Yeast, 1996 Dec, 12(15), 1555 - 62 Sequence of a 39,411 bp DNA fragment covering the left end of chromosome VII of Saccharomyces cerevisiae; Coissac E et al.; We have sequenced a DNA fragment of 39,411 bp which includes part of the left telomere of chromosome VII of Saccharomyces cerevisiae . We have identified 19 open reading frames (ORFs); six correspond to known yeast genes (ADH4, FZF1, HKB, RTG2, HFM1 and PDE1), nine have similarity with other genes and four exhibit no significant similarity with any known gene . The average size of these ORFs seems to be related to their location, the eight ORF's nearest the telomere being shorter than the 11 others . These two groups of genes are separated by a region of 4.5 kb devoid of significant ORFs . One ORF, NRF120, is a new member of the seripauperine family, represented once in all sequenced yeast chromosomes, in a subtelomeric location. Mol Biol Cell, 1996 Dec, 7(12), 1953 - 66 A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6; Liang H et al.; We show that cells deleted for SNF3, HXT1, HXT2, HXT3, HXT4, HXT6, and HXT7 do not take up glucose and cannot grow on media containing glucose as a sole carbon source . The expression of Hxt1, Hxt2, Hxt3, Hxt6, or Gal2 in these cells resulted in glucose transport and allowed growth on glucose media . In contrast, the expression of Snf3 failed to confer glucose uptake or growth on glucose . HXT6 is highly expressed on raffinose, low glucose, or nonfermentable carbon sources but is repressed in the presence of high concentrations of glucose . The maintenance of HXT6 glucose repression is strictly dependent on Snf3 and not on intracellular glucose . In snf3 delta cells expression of HXT6 is constitutive even when the entire repertoire of HXT genes is present and glucose uptake is abundant . In addition, glucose repression of HXT6 does not require glucose uptake by HXT1, HXT2, HXT3 or HXT4 . We show that a signal transduction pathway defined by the Snf3-dependent hexose regulation of HXT6 is distinct from but also overlaps with general glucose regulation pathways in Saccharomyces cerevisiae . Finally, glucose repression of ADH2 and SUC2 is intact in snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta hxt6 delta hxt7 delta gal2 cells, suggesting that the sensing and signaling mechanism for general glucose repression is independent from glucose uptake. Mol Biol Cell, 1996 Dec, 7(12), 1909 - 19 Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway; Ziman M et al.; In Saccharomyces cerevisiae, the synthesis of chitin, a cell-wall polysaccharide, is temporally and spatially regulated with respect to the cell cycle and morphogenesis . Using immunological reagents, we found that steady-state levels of Chs1p and Chs3p, two chitin synthase enzymes, did not fluctuate during the cell cycle, indicating that they are not simply regulated by synthesis and degradation . Previous cell fractionation studies demonstrated that chitin synthase I activity (CSI) exists in a plasma membrane form and in intracellular membrane-bound particles called chitosomes . Chitosomes were proposed to act as a reservoir for regulated transport of chitin synthase enzymes to the division septum . We found that Chs1p and Chs3p resided partly in chitosomes and that this distribution was not cell cycle regulated . Pulse-chase cell fractionation experiments showed that chitosome production was blocked in an endocytosis mutant (end4-1), indicating that endocytosis is required for the formation or maintenance of chitosomes . Additionally, Ste2p, internalized by ligand-induced endocytosis, cofractionated with chitosomes, suggesting that these membrane proteins populate the same endosomal compartment . However, in contrast to Ste2p, Chs1p and Chs3p were not rapidly degraded, thus raising the possibility that the temporal and spatial regulation of chitin synthesis is mediated by the mobilization of an endosomal pool of chitin synthase enzymes. FEMS Microbiol Lett, 1996 Dec 1, 145(2), 255 - 9 Formation of cadaverine derivatives in Saccharomyces cerevisiae; Walters DR et al.; The higher homologues of cadaverine, aminopropylcadaverine (APC) and N,N-bis(3-aminopropyl)cadaverine (3APC) were formed by a wild-type strain of Saccharomyces cerevisiae, and by two mutant strains, spe 3-1 and spe 4-1, exhibiting point mutations in the genes for spermidine synthase and spermine synthase, respectively . This, together with the incomplete inhibition of APC and 3APC formation in the presence of inhibitors of S-adenosylmethionine decarboxylase and spermidine synthase, suggests that the cadaverine derivatives are formed partly by the operation of a different route . However, the yeast strains were unable to utilise {14C}aspartate and lysine to form APC and 3APC . Since the ornithine decarboxylase inhibitor alpha-difluoromethylornithine (DFMO) greatly reduced the formation of APC and 3APC, it is suggested that these compounds are formed preferentially in these yeast strains from cadaverine formed by ODC . APC and 3APC formation in the yeast strains was increased substantially following exposure to 37 degrees C for 2 h. J Bacteriol, 1996 Dec, 178(24), 7197 - 205 Amino acid substitutions in membrane-spanning domains of Hol1, a member of the major facilitator superfamily of transporters, confer nonselective cation uptake in Saccharomyces cerevisiae; Wright MB et al.; Selection for the ability of Saccharomyces cerevisiae cells to take up histidinol, the biosynthetic precursor to histidine, results in dominant mutations at HOL1 . The DNA sequence of HOL1 was determined, and it predicts a 65-kDa protein related to the major facilitator family (drug resistance subfamily) of putative transport proteins . Two classes of mutations were obtained: (i) those that altered the coding region of HOL1, conferring the ability to take up histidinol; and (ii) cis-acting mutations (selected in a mutant HOL1-1 background) that increased expression of the Hol1 protein . The ability to transport histidinol and other cations was conferred by single amino acid substitutions at any of three sites located within putative membrane-spanning domains of the transporter . These mutations resulted in the conversion of a small hydrophobic amino acid codon to a phenylalanine codon . Selection for spontaneous mutations that increase histidinol uptake by such HOL1 mutants resulted in mutations that abolish the putative start codon of a six-codon open reading frame located approximately 171 nucleotides downstream of the transcription initiation site and 213 nucleotides upstream of the coding region of HOL1 . This single small upstream open reading frame (uORF) confers translational repression upon HOL1; genetic disruption of the putative start codon of the uORF results in a 5- to 10-fold increase in steady-state amounts of Hol1 protein without significantly affecting the level of HOL1 mRNA expression. Protein Expr Purif, 1996 Dec, 8(4), 447 - 55 Production, purification, and characterization of recombinant human hemoglobin rainier expressed in Saccharomyces cerevisiae; Motwani N et al.; Hemoglobin Rainier is a naturally occurring hemoglobin variant in which the beta 145 tyrosine is substituted with cysteine . The alpha and beta Rainier globin cDNAs were cloned in a high copy number vector and expressed in Saccharomyces cerevisiae under the control of galactose-regulated hybrid promoters . Using this system, we have expressed individual alpha and beta Rainier globin chains . Coexpression of both alpha and beta Rainier cDNAs resulted in the production of a functