DNA was mainly due to conversion of the chromosomal allele to the plasmid DNA. Nucleic Acids Res, 1995 May 11, 23(9), 1454 - 60 Protein-DNA interactions in soluble telosomes from Saccharomyces cerevisiae; Wright JH et al.; Telomeric DNA in Saccharomyces is organized into a non-nucleosomal chromatin structure called the telosome that can be released from chromosome ends in soluble form by nuclease digestion (Wright, J . H., Gottschling, D . E . and Zakian, V . A . (1992) Genes Dev . 6, 197-210) . The protein-DNA interactions of soluble telosomes were investigated by monitoring isolated telomeric DNA fragments for the retention of bound protein using both gel mobility shift and nitrocellulose filter-binding assays . Telosomal proteins remained associated with telomeric DNA at concentrations of ethidium bromide that dissociated nucleosomes . The protein-DNA interactions in the yeast telosome were also disrupted by much lower salt concentrations than those known to disrupt either the interactions of ciliate terminus-binding proteins with telomeric DNA or the interactions of histones with DNA in nucleosomes . Taken together, these data corroborate previously published nuclease mapping data indicating that telosomes are distinct in structure from conventional nucleosomes . These data also indicate that yeast do not possess telomere binding proteins similar to those detected in ciliates that remain tightly bound to telomeric DNA even in high salt . In addition, the characteristic gel mobility shift of soluble telosomes could be mimicked by complexes formed in vitro with yeast telomeric DNA and recombinant Rap1p suggesting that Rap1p, a known component of soluble yeast telosomes (Wright, J . H., Gottschling, D . E . and Zakian, V . A . (1992) Genes Dev . 6, 197-210; Conrad, M . N., Wright, J . H., Wolf, A . J . and Zakian, V . A . (1990) Cell 63, 739-750), is likely to be the major structural protein bound directly to yeast telomeric DNA. Mol Gen Genet, 1995 May 10, 247(3), 265 - 74 Structure-function analysis of small G proteins from Volvox and Chlamydomonas by complementation of Saccharomyces cerevisiae YPT/SEC mutations; Fabry S et al.; cDNAs representing nine small G protein genes encoding Ypt proteins from the green algae Volvox carteri (YptV) and Chlamydomonas reinhardtii (YptC) were tested for their ability to complement mutations in the YPT1, SEC4, and YPT7 genes of Saccharomyces cerevisiae strains defective in different steps of intracellular vesicle transport . None of the heterologously expressed algal genes was able to complement mutations in SEC4 or YPT7, but three of them, yptV1, yptC1, and yptV2, restored a YPT1 null mutation . On the amino acid sequence level, and particularly with respect to known small G protein specificity domains, YptV1p and YptC1p are the closest algal analogs of yeast Ypt1p, with 70% overall identity and identical effector regions, but YptV2p is only 55% identical to Ypt1p, and its effector domain resembles that of Sec4p . To define more precisely the regions that supply Ypt1p function, six chimeras were constructed by reciprocal exchange of 68/72-, 122/123-, and 162/163-amino acid segments of the C-terminal regions between YptV1p (complementing) and YptV3p (non-complementing) . Segments containing 68 amino acids of the hypervariable C-terminal, and 41 residues of the N-terminal region including the effector region, of YptV1p could be replaced by the corresponding parts of YptV3p without loss of function in yeast, but exchanges within the central core destroyed the ability to rescue the YPT1 mutation . Sequence analysis of ypt1-complementing and -noncomplementing Ypt types suggests that surface loop3 represents a novel specificity domain of small G proteins. J Mol Biol, 1995 May 5, 248(3), 639 - 52 Allosteric regulation of carbamoylphosphate synthetase-aspartate transcarbamylase multifunctional protein of Saccharomyces cerevisiae: selection, mapping and identification of missense mutations define three regions involved in feedback inhibition by UTP; Jaquet L et al.; The positive screening procedure previously described was used in order to select, clone and characterize mutants defective in negative feedback control by UTP of the yeast carbamoylphosphate synthetase-aspartate transcarbamylase protein (CPSase-ATCase) . The selection procedure was improved by adding a general mapping method for dominant mutations in order to avoid sequencing the whole URA2 allele (7 kb) . All 16 mutants obtained carry missense mutations leading to single amino acid replacements: five of them are located in the CPSase domain while the other 11 are in the ATCase domain . In these 16 mutants, ATCase is no longer inhibited by UTP although CPSase retains full sensitivity to the effector, suggesting that the regulation of the two activities involve distinct mechanisms . Amino acid replacements in the ATCase domain were located on a three-dimensional model structure of the yeast ATCase domain . They are clustered in two regions of this domain which must be directly involved in the feedback process. Eur J Biochem, 1995 May 1, 229(3), 651 - 7 Expression of the AAC2 gene encoding the major mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is controlled at the transcriptional level by oxygen, heme and HAP2 factor; Betina S et al.; Expression of the Saccharomyces cerevisiae AAC2 gene encoding the major mitochondrial ADP/ATP carrier was examined . The intracellular level of the carrier protein, as well as the level of the AAC2-gene-specific mRNA, is influenced by the presence or absence of oxygen or of heme, and it is subject to carbon-source control . In addition, the expression of AAC2 gene requires the products of the HAP2 and HAP3 genes, but not that of the HAP1 gene . The 5'-flanking region of the gene was isolated, sequenced and fused to the lacZ reporter gene in order to study the effect of carbon sources and of specific deletion mutations on expression of the gene in yeast transformants . The expression of the reporter gene reveals that the AAC2 gene possesses a strong inducible promoter . The promoter analysis, combined with expression studies in the wild-type as well as in various mutant strains, identified an upstream activation site (UAS) contained within a sequence between -393 bp and -268 bp, and several major initiation sites of AAC2 mRNA between -105bp and -95 bp . Deletion analysis also shows that the TATA boxes located 45 bp and 104 bp upstream of the 5'-ends of AAC2 mRNA are not essential for the transcription . The UAS of the AAC2 gene is required for activation by HAP2 and heme and for release from glucose repressin . A restriction fragment containing the UAS conferred oxygen and carbon source regulation when placed upstream of another yeast gene encoding ADP/ATP carrier (AAC3), deleted of its regulatory sequences . The UAS of the AAC2 gene contains at least two distinct motifs for DNA-binding transcriptional activators, including one which is identical with the core HAP2/3/4 binding motif, and a second one with the ABF1 consensus binding sequence . Our results indicate that these sequences mediate the effects of the respective transactivator on the oxygen- and carbon-source-dependent transcription of the AAC2 gene. FEBS Lett, 1995 May 1, 364(1), 91 - 7 A C-terminal domain, which prevents secretion of the neuroendocrine protein 7B2 in Saccharomyces cerevisiae, inhibits Kex2 yet is processed by the Yap3 protease; Chaudhuri B et al.; Recent reports reveal that the C-terminal half of the neuroendocrine polypeptide 7B2 selectively inhibits and binds PC2, a mammalian prohormone converting enzyme that is homologous to the yeast pro-alpha-factor processing protease Kex2 . During attempted secretion of the 185 amino-acid human 7B2 in Saccharomyces cerevisiae, we observe that the protein is mostly retained inside the cell . However a mutant polypeptide (7B2 delta 1), where the C-terminal 48 amino acids of 7B2 are deleted, is efficiently secreted . Two shorter C-terminal truncations either permit poor secretion or no secretion at all . Surprisingly, full-length 7B2 but not 7B2 delta 1 abolishes the catalytic activity of Kex2, indicating that C-terminal residues of 7B2 might also be important for inhibition of the yeast protease . When the KEX2 gene is disrupted, yeast cells unexpectedly secrete a 7B2 variant similar in size to 7B2 delta 1, suggesting involvement of the alternate yeast prohormone convertase Yap3 in processing . Secretion is enhanced by overexpression of Yap3 and by the presence of a Lys-Arg residue at the processing site of precursor 7B2 . These results purport that, in neuroendocrine cells too, secretion of 7B2 could be mediated by a homologue of Yap3. DNA Cell Biol, 1995 May, 14(5), 411 - 8 An essential gene pair in Saccharomyces cerevisiae with a potential role in mating; Yu Y et al.; In the yeast Saccharomyces cerevisiae, the signal generated by extracellular pheromone is transmitted through the beta and gamma subunits of a trimeric G-protein to downstream signaling molecules that mediate the cellular responses associated with mating . To isolate potential downstream signaling components, a yeast genomic library on a multicopy plasmid was screened for genes that increased the mating efficiency of a strain containing a temperature-sensitive G beta subunit mutation . Overexpression of STE5, STE18 (which encodes the G gamma subunit), and a previously unidentified gene, termed SSF1, partially suppressed the mating defect of a G beta mutant . Hybridization of yeast genomic DNA with an SSF1 probe revealed a closely related homolog, termed SSF2, which was isolated and also found to test positively in the assay for suppression . Null mutations in either SSF1 or SSF2 had no obvious phenotype, but disruption of both genes was lethal . Depletion of SSF gene products from growing cultures caused both an arrest of cell division and a significant decrease in the ability of cells to mate . Because mating efficiency was increased by extra copies of the SSF genes and decreased by elimination of the gene products, it is likely that these genes play a role in mating as well as in an essential function. Mol Cell Biol, 1995 May, 15(5), 2719 - 27 Synthetic lethality of sep1 (xrn1) ski2 and sep1 (xrn1) ski3 mutants of Saccharomyces cerevisiae is independent of killer virus and suggests a general role for these genes in translation control; Johnson AW et al.; Strand exchange protein 1 (Sep1) (also referred to as exoribonuclease I {Xrn1}) from Saccharomyces cerevisiae has been implicated in DNA recombination, RNA turnover, karyogamy, and G4 DNA pairing among other disparate cellular processes . Using a genetic approach to study the role of SEP1/XRN1 in mitotic yeast cells, we identified mutations in the genes superkiller 2 (SKI2) and superkiller 3 (SKI3) as synthetically lethal with an sep1 null mutation . The SKI genes are thought to comprise an intracellular antiviral system controlling the expression of killer toxin from double-stranded RNA virus found in many yeast strains . However, the lethality of sep1 ski2 and sep1 ski3 mutants was independent of the L-A and M viruses, suggesting that the SKI genes act in a general cellular process in addition to virus control . We propose that Sep1/Xrn1 and Ski2 both act to block translation on transcripts targeted for degradation . Using a temperature-sensitive allele of SEP1/XRN1, we show that double mutants display a synthetic cell cycle arrest in late G1 at Start. Mol Cell Biol, 1995 May, 15(5), 2509 - 16 FAR1 and the G1 phase specificity of cell cycle arrest by mating factor in Saccharomyces cerevisiae; McKinney JD et al.; Significant accumulation of Far1p is restricted to the G1 phase of the Saccharomyces cerevisiae cell cycle . Here we demonstrate yeast cell cycle regulation of Far1p proteolysis . Deletions within the 50 N-terminal amino acids of Far1p increase stability and reduce cell cycle regulation of Far1p abundance . Whereas wild-type Far1p specifically and exclusively promotes G1 phase arrest in response to mating factor, stabilized Far1p promoted arrest both during and after G1 . The loss of the G1 specificity of Far1p action requires elimination of FAR1 transcriptional regulation (by means of the GAL1 promoter) as well as N-terminal truncation . Thus, the cell cycle specificity of mating factor arrest may be largely due to cell cycle regulation of FAR1 transcription and protein stability. Mol Cell Biol, 1995 May, 15(5), 2466 - 73 A complex unidirectional signal element mediates GCN4 mRNA 3' end formation in Saccharomyces cerevisiae; Egli CM et al.; The yeast GCN4 3' element represents a class of polyadenylation sites which function unidirectionally and efficiently in test systems in vivo as well as in vitro . A complex signal element is required for polyadenylation activity with a minimal size of 116 nucleotides for the functional element . We subdivided this element into five regions (EL1 to EL5) of 16 to 26 nucleotides each . Each region was characterized by deletion analysis in an in vivo test system . Two TTTTTAT motifs are located in different regions (EL1 and EL4) upstream of the poly(A) site . The 3' end processing activity was significantly reduced when both motifs were mutated by site-directed mutagenesis and abolished when EL1 and EL4 were deleted . The major poly(A) site is located in EL5, 3 nucleotides downstream of the second TTTTTAT motif . Additional minor poly(A) sites are used in less than 10% of the mRNA 3' ends . Deletion of EL3 resulted in a changed pattern of mRNA 3' ends by increased usage of the minor poly(A) addition sites . The major poly(A) site in EL5 can be removed without loss of function when sequences upstream of EL1 are present . The tripartite TAG...TATGT...TTT sequence located downstream of EL5 is not required for function. Mol Cell Biol, 1995 May, 15(5), 2420 - 8 A polymerase switch in the synthesis of rRNA in Saccharomyces cerevisiae; Conrad-Webb H et al.; Transcription of ribosomal DNA by RNA polymerase I is believed to be the sole source of the 25S, 18S, and 5.8S rRNAs in wild-type cells of Saccharomyces cerevisiae . Here we present evidence for a switch from RNA polymerase I to RNA polymerase II in the synthesis of a substantial fraction of those rRNAs in respiratory-deficient (petite) cells . The templates for the RNA polymerase II transcripts are largely, if not exclusively, episomal copies of ribosomal DNA arising from homologous recombination events within the ribosomal DNA repeat on chromosome XII . Ribosomal DNA contains a cryptic RNA polymerase II promoter that is activated in petites; it overlaps the RNA polymerase I promoter and produces a transcript equivalent to the 35S precursor rRNA made by RNA polymerase I . Yeast cells that lack RNA polymerase I activity, because of a disruption of the RPA135 gene that encodes subunit II of the enzyme, can survive by using the RNA polymerase II promoter in ribosomal DNA to direct the synthesis of the 35S rRNA precursor . This polymerase switch could provide cells with a mechanism to synthesize rRNA independent of the controls of RNA polymerase I transcription. J Cell Biol, 1995 May, 129(3), 751 - 65 Patterns of bud-site selection in the yeast Saccharomyces cerevisiae; Chant J et al.; Cells of the yeast Saccharomyces cerevisiae select bud sites in either of two distinct spatial patterns, known as axial (expressed by a and alpha cells) and bipolar (expressed by a/alpha cells) . Fluorescence, time-lapse, and scanning electron microscopy have been used to obtain more precise descriptions of these patterns . From these descriptions, we conclude that in the axial pattern, the new bud forms directly adjacent to the division site in daughter cells and directly adjacent to the immediately preceding division site (bud site) in mother cells, with little influence from earlier sites . Thus, the division site appears to be marked by a spatial signal(s) that specifies the location of the new bud site and is transient in that it only lasts from one budding event to the next . Consistent with this conclusion, starvation and refeeding of axially budding cells results in the formation of new buds at nonaxial sites . In contrast, in bipolar budding cells, both poles are specified persistently as potential bud sites, as shown by the observations that a pole remains competent for budding even after several generations of nonuse and that the poles continue to be used for budding after starvation and refeeding . It appears that the specification of the two poles as potential bud sites occurs before a daughter cell forms its first bud, as a daughter can form this bud near either pole . However, there is a bias towards use of the pole distal to the division site . The strength of this bias varies from strain to strain, is affected by growth conditions, and diminishes in successive cell cycles . The first bud that forms near the distal pole appears to form at the very tip of the cell, whereas the first bud that forms near the pole proximal to the original division site (as marked by the birth scar) is generally somewhat offset from the tip and adjacent to (or overlapping) the birth scar . Subsequent buds can form near either pole and appear almost always to be adjacent either to the birth scar or to a previous bud site . These observations suggest that the distal tip of the cell and each division site carry persistent signals that can direct the selection of a bud site in any subsequent cell cycle. J Cell Biol, 1995 May, 129(3), 725 - 37 The Drosophila cell cycle gene fizzy is required for normal degradation of cyclins A and B during mitosis and has homology to the CDC20 gene of Saccharomyces cerevisiae; Dawson IA et al.; The Drosophila cell cycle gene fizzy (fzy) is required for normal execution of the metaphase-anaphase transition . We have cloned fzy, and confirmed this by P-element mediated germline transformation rescue . Sequence analysis predicts that fzy encodes a protein of 526 amino acids, the carboxy half of which has significant homology to the Saccharomyces cerevisiae cell cycle gene CDC20 . A monoclonal antibody against fzy detects a single protein of the expected size, 59 kD, in embryonic extracts . In early embryos fzy is expressed in all proliferating tissues; in late embryos fzy expression declines in a tissue-specific manner correlated with cessation of cell division . During interphase fzy protein is present in the cytoplasm; while in mitosis fzy becomes ubiquitously distributed throughout the cell except for the area occupied by the chromosomes . The metaphase arrest phenotype caused by fzy mutations is associated with failure to degrade both mitotic cyclins A and B, and an enrichment of spindle microtubules at the expense of astral microtubules . Our data suggest that fzy function is required for normal cell cycle-regulated proteolysis that is necessary for successful progress through mitosis. Bone Marrow Transplant, 1995 May, 15(5), 785 - 6 Saccharomyces cerevisiae fungemia with granulomas in the bone marrow in a patient undergoing BMT; Cairoli R et al.; A 48-year-old woman underwent allogeneic BMT for CML in chronic phase . One day +180 she experienced fever (37.8 degrees C) and skin rash . Blood cultures from the Hickman catheter and peripheral veins were positive for Saccharomyces cerevisiae . The clinical course of this patient indicates that Saccharomyces should be considered as a possible cause of fever of otherwise unknown origin. Anal Biochem, 1995 May 1, 227(1), 32 - 9 Pulsed field gel electrophoresis labeling method to study the pattern of Saccharomyces cerevisiae chromosomal DNA synthesis during the G1/S phase of the cell cycle; Jong AY et al.; Yeast Saccharomyces cerevisiae is an excellent model to study eukaryotic DNA replication . Since yeast lacks thymidine kinase, it is difficult to assay DNA synthesis . In this report, a novel approach called the pulsed field gel electrophoresis (PFGE) labeling method is used to investigate yeast chromosomal DNA synthesis . In this method, yeast cells are first labeled by 32P in vivo and chromosomal DNA molecules are then resolved by pulsed field gel electrophoresis . A linear 32P labeling of chromosomal-size DNA molecules can be observed up to 100 min in an asynchronized culture . In an alpha-factor arresting-and-releasing synchronized culture, we observed that 32P can be rapidly taken up in the S phase . Our results show that all of the chromosomes are labeled at approximately the same time (within a 15-min interval), suggesting that the temporal order of all chromosomal DNA synthesis is synchronized in the S phase . Cell cycle blockers were used to arrest the yeast cultures to study DNA synthesis that coincides with cell cycle analysis . Our results show that alpha-factor and hydroxyurea block the cell cycle at the late G1 and S phases, respectively, resulting in the failure of DNA synthesis . Nocodazole blocks the cell cycle after the S phase and thus shows no effect on chromosomal DNA synthesis . Since the PFGE labeling method is highly specific to chromosomal DNA synthesis, we used it to examine the DNA synthesis pattern in various cell cycle mutants . All of the G1/S cdc mutants tested, cdc4, 6, 7, 8, 17, 2, and 9, cannot label the chromosomal size of DNA molecules; but the G2/M cdc mutant cdc13 can.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Mol Biol Int, 1995 May, 36(1), 137 - 44 Segment selection during 'phase variation'-type regulation of gene replacement mediated by FLP recombinase in the yeast Saccharomyces cerevisiae; Fessing MY et al.; FLP recombinase has recently been used as a tool to direct the exchange between invertible DNA segments, called 'Phase variation'-type regulation of gene replacement in eukaryotic cells . Using an appropriate selective medium, positive segment selection was shown to be efficient during the regulation of gene replacement . The efficiency was determined from the copy number ratio of invertible segments with the use of the neomycinphosphotransferase II (NPTII) gene bearing invertible segments located on the episomal yeast plasmid, and the resident 2-microns circle . Without the selection the segments copy number ratio was retained in growing cells . The results obtained are an evidence for the efficiency of positive segment selection during the 'Phase variation'-type regulation of gene replacement in eukaryotic cells. Appl Environ Microbiol, 1995 May, 61(5), 2043 - 5 Xylitol formation and reduction equivalent generation during anaerobic xylose conversion with glucose as cosubstrate in recombinant Saccharomyces cerevisiae expressing the xyl1 gene; Thestrup HN et al.; Glucose was used as a cosubstrate under anaerobic conditions in the conversion of xylose to xylitol by a recombinant Saccharomyces cerevisiae strain expressing the xyl1 gene . Glucose was metabolized mainly through glycolysis, with carbon dioxide, acetate, and ethanol as end products and with reduction equivalents generated in the glyceraldehyde-3-phosphate dehydrogenase and acetaldehyde dehydrogenase reactions . At a high glucose supply rate, generation of surplus reduction equivalents resulted in simultaneous ethanol formation . On the other hand, at a low glucose supply rate, additional reduction equivalents were generated by simultaneous ethanol consumption . A significantly lower xylitol formation rate was observed. Appl Environ Microbiol, 1995 May, 61(5), 1904 - 9 Activation of plasma membrane H(+)-ATPase and expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cells grown at supraoptimal temperatures; Viegas CA et al.; During exponential growth at temperatures of 30 to 39 degrees C, the specific activity of H(+)-ATPase in the plasma membrane of Saccharomyces cerevisiae (assayed at the standard temperature 30 degrees C) increased with increases in growth temperature . In addition, the optimal temperature for in vitro activity of this ATPase was 42 degrees C . Therefore, the maximum values of ATPase activity were expected to occur in cells that grew within the supraoptimal range of temperatures . Activation induced by supraoptimal temperatures was not the result of increased synthesis of this membrane enzyme . When the growth temperature increased from 30 to 40 degrees C, expression of the essential PMA1 gene, monitored either by the level of PMA1 mRNA or the beta-galactosidase activity of the lacZ-PMA1 fusion, was reduced . Consistently, quantitative immunoassays showed that the ATPase content in the plasma membrane decreased . Like ATPase activity, the efficiency of the PMA2 promoter increased with increases in growth temperature in cells that had been grown at 30 to 39 degrees C, but its level of expression was several hundred-fold lower than that of PMA1 . These results suggest that the major PMA1 ATPase is activated at supraoptimal temperatures. Yeast, 1995 May, 11(6), 587 - 91 The complete sequence of a 9037 bp DNA fragment of the right arm of Saccharomyces cerevisiae chromosome VII; Arroyo J et al.; We report the sequence of a 9037 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII . Analysis of the sequence revealed four complete open reading frames (ORFs), namely G7572, G7576, G7579 and G7584 . The first three corresponded, respectively, to the previously cloned genes: HIP1, coding for a high-affinity histidine-specific permease, TDH1, one of the known genes coding for glyceraldehyde-3-phosphate dehydrogenase and ODPX, which encodes a precursor of protein X, a component of the pyruvate dehydrogenase complex . The ORF G7584 showed 35.8% identity with a hypothetical protein of Caenorhabditis elegans chromosome 3 . The reported sequence has been deposited in the EMBL data library under Accession Number X82408. Yeast, 1995 May, 11(6), 573 - 80 The upstream sequences of the HSP82 and HSC82 genes of Saccharomyces cerevisiae: regulatory elements and nucleosome positioning motifs; Erkine AM et al.; We present the upstream sequences of HSP82 and HSC82, two closely related, but differentially regulated, heat-shock genes of Saccharomyces cerevisiae . Several dozen potential regulatory elements are identified within each upstream region; interestingly, only a few are conserved between the two genes . These include a consensus heat-shock element, an upstream repressor element, and a consensus TATA element . A search for motifs known actively to position nucleosomes in vitro revealed that such sequences are three- to seven-fold enriched within each promoter; a comparable enrichment is seen near the 3' end of each transcription unit . Located approximately 1100 bp upstream of HSC82 is an open reading frame (ORF) of 255 amino acids; approximately 800 bp upstream of HSP82 is an ORF of 132 amino acids . The latter ORF contains several conserved ankyrin motifs and appears to be expressed under normal growth conditions . Finally, we show by clamped homogeneous electric field gel electrophoresis that the two genetic loci map to different chromosomes: HSP82 to chromosome XVI and HSC82 to chromosome XIII . The sequences have been deposited in the GenBank database under Accession Numbers U20323 and U20349. Yeast, 1995 May, 11(6), 555 - 65 Automatic intron detection in nuclear DNA sequences of Saccharomyces cerevisiae; Kalogeropoulos A; The goal of the present work is the construction of software (EXPLORA) which automatically detects Open Reading Frames (ORF), intron-containing or not, in the nuclear sequences of the yeast Saccharomyces cerevisiae . In order to detect introns within (or preceding) ORFs, one must define precise rules of how an intron is identified . These rules are described here . The software is able to detect correctly an intron-associated ORF in at least 88% of cases . We tested all yeast nuclear entries of the EMBL database with the software, and found, in addition to the known intron-associated ORFs, some others which may include an intron . Finally the software was applied to the DNA sequences of chromosomes III and XI, in which it detected eight new intron-associated ORFs. Yeast, 1995 May, 11(6), 549 - 54 The peculiarities of the interaction of radiation and hyperthermia in Saccharomyces cerevisiae irradiated with various dose rates; Petin VG et al.; In experiments with yeast cells it was shown that the synergistic effect of a simultaneous action of ionizing radiation and hyperthermia was a function of dose rate . It was demonstrated that, with increasing dose rate to obtain maximum synergistic effect, the temperature at which radiation is delivered should by elevated . Possible explanation of this effect is discussed. Prikl Biokhim Mikrobiol, 1995 May-Jun, 31(3), 311 - 5 {Increase in the yield of recombinant proteins in Saccharomyces cerevisiae yeasts as a result of optimizing their culture conditions}; Krupnova OF et al.; The optimal one-stage scheme of the cultivation of the recombinant yeast strains was developed . In these strains, the expression of the foreign gene is induced at the medium depletion by phosphorus . The scheme was tested for the producers of human interleukin-2 and bovine gamma-interferon . In all the strains, the yield of cell biomass and the production of the foreign proteins increased as compared to the standard two-stage cultivation . The use of the developed scheme resulted in the threefold increase in the yield of the target proteins and in the productivity of the biotechnological process. Genetics, 1995 May, 140(1), 91 - 101 Genetic analysis of the fimbrin-actin binding interaction in Saccharomyces cerevisiae; Brower SM et al.; Yeast fimbrin is encoded by the SAC6 gene, mutations of which suppress temperature-sensitive mutations in the actin gene (ACT1) . To examine the mechanism of suppression, we have sequenced 17 sac6 suppressor alleles, and found that they change nine different residues, all of which cluster in three regions of one of the two actin-binding domains of Sac6p . Two of these clusters occur in highly conserved regions (ABS1 and ABS3) that have been strongly implicated in the binding of related proteins to actin . The third cluster changes residues not previously implicated in the interaction with actin . As changes in any of nine different residues can suppress several different act1 alleles, it is likely that the suppressors restore the overall affinity, rather than specific lost interactions, between Sac6p and actin . Using mutagenesis, we have identified two mutations of the second actin-binding domain that can also suppress the act1 mutations of interest . This result suggests the two actin-binding domains of Sac6p interact with the same region of the actin molecule . However, differences in strength of suppression of temperature-sensitivity and sporulation indicate that the two actin-binding domains are distinct, and explain why second-domain mutations were not identified previously. Genetics, 1995 May, 140(1), 79 - 90 Identification of genes required for alpha 2 repression in Saccharomyces cerevisiae; Wahi M et al.; Transcriptional repression of the a-specific genes in Saccharomyces cerevisiae alpha cells involves the concerted action of several proteins . The homeodomian protein alpha 2, together with MCM1, recruits two general transcriptional repressors, SSN6 and TUP1, to the promoters of a-specific genes . SSN6 and TUP1 then mediate repression of the a-specific genes . SIN4, another general negative regulator, is required for this repression, but unlike tup1 or ssn6 deletions, sin4 deletions cause only partial loss of repression . We have screened for other genes required for a-specific gene repression in alpha cells . In addition to recovering multiple alleles of previously identified genes required for this process (referred to as alpha 2 repression), we have identified four other genes, designated ARE1, ARE2, ARE3, and ARE4 (for alpha 2 repression) . Recessive mutations in the ARE genes cause partial loss of a-specific gene repression and cause pleiotropic phenotypes similar to those resulting from mutations in SSN6, TUP1, or SIN4, suggesting that the ARE genes are general negative regulators . Based on our initial analysis, we propose that two distinct classes of general negative regulators cooperate to bring about full levels of alpha 2 repression . The sequence of ARE1 revealed that it encodes a CDC28-related protein kinase, identical to UME5, and thus suggests that protein phosphorylation plays a role in alpha 2 repression. Genetics, 1995 May, 140(1), 55 - 66 Factors that affect the location and frequency of meiosis-induced double-strand breaks in Saccharomyces cerevisiae; Wu TC et al.; Double-strand DNA breaks (DSBs) initiate meiotic recombination in Saccharomyces cerevisiae . DSBs occur at sites that are hypersensitive in nuclease digests of chromatin, suggesting a role for chromatin structure in determining DSB location . We show here that the frequency of DSBs at a site is not determined simply by DNA sequence or by features of chromatin structure . An arg4-containing plasmid was inserted at several different locations in the yeast genome . Meiosis-induced DSBs occurred at similar sites in pBR322-derived portions of the construct at all insert loci, and the frequency of these breaks varied in a manner that mirrored the frequency of meiotic recombination in the arg4 portion of the insert . However, DSBs did not occur in the insert-borne arg4 gene at a site that is frequently broken at the normal ARG4 locus, even though the insert-borne arg4 gene and the normal ARG4 locus displayed similar DNase I hypersensitivity patterns . Deletions that removed active DSB sites from an insert at HIS4 restored breaks to the insert-borne arg4 gene and to a DSB site in flanking chromosomal sequences . We conclude that the frequency of DSB at a site can be affected by sequences several thousand nucleotides away and suggest that this is because of competition between DSB sites for locally limited factors. Genetics, 1995 May, 140(1), 103 - 14 Regulation of HIS4 expression by the Saccharomyces cerevisiae SIN4 transcriptional regulator; Jiang YW et al.; The yeast SIN4 gene functions in the transcriptional activation and repression of diverse yeast genes . Previous experiments suggest a sin4 mutation affects chromatin structure and thus alters transcriptional regulation . In this report we show that SIN4 is required for full expression of the HIS4, Ty1, and MAT alpha genes, in addition to the previously described SIN4-dependence of CTS1 expression . All of these genes contain within their promoters a binding site for the Rap1p transcriptional regulator . However, SIN4 does not play a direct role either in transcriptional activation or repression by Rap1p . The HIS4 gene can be activated by either of two pathways, the basal or the inducible pathway, and experiments are described that show that a sin4 mutation affects both pathways . It was shown previously that mutation of the Rap1p binding site in the HIS4 promoter causes a similar effect on HIS4 expression and that this promoter mutation also causes a change in chromatin structure . The SNF2/SWI2 gene is also required for full HIS4 expression, and we show that a sin4 snf2 double mutant is not synergistic compared to either single mutant . We show that nucleosomes are positioned at the HIS4 promoter and that this positioning is disrupted in a snf2 mutant but not in a sin4 mutant . These findings suggest that SIN4 plays a distinct role in transcriptional regulation. Biotechnol Prog, 1995 May-Jun, 11(3), 342 - 7 Tracking of individual cell cohorts in asynchronous Saccharomyces cerevisiae populations; Porro D et al.; A novel flow cytometric procedure has been developed with the aim to obtain the growth properties of individual Saccharomyces cerevisiae cells in asynchronous culture . The method is based on labeling of the cell surface with FITC-conjugated concanavalin A and detection of the single-cell fluorescence with flow cytometry after cell exposure to growth conditions . Because the formation of new cell wall material in budded cells is restricted to the bud tip, exposure of the stained cells to growth conditions results in three cell types: (i) stained cells, (ii) partially stained cells, and (iii) unstained cells . Analysis of the staining pattern over time permits the determination of the specific growth rate of the cell population, the length of the budded cell cycle phase, and the growth pattern during the cell cycle of newly formed, partially stained daughter cells . The procedure has been tested with yeast cell populations growing at different rates . The data suggest an exponential increase in the size of individual cells during the cell cycle, as reflected by the forward angle light scattering (FALS) signals . It has been found that the apparent single-cell specific cell size growth rates, determined by FALS intensity, are significantly lower than the specific growth rates of the overall population . This could indicate that the tracking of a cohort of cells is significantly perturbed by a distribution of staining levels of daughter cells at cell division and that FALS may not be a good indicator of the cell size. Mol Microbiol, 1995 May, 16(3), 415 - 23 The role of the YAP1 and YAP2 genes in the regulation of the adaptive oxidative stress responses of Saccharomyces cerevisiae; Stephen DW et al.; The YAP1 and YAP2 genes encode yeast transcription factors of the c-jun family . We show that yeast mutants deleted for either the YAP1 or the YAP2 genes are hypersensitive to oxidants, particularly H2O2, and that these genes play a role in regulating the induction of the H2O2 adaptive stress response in Saccharomyces cerevisiae . They do not significantly affect the regulation of the superoxide adaptive stress response . The intrinsic resistance of stationary-phase and respiring yeast cells towards superoxide anions is unaffected by deletion of the YAP1 and YAP2 genes . However, resistance towards H2O2 under these conditions is significantly reduced . We show that expression of the yeast GSH1 gene (encoding gamma-glutamylcysteine synthetase) and the SSA1 gene (encoding an HSP70 isoform) are induced by oxidants . Unlike the SSA1 and thioredoxin (TRX2) genes, expression of the GSH1 gene is more strongly induced by superoxide anions than by H2O2 . In the absence of added oxidants, transcription of the GSH1 gene is reduced in strains carrying the yap1 deletion . However, we show that Yap1 is not required for the superoxide anion-mediated induction of GSH1 gene expression . Furthermore, while the H2O2-mediated induction of SSA1 expression is shown to by YAP1 dependent, the heat-shock-mediated induction of the SSA1 gene does not require YAP1 . We also present evidence to show that the YAP2 gene does not regulate the expression of the TRX2, SSA1 or GSH1 genes. Curr Genet, 1995 May, 27(6), 496 - 500 Failure to detect an antimutator phenotype following disruption of the Saccharomyces cerevisiae DDR48 gene; Roche H et al.; The antimutator phenotype, reportedly conferred by disruption of the Saccharomyces cerevisiae DDR48 gene, was suggested to affect only a specific spontaneous mutational pathway . We attempted to identify the types of mutation that are DDR48-dependent by determining the specificity of the ddr48 antimutator . However, disruption of DDR48 did not decrease the rates of spontaneous forward mutation in a plasmid-borne copy of the yeast SUP4-o gene, the reversion or suppression of the lys2-1 allele, or forward mutation at the CAN1 locus . Interestingly, the latter gene had been reported previously to be subject to the antimutator effect . DNA sequence analysis of spontaneous SUP4-o mutations arising in DDR48 and ddr48 backgrounds provided no evidence for a reduction in the rates of individual mutational classes . Thus, we were unable to verify that disruption of DDR48 causes an antimutator phenotype. RNA, 1995 May, 1(3), 260 - 72 Mammalian splicing factor SF3a120 represents a new member of the SURP family of proteins and is homologous to the essential splicing factor PRP21p of Saccharomyces cerevisiae; Kramer A et al.; Mammalian splicing factor SF3a consists of three subunits of 60, 66, and 120 kDa and functions early during pre-mRNA splicing by converting the U2 snRNP into its active form . A cDNA encoding the 120-kDa subunit of SF3a has been cloned . The SF3a120 gene was localized to human chromosome 22, and three mRNAs of 3.2, 3.8, and 5.7 kb are ubiquitously expressed . The N-terminal half of the deduced SF3a120 amino acid sequence contains a tandemly repeated motif (the SURP module) that has recently been identified in the essential splicing factor PRP21p of Saccharomyces cerevisiae, the Drosophila alternative splicing regulator suppressor-of-white-apricot, and four proteins from nematodes and mammals; the C-terminal half is organized into a proline-rich region and a ubiquitin-like domain . The spacing between the SURP modules and the protein's essential function in constitutive splicing identify SF3a120 as the mammalian homologue of yeast PRP21p . Binding studies with truncated derivatives of SF3a120 revealed that the SURP domains function in binding to SF3a60, whereas a region of 130 amino acids C-terminal to these domains is essential for contacts with SF3a66. RNA, 1995 May, 1(3), 246 - 59 The non-RNase H domain of Saccharomyces cerevisiae RNase H1 binds double-stranded RNA: magnesium modulates the switch between double-stranded RNA binding and RNase H activity; Cerritelli SM et al.; Eukaryotic ribonucleases H of known sequence are composed of an RNase H domain similar in size and sequence to that of Escherichia coli RNase HI and additional domains of unknown function . The RNase H1 of Saccharomyces cerevisiae has such an RNase H domain at its C-terminus . Here we show that the N-terminal non-RNase H portion of the yeast RNase H1 binds tightly to double-stranded RNA (dsRNA) and RNA-DNA hybrids even in the absence of the RNase H domain . Two copies of a sequence with limited similarity to the dsRNA-binding motif are present in this N-terminus . When the first of these sequences is altered, the protein no longer binds tightly to dsRNA and exhibits an increase in RNase H activity . Unlike other dsRNA-binding proteins, increasing the Mg2+ concentration from 0.5 mM to 5 mM inhibits binding of RNase H1 to dsRNA; yet a protein missing the RNase H domain binds strongly to dsRNA even at the higher Mg2+ concentration . These results suggest that binding to dsRNA and RNase H activity are mutually exclusive, and the Mg2+ concentration is critical for switching between the activities . Changes in the Mg2+ concentration or proteolytic severing of the dsRNA-binding domain could alter the activity or location of the RNase H and may govern access of the enzyme to the substrate . Sequences similar to the dsRNA-binding motif are present in other eukaryotic RNases H and the transactivating protein of cauliflower mosaic virus, suggesting that these proteins may also bind to dsRNA. Yeast, 1995 Apr 30, 11(5), 481 - 3 Genetic mapping of the alpha-galactosidase MEL gene family on right and left telomeres of Saccharomyces cerevisiae; Naumov GI et al.; The alpha-galactosidase MEL2-MEL10 genes have been genetically mapped to right and left telomere regions of the following chromosomes of Saccharomyces cerevisiae: MEL2 at VII L, MEL3 at XVI L, MEL4 at XI L, MEL5 at IV L, MEL6 at XIII R, MEL7 at VI R, MEL8 at XV R, MEL9 at X R and MEL10 at XII R . A set of tester strains with URA3 inserted into individual telomeres and no MEL genes was used for mapping. Yeast, 1995 Apr 30, 11(5), 475 - 9 Sequence and analysis of 24 kb on chromosome II of Saccharomyces cerevisiae; Aljinovic G et al.; In the course of the European yeast genome sequencing project, we determined 23,920 bp of a continuous chromosome II right arm sequence . Analysis of data revealed 13 open reading frames (ORFs), three of which corresponded to previously identified genes; two tRNA genes and one repetitive element . One ORF showed considerable homology (46%) to a hypothetical chromosome III gene; another, putatively very hydrophobic gene product, was 30% identical to the heat-shock protein HSP30 . Two ORFs were homologous to human genes. Yeast, 1995 Apr 30, 11(5), 435 - 46 Transcriptional regulation of flocculation genes in Saccharomyces cerevisiae; Teunissen AW et al.; Northern analysis showed that DNA from the flocculation gene FLO1 hybridized to mRNA molecules of 4.8 kb . This transcript was specific for the FLO1 gene at the right end of chromosome I since disruption of this gene resulted in the disappearance of the transcript . We further found an absolute correlation between flocculation and the presence of transcripts hybridizing to FLO1 DNA, both in various flocculent and non-flocculent strains and in cells from the non-flocculating and flocculating stages of growth . In all cases transcripts were present in flocculating and absent from non-flocculating cultures . From these results we conclude that the FLO1 gene is transcriptionally regulated . Mutations in TUP1 or SSN6 cause flocculation . Several transcripts hybridizing to FLO1 DNA were present in the mutants but not in the corresponding wild-type strains . Disruption of the FLO1 gene in the tup1 and ssn6 strains showed that one of the transcripts corresponded to the FLO1 gene . Disruption of FLO1 did not abolish flocculation completely but only reduced it, indicating that at least two flocculation genes, including FLO1, are activated or derepressed by mutations in the TUP1/SSN6 regulatory cascade. Yeast, 1995 Apr 30, 11(5), 419 - 24 Isolation of the gene HEM4 encoding uroporphyrinogen III synthase in Saccharomyces cerevisiae; Amillet JM et al.; We have isolated a genomic DNA fragment that complements the yeast temperature-sensitive cyt mutation, causing respiratory deficiency and accumulation of porphyrins (Sugimura et al., 1966) . Partial DNA sequencing of the complementing region and search for similarity in the DNA and protein databases revealed that (1) the gene had been previously isolated by complementation of the mutation ts2326 (Langgut et al., 1986; accession number X04694), and (2) it encodes a protein with 18-23% identity to uroporphyrinogen III synthases from different sources . This enzyme catalyses the fourth step in the heme biosynthetic pathway and we named its gene HEM4 . A hem4 delta disruption mutation was constructed which had phenotypes identical to the cyt mutation . Biochemical analysis confirmed the absence of uroporphyrinogen III synthase activity in both hem4 delta and cyt mutant strains. J Mol Biol, 1995 Apr 28, 248(2), 255 - 63 Functional interaction between the CSE2 gene product and centromeres in Saccharomyces cerevisiae; Xiao ZX et al.; The cse2-1 allele was identified through a genetic screen for mutations affecting chromosome segregation in Saccharomyces cerevisiae . This mutation confers cold and temperature sensitivity and causes increases in mitotic chromosome non-disjunction and loss . The CSE2 gene encodes a 17 kDa protein with a basic region-leucine zipper motif . Disruption of CSE2 is not lethal but results in the accumulation of large-budded cells . Here, we report that disruption of CSE2 results in a significant increase in chromosome missegregation, slower growth and defective meiosis . The combination of the CSE2 disruption and a mutant centromere results in a synergistic effect on both cell growth and cell viability . These data suggest a functional interaction between the CSE2 protein and the yeast centromere. J Biol Chem, 1995 Apr 28, 270(17), 10193 - 8 Expression and function of the trehalase genes NTH1 and YBR0106 in Saccharomyces cerevisiae; Nwaka S et al.; The biological function of the trehalose-degrading yeast enzyme neutral trehalase consists of the control of the concentration of trehalose, which is assumed to play a role in thermotolerance, in germination of spores, and in other life functions of yeast . Resequencing of the neutral trehalase gene NTH1 on chromosome IV resulted in the observation of two possible start codons (Kopp, M., Nwaka, S., and Holzer, H . (1994) Gene (Amst.) 150, 403-404) . We show here that only the most upstream start codon which initiates translation of the longest possible ORF is used for expression of NTH1 in vivo . A gene with 77% identity with NTH1, YBR0106, which was discovered during sequencing of chromosome II (Wolfe, K . H., and Lohan, A . J . E . (1994) Yeast 10, S41-S46), is shown here to be expressed into mRNA . Experiments with a mutant disrupted in the YBR0106 ORF showed, in contrast to a NTH1 deletion mutant, no changes in trehalase activity and in trehalose concentration . However, similar to the NTH1 gene a requirement of the intact YBR0106 gene for thermotolerance is demonstrated in experiments with the respective mutants . This indicates that the products of the likely duplicated YBR0106 gene and the NTH1 gene serve a heat shock protein function . In case of the YBR0106 gene, this is the only phenotypic feature found at present. Nucleic Acids Res, 1995 Apr 25, 23(8), 1426 - 33 INO1-100: an allele of the Saccharomyces cerevisiae INO1 gene that is transcribed without the action of the positive factors encoded by the INO2, INO4, SWI1, SWI2 and SWI3 genes; Swift S et al.; A dominant allele of the INO1 locus, INO1-100, does not require the positive regulators encoded by INO2 and INO4 for expression . Sequence analysis showed that INO1-100 had a 239 bp deletion in the INO1 promoter . INO1-100 suppressed the inositol auxotrophy of ino2, ino4, swi1, swi2 and swi3 mutants . Transcription of INO1-100 was constitutive and independent of these regulators . A 20 bp deletion from -247 to -228 caused a similar phenotype . A 38 bp deletion from -245 to -208 suppressed the inositol auxotrophy of an ino2 mutant, but not an ino4 mutant, indicating that Ino2p and Ino4p may function alone as well as in a complex . A 40 bp deletion from -287 to -248 that removed a URS1 site caused constitutive transcription that required INO2 and INO4 . A deletion from -167 to -128 suppressed the inositol auxotrophy of swi, ino2 and ino4 mutants, indicating the presence of a previously unidentified URS1 . Mutation of the specific negative regulator of phospholipid synthesis encoded by OPI1 suppressed the inositol auxotrophy of swi2 mutants . This study indicates that negative regulation of INO1 is chromatin mediated and provides in vivo information on the interaction of both general and specific regulatory factors that function to accomplish negative and positive regulation of the INO1 promoter in response to inositol. Proc Natl Acad Sci U S A, 1995 Apr 25, 92(9), 3809 - 13 The nucleotide sequence of chromosome I from Saccharomyces cerevisiae; Bussey H et al.; Chromosome I from the yeast Saccharomyces cerevisiae contains a DNA molecule of approximately 231 kbp and is the smallest naturally occurring functional eukaryotic nuclear chromosome so far characterized . The nucleotide sequence of this chromosome has been determined as part of an international collaboration to sequence the entire yeast genome . The chromosome contains 89 open reading frames and 4 tRNA genes . The central 165 kbp of the chromosome resembles other large sequenced regions of the yeast genome in both its high density and distribution of genes . In contrast, the remaining sequences flanking this DNA that comprise the two ends of the chromosome and make up more than 25% of the DNA molecule have a much lower gene density, are largely not transcribed, contain no genes essential for vegetative growth, and contain several apparent pseudogenes and a 15-kbp redundant sequence . These terminally repetitive regions consist of a telomeric repeat called W', flanked by DNA closely related to the yeast FLO1 gene . The low gene density, presence of pseudogenes, and lack of expression are consistent with the idea that these terminal regions represent the yeast equivalent of heterochromatin . The occurrence of such a high proportion of DNA with so little information suggests that its presence gives this chromosome the critical length required for proper function. Proc Natl Acad Sci U S A, 1995 Apr 25, 92(9), 3784 - 8 The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity; Lin SJ et al.; In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as superoxide dismutase (SOD) and catalase . Here we describe the isolation and characterization of another gene in the yeast Saccharomyces cerevisiae that plays a critical role in detoxification of reactive oxygen species . This gene, named ATX1, was originally isolated by its ability to suppress oxygen toxicity in yeast lacking SOD . ATX1 encodes a 8.2-kDa polypeptide exhibiting significant similarity and identity to various bacterial metal transporters . Potential ATX1 homologues were also identified in multicellular eukaryotes, including the plants Arabidopsis thaliana and Oryza sativa and the nematode Caenorhabditis elegans . In yeast cells, ATX1 evidently acts in the transport and/or partitioning of copper, and this role in copper homeostasis appears to be directly relevant to the ATX1 suppression of oxygen toxicity: ATX1 was incapable of compensating for SOD when cells were depleted of exogenous copper . Strains containing a deletion in the chromosomal ATX1 locus were generated . Loss of ATX1 function rendered both mutant and wild-type SOD strains hypersensitive toward paraquat (a generator of superoxide anion) and was also associated with an increased sensitivity toward hydrogen peroxide . Hence, ATX1 protects cells against the toxicity of both superoxide anion and hydrogen peroxide. J Biol Chem, 1995 Apr 21, 270(16), 9607 - 14 Amplification of a circular episome carrying an inverted repeat of the DFR1 locus and adjacent autonomously replicating sequence element of Saccharomyces cerevisiae; Huang T et al.; Lack of suitable amplification markers has hindered the use of the yeast system for investigating the mechanism of gene amplification in a eukaryote with a simple genome and well defined genetic system . Recently, methotrexate has been used to select for Saccharomyces cerevisiae mutants with de novo amplification of the dihydrofolate reductase gene (DFR1) (Huang, T . (1993) In Vivo Disruption and de Novo Amplification of the DFR1 Gene Encoding Dihydrofolate Reductase in Saccharomyces cerevisiae . Ph . D . thesis, University of Alberta, Edmonton, Canada) . We report here the detailed structure of a DFR1 episome amplified in methotrexate-resistant strain 25-1 . The extrachromosomal DNA is found predominantly as a single 11-kilobase circular molecule . It consists of a 5.5-kilobase inverted duplication that contains the DFR1 locus and adjacent ARS (autonomously replicating sequence) element . This molecular configuration mimics the inferred structure of double minute chromosomes observed in a number of mammalian amplification systems and suggests that mechanisms that generate amplified DNAs are conserved from yeast to mammals. Mol Gen Genet, 1995 Apr 20, 247(2), 148 - 56 Isolation and characterization of temperature-sensitive plc1 mutants of the yeast Saccharomyces cerevisiae; Yoko-o T et al.; The PLC1 gene of the yeast Saccharomyces cerevisiae has been discovered to encode a homolog of mammalian phosphoinositide-specific phospholipase C (PLC) . Five temperature-sensitive plc1 mutants were isolated by in vitro mutagenesis with subsequent plasmid shuffling . All of the amino acid substitutions that caused a temperature-sensitive growth phenotype were located in the X or the Y region, both of which are conserved among PLC isoenzymes . The PLC activity of all products of mutant plc1 genes was dramatically lower than that of the wild-type product, indicating that PLC activity itself is important for cell growth . At the restrictive temperature, plc1 mutant cells ceased growth at random times during the cell cycle, a result that suggests that PLC1 is required at several or all stages of the cell cycle. Mol Gen Genet, 1995 Apr 20, 247(2), 123 - 36 The role of Saccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance; Vaisman N et al.; Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication . In this report we present evidence suggesting that, in Saccharomyces cerevisiae, the CDC40 gene product is required to regulate both DNA replication and mitotic spindle formation . The deduced amino acid sequence of CDC40 (455 amino acids) contains four copies of a beta-transducin-like repeat . Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in the cdc40-1 allele) results in normal vegetative growth at 23 degrees C, and cell cycle arrest at 36 degrees C . In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36 degrees C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea) . The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed. FEBS Lett, 1995 Apr 17, 363(1-2), 17 - 21 Interaction of murine monoclonal subunit-specific antibodies with phosphofructokinase-1 from Saccharomyces cerevisiae; Kopperschlager G et al.; Murine monoclonal subunit-specific antibodies against heterooctameric phosphofructokinase-1 from Saccharomyces cerevisiae exhibiting no cross-reactivity were purified and characterized regarding complex formation with the native enzyme, immunological reactivity to the SDS-denatured subunits originating from native and proteolytically truncated phosphofructokinase, and protection against proteolytic degradation . Strong complex formation was found with one alpha-specific antibody . Western blotting employing different enzyme forms allowed to localize epitope sites of the alpha-polypeptide chain . Monoclonal antibodies protect phosphofructokinase-1 against chymotryptic degradation. Yeast, 1995 Apr 15, 11(4), 383 - 9 Nucleotide sequence and characterization of the Saccharomyces cerevisiae RPL19A gene encoding a homolog of the mammalian ribosomal protein L19; Song JM et al.; A gene designated RPL19A has been identified in the region downstream from the 3'-end of the Saccharomyces cerevisiae MIS1 gene encoding the mitochondrial C1-tetrahydrofolate synthase . The gene codes for the yeast ribosomal protein YL19 which exhibits 57.5% identify with the mammalian ribosomal protein L19 . RPL19A is one of two functional copies of the YL19 gene located on chromosome II . The disruption of RPL19A has no effect on the growth of the yeast . The RPL19A gene contains an intron located near the 5'-end . The 5'-flanking region contains one similar and one complete UASrpg upstream activating sequence . RPL19A was also found to be adjacent to the chromosome II AAC3 gene, encoding the mitochondrial ADP/ATP carrier protein. Yeast, 1995 Apr 15, 11(4), 361 - 9 Production of senescent cells of Saccharomyces cerevisiae by centrifugal elutriation; Woldringh CL et al.; The centrifugal elutriator has been used as a baby machine by loading the chamber with a population of mixed-generation daughter cells and allowing this population to grow, divide and age under continuous washing-out of newborn daughter cells . Clear peaks in the number of elutriated cells were reproducibly obtained for at least ten generations . The parent cells growing in the chamber continued to divide at the steady-state generation time of 95-100 min, showing no change in cycle time during aging . The washed-out daughter cells increased in volume during the first five generations from their steady-state value of 17 micro3 to a maximum of 34 micro3 . As to be expected, the generation times of these large daughters, determined in a synchronous batch culture, were shorter (130 min) than that of the steady-state daughters (240 min), even when derived from 15-generation parents . No indication for a volume increase of daughter cells without bud was observed when a population was allowed to grow in the chamber without washing-out the smaller daughter cells . The 15-generation parent population, recovered from the chamber, had an average volume of 80 micro3 and consisted of: (i) 71% cells with more than ten scars, (ii) 13% cells with one to nine scars, and (iii) 17% daughter cells . The production of senescent cells by undisturbed growth in the elutriator chamber has been prolonged to 29 generations . The method is therefore suitable to examine what factors determine the life span of budding yeast. Gene, 1995 Apr 14, 156(1), 19 - 25 Cloning and inducible synthesis of poliovirus non-structural proteins in Saccharomyces cerevisiae; Barco A et al.; Several coding regions of the poliovirus (PV) genome were cloned in the yeast Saccharomyces cerevisiae, and placed under the control of the inducible hybrid promoter pGAL/CYC, such that expression was triggered by incubating the cells in galactose (Gal)-containing medium . A number of PV genes encoding non-structural proteins, including 2Apro, 2B, 2C, 3A, 3AB and 3Cpro, were cloned and expressed in this eukaryotic system . The presence of these proteins after induction was detected by immunoblot analysis using specific antisera against each protein . The levels and the kinetics of protein synthesis after induction varied according to the PV protein analyzed . Thus, 2C was detected soon after Gal addition (3-5 h) and was one of the major polypeptides synthesized by yeast cells after 16 h of induction . In contrast, only low levels of synthesis were observed for 3A or 3AB, and then only after several hours of growth in Gal . The induction of the PV protease, 2Apro, was highly toxic for the cells such that growth was arrested after 5 h of induction and cell survival sharply declined. J Biol Chem, 1995 Apr 14, 270(15), 8936 - 43 The relationship between eukaryotic translation and mRNA stability . A short upstream open reading frame strongly inhibits translational initiation and greatly accelerates mRNA degradation in the yeast Saccharomyces cerevisiae; Oliveira CC et al.; A new strategy was developed to study the relationship between the translation and degradation of a specific mRNA in the yeast Saccharomyces cerevisiae . A series of 5'-untranslated regions (UTR) was combined with the cat gene from the bacterial transposon Tn9, allowing us to test the influence of upstream open reading frames (uORFs) on translation and mRNA stability . The 5'-UTR sequences were designed so that the minimum possible sequence alteration, a single nucleotide substitution, could be used to create a 7-codon ORF upstream of the cat gene . The uORF was translated efficiently, but at the same time inhibited translation of the cat ORF and destabilized the cat mRNA . Investigations of various derivatives of the 5'-UTR indicated that cat translation was primarily attributable to leaky scanning of ribosomes past the uORF rather than to reinitiation . Therefore, these data directly demonstrate destabilization of a specific mRNA linked to changes in translational initiation on the same transcript . In contrast to the previously proposed nonsense-mediated mRNA decay pathway, destabilization was not triggered by premature translational termination in the main ORF and was not discernibly dependent upon a reinitiation-driven mechanism . This suggests the existence of an as yet not described pathway of translation-linked mRNA degradation. Biochim Biophys Acta, 1995 Apr 12, 1235(1), 52 - 6 Effect of the kinetics of temperature variation on Saccharomyces cerevisiae viability and permeability; Gervais P et al.; The variation rate of the temperature increase was found to have a great effect on the viability of Saccharomyces cerevisiae subjected to heat perturbations between 25 degrees C and 50 degrees C . A low intensity of the increase rate of temperature could maintain an important viability of the cells (about 34% of the initial population) with regard to the corresponding viability (about 1%) observed after a sudden step change for the same final temperature level of 50 degrees C . A cell volume reduction more important (22% of the initial volume) has been observed in cells submitted to a heat shock than for the cells which have been submitted to a slow kinetic of temperature increase (9%) . Such an observation allowed to propose a relation between the membrane permeability and the kinetics of temperature variation. Nucleic Acids Res, 1995 Apr 11, 23(7), 1127 - 32 A human protein with homology to Saccharomyces cerevisiae SNF5 interacts with the potential helicase hbrm; Muchardt C et al.; In yeast, the SNF/SWI complex is involved in transcriptional activation of several inducible promoters, possibly by causing a local modification of the chromatin structure . Recently, two human homologues of the SNF2/SWI2 protein have been isolated, hbrm and BRG-1 . In addition, a complex containing one of the SNF2/SWI2 homologues and having an in vitro activity similar to the yeast complex has been partially purified from HeLa cells . Here we describe the characterization of a cDNA encoding a human nuclear protein containing a large domain of homology with SNF5, another member of the yeast SNF/SWI complex . This protein can be co-immunoprecipitated with hbrm and the interaction between the two proteins is dependent on the region conserved between the human and the yeast SNF5 . These findings suggest that the cDNA we have cloned encodes one of the members of the human SNF/SWI complex. Proc Natl Acad Sci U S A, 1995 Apr 11, 92(8), 3229 - 33 Non-clathrin-coat protein alpha is a conserved subunit of coatomer and in Saccharomyces cerevisiae is essential for growth; Gerich B et al.; To complete the molecular characterization of coatomer, the preformed cytosolic complex that is involved in the formation of biosynthetic transport vesicles, we have cloned and characterized the gene for non-clathrin-coat protein alpha (alpha-COP) from Saccharomyces cerevisiae . The derived protein, molecular weight of 135,500, contains four WD-40 repeated motifs (Trp/Asp-containing motifs of approximately 40 amino acids) . Disruption of the yeast alpha-COP gene is lethal . Comparison of the DNA-derived primary structure with peptides from bovine alpha-COP shows a striking homology . alpha-COP is localized to coated transport vesicles and coated buds of Golgi membranes derived from CHO cells. Proc Natl Acad Sci U S A, 1995 Apr 11, 92(8), 3127 - 31 Identification of the gene (SSU71/TFG1) encoding the largest subunit of transcription factor TFIIF as a suppressor of a TFIIB mutation in Saccharomyces cerevisiae; Sun ZW et al.; Mutations in the Saccharomyces cerevisiae SSU71 gene were isolated as suppressors of a transcription factor TFIIB defect that confers both a cold-sensitive growth defect and a downstream shift in transcription start-site selection at the cyc1 locus . The ssu71-1 suppressor not only suppresses the conditional phenotype but also restores the normal pattern of transcription initiation at cyc1 . In addition, the ssu71-1 suppressor confers a heat-sensitive phenotype that is dependent upon the presence of the defective form of TFIIB . Molecular and genetic analysis of the cloned SSU71 gene demonstrated that SSU71 is a single-copy essential gene encoding a highly charged protein with a molecular mass of 82,194 daltons . Comparison of the deduced Ssu71 amino acid sequence with the protein data banks revealed significant similarity to RAP74, the larger subunit of the human general transcription factor TFIIF . Moreover, Ssu71 is identical to p105, a component of yeast TFIIF . Taken together, these data demonstrate a functional interaction between TFIIB and the large subunit of TFIIF and that this interaction can affect start-site selection in vivo. Biochemistry, 1995 Apr 11, 34(14), 4577 - 82 Purification and properties of the alkylation repair DNA glycosylase encoded the MAG gene from Saccharomyces cerevisiae; Bjoras M et al.; The MAG gene of Saccharomyces cerevisiae encodes an alkylation repair DNA glycosylase whose sequence is homologous to the AlkA DNA glycosylase from Escherichia coli . To investigate the biochemical properties of MAG in comparison to AlkA, MAG was expressed in E . coli and purified to electrophoretic homogeneity . N-Terminal sequencing of the purified protein identified the translational start site which corresponded to that predicted previously from the nucleotide sequence . Polyclonal antibodies raised against MAG inhibited the enzymatic activity of MAG, but not that of AlkA, and vice versa, implying that the structures of the active site regions of these enzymes are antigenic, but sufficiently different to have different epitopes . Kinetic analysis of base excision from DNA exposed to {3H}methyl-N-nitrosourea and {3H}dimethyl sulfate showed that MAG was as effective as AlkA in removing 3-methyladenine, 7-methylguanine, and 7-methyladenine . However, the purified MAG enzyme did not catalyze the excision of O2-methylthymine, which is a major substrate for AlkA . Furthermore, 3-methylguanine was excised 20-40 times more slowly by MAG than by AlkA . The kinetics of 3-methylguanine excision by MAG were found to be similar to the low rate of 3-methylguanine excision catalyzed by 3-methyladenine DNA glycosylase I (Tag) of E . coli . Expression of MAG in alkA mutant cells did not effectively restore alkylation resistance of the mutant as did AlkA itself . It thus appears that MAG is a less versatile enzyme than AlkA in spite of the sequence relationship and may have a similar function in yeast as the nonhomologous Tag enzyme in E . coli. Mol Gen Genet, 1995 Apr 10, 247(1), 55 - 60 Influence of non-homology between recombining DNA sequences on double-strand break repair in Saccharomyces cerevisiae; Glasunov A et al.; In this paper we study the influence of non-homology between plasmid and chromosomal DNA on the efficiency of recombinational repair of plasmid double-strand breaks and gaps in yeast . For this purpose we used different combinations of plasmids and yeast strains carrying various deletions within the yeast LYS2 gene . A 400 bp deletion in plasmid DNA had no effect on recombinational plasmid repair . However, a 400 bp deletion in chromosomal DNA dramatically reduced the efficiency of this repair mechanism, but recombinational repair of plasmids linearized by a double-strand break with cohesive ends still remained the dominant repair process . We have also studied the competition between recombination and ligation in the repair of linearized plasmids . Our experimental evidence suggests that recombinational repair is attempted but aborted if only one recombinogenic end with homology to chromosomal DNA is present in plasmid DNA . This situation results in a decreased probability of non-recombinational (i.e . ligation) repair of linearized plasmid DNA. Mol Gen Genet, 1995 Apr 10, 247(1), 48 - 54 The glucose repression and RAS-cAMP signal transduction pathways of Saccharomyces cerevisiae each affect RNA processing and the synthesis of a reporter protein; Tung KS et al.; Previously we reported that mutations in the Saccharomyces cerevisiae REG1 gene encoding a negative regulator of glucose-repressible genes, suppress the RNA processing defects and temperature-sensitive growth of rna1-1 and prp cells . This result and the fact that growth on non-glucose carbon sources also suppresses rna1-1 led us to propose that RNA processing and export of RNA from the nucleus are responsive to carbon source regulation . To understand how carbon source affects these processes, we used p70, an antigen regulated by REG1 and by glucose availability, as a reporter . We found that the response of p70 to glucose availability is mediated by both the SNF1-SSN6-dependent glucose repression and the RAS-cAMP pathways . These results led us to test whether the RAS-cAMP pathway interacts with RNA1 . We found that suppression of rna1-1 appears to be mediated, at least in part, by the RAS-cAMP pathway. Biochem Biophys Res Commun, 1995 Apr 6, 209(1), 322 - 6 The Saccharomyces cerevisiae processing alpha 1,2-mannosidase is an inverting glycosidase; Lipari F et al.; The alpha 1,2-mannosidase from Saccharomyces cerevisiae, which removes one specific alpha 1,2-linked mannose residue from Man9GlcNAc2, is a member of the Class 1 alpha 1,2-mannosidase family conserved from yeast to mammals . Although Class 1 alpha 1,2-mannosidases are essential for the maturation of N-linked oligosaccharides in mammalian cells, nothing is known about their mechanism of action . The availability of sufficient quantities of recombinant yeast alpha 1,2-mannosidase and its homology with the mammalian enzymes make it a good model to study the catalytic mechanism of this family of alpha 1,2-mannosidases . The stereochemical course of hydrolysis of Man9GlcNAc by the yeast enzyme was followed by proton nuclear magnetic resonance spectroscopy . It was observed that beta-D-mannose is related from the oligosaccharide substrate, thereby demonstrating that the enzyme is of the inverting type. Can J Microbiol, 1995 Apr-May, 41(4-5), 388 - 98 A reevaluation of the thermodynamics of growth of Saccharomyces cerevisiae on glucose, ethanol, and acetic acid; Battley EH; Statistically determined values for the free energy and enthalpy change per equivalent accompanying the combustion of organic substances have been used to test the hypothesis that the free energy, enthalpy, and entropy changes accompanying anabolism are zero or nearly so . It is found that while this can be true in some cases, it is not always so, because the free energy and entropy changes per available electron accompanying biological combustion are different for different small molecular weight substrates although they are constant for cellular biomass . If the average energy change per equivalent accompanying the biological combustion of the cells is markedly different from that of the substrate, there will be a corresponding change in energy accompanying anabolism that is determined by the difference between them . The efficiency of available electron conservation is proposed as being preferable to free energy, enthalpy, and entropy efficiencies in that the latter parameters all have different values, whereas the former is common to all three of the latter . Free energy, enthalpy, and entropy efficiencies can be calculated from available electron conservation efficiencies if the biological combustion energies of the substrate, cells, and products are known per electron transferred . The methods described are applicable equally to aerobic and anaerobic growth process systems. Mol Cell Biol, 1995 Apr, 15(4), 2321 - 30 Roles of URE2 and GLN3 in the proline utilization pathway in Saccharomyces cerevisiae; Xu S et al.; The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as arginine, urea, allantoin, gamma-aminobutyrate, or proline when preferred nitrogen sources like glutamine, asparagine, or ammonium ions are unavailable in the environment . Utilization of alternative nitrogen sources requires the relief of nitrogen repression and induction of specific permeases and enzymes . The products of the GLN3 and URE2 genes are required for the appropriate transcription of many genes in alternative nitrogen assimilatory pathways . GLN3 appears to activate their transcription when good nitrogen sources are unavailable, and URE2 appears to repress their transcription when alternative nitrogen sources are not needed . The participation of nitrogen repression and the regulators GLN3 and URE2 in the proline utilization pathway was evaluated in this study . Comparison of PUT gene expression in cells grown in repressing or derepressing nitrogen sources, in the absence of the inducer proline, indicated that both PUT1 and PUT2 are regulated by nitrogen repression, although the effect on PUT2 is comparatively small . Recessive mutations in URE2 elevated expression of the PUT1 and PUT2 genes 5- to 10-fold when cells were grown on a nitrogen-repressing medium . Although PUT3, the proline utilization pathway transcriptional activator, is absolutely required for growth on proline as the sole nitrogen source, a put3 ure2 strain had somewhat elevated PUT gene expression, suggesting an effect of the ure2 mutation in the absence of the PUT3 product . PUT1 and PUT2 gene expression did not require the GLN3 activator protein for expression under either repressing or derepressing conditions . Therefore, regulation of the PUT genes by URE2 does not require a functional GLN3 protein . The effect of the ure2 mutation on the PUT genes is not due to increased internal proline levels . URE2 repression appears to be limited to nitrogen assimilatory systems and does not affect genes involved in carbon, inositol, or phosphate metabolism or in mating-type control and sporulation. Mol Cell Biol, 1995 Apr, 15(4), 2245 - 51 RAD1 and RAD10, but not other excision repair genes, are required for double-strand break-induced recombination in Saccharomyces cerevisiae; Ivanov EL et al.; HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous sequences . We have shown previously (J . Fishman-Lobell and J . E . Haber, Science 258:480-484, 1992) that during the repair of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends . In this report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required for removal of nonhomologous sequences from the DSB ends . rad1 delta and rad10 delta mutants displayed a 20-fold reduction in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination . Mutations in RAD2, RAD3, and RAD14 reduced HO-induced recombination by about twofold . We also show that RAD7 and RAD16, which are required to remove UV photodamage from the silent HML, locus, are not required for MAT switching with HML or HMR as a donor . Our results provide a molecular basis for understanding the role of yeast nucleotide excision repair gene and their human homologs in DSB-induced recombination and repair. Mol Cell Biol, 1995 Apr, 15(4), 2173 - 9 DNA polymerases required for repair of UV-induced damage in Saccharomyces cerevisiae; Budd ME et al.; The ability of yeast DNA polymerase mutant strains to carry out repair synthesis after UV irradiation was studied by analysis of postirradiation molecular weight changes in cellular DNA . Neither DNA polymerase alpha, delta, epsilon, nor Rev3 single mutants evidenced a defect in repair . A mutant defective in all four of these DNA polymerases, however, showed accumulation of single-strand breaks, indicating defective repair . Pairwise combination of polymerase mutations revealed a repair defect only in DNA polymerase delta and epsilon double mutants . The extent of repair in the double mutant was no greater than that in the quadruple mutant, suggesting that DNA polymerases alpha and Rev3p play very minor, if any, roles . Taken together, the data suggest that DNA polymerases delta and epsilon are both potentially able to perform repair synthesis and that in the absence of one, the other can efficiently substitute . Thus, two of the DNA polymerases involved in DNA replication are also involved in DNA repair, adding to the accumulating evidence that the two processes are coupled. Mol Cell Biol, 1995 Apr, 15(4), 1999 - 2009 A new class of histone H2A mutations in Saccharomyces cerevisiae causes specific transcriptional defects in vivo; Hirschhorn JN et al.; Nucleosomes have been shown to repress transcription both in vitro and in vivo . However, the mechanisms by which this repression is overcome are only beginning to be understood . Recent evidence suggests that in the yeast Saccharomyces cerevisiae, many transcriptional activators require the SNF/SWI complex to overcome chromatin-mediated repression . We have identified a new class of mutations in the histone H2A-encoding gene HTA1 that causes transcriptional defects at the SNF/SWI-dependent gene SUC2 . Some of the mutations are semidominant, and most of the predicted amino acid changes are in or near the N- and C-terminal regions of histone H2A . A deletion that removes the N-terminal tail of histone H2A also caused a decrease in SUC2 transcription . Strains carrying these histone mutations also exhibited defects in activation by LexA-GAL4, a SNF/SWI-dependent activator . However, these H2A mutants are phenotypically distinct from snf/swi mutants . First, not all SNF/SWI-dependent genes showed transcriptional defects in these histone mutants . Second, a suppressor of snf/swi mutations, spt6, did not suppress these histone mutations . Finally, unlike in snf/swi mutants, chromatin structure at the SUC2 promoter in these H2A mutants was in an active conformation . Thus, these H2A mutations seem to interfere with a transcription activation function downstream or independent of the SNF/SWI activity . Therefore, they may identify an additional step that is required to overcome repression by chromatin. Mol Cell Biol, 1995 Apr, 15(4), 1968 - 73 Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae; Choulika A et al.; The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes . We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous regions flanking the breaks . This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter . As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break . This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells . These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements. Mol Cell Biol, 1995 Apr, 15(4), 1915 - 22 CAT8, a new zinc cluster-encoding gene necessary for derepression of gluconeogenic enzymes in the yeast Saccharomyces cerevisiae; Hedges D et al.; The expression of gluconeogenic fructose-1,6-bisphosphatase (encoded by the FBP1 gene) depends on the carbon source . Analysis of the FBP1 promoter revealed two upstream activating elements, UAS1FBP1 and UAS2FBP1, which confer carbon source-dependent regulation on a heterologous reporter gene . On glucose media neither element was activated, whereas after transfer to ethanol a 100-fold derepression was observed . This gene activation depended on the previously identified derepression genes CAT1 (SNF1) (encoding a protein kinase) and CAT3 (SNF4) (probably encoding a subunit of Cat1p {Snf1p}) . Screening for mutations specifically involved in UAS1FBP1 derepression revealed the new recessive derepression mutation cat8 . The cat8 mutants also failed to derepress UAS2FBP1, and these mutants were unable to grow on nonfermentable carbon sources . The CAT8 gene encodes a zinc cluster protein related to Saccharomyces cerevisiae Gal4p . Deletion of CAT8 caused a defect in glucose derepression which affected all key gluconeogenic enzymes . Derepression of glucose-repressible invertase and maltase was still normally regulated . A CAT8-lacZ promoter fusion revealed that the CAT8 gene itself is repressed by Cat4p (Mig1p) . These results suggest that gluconeogenic genes are dere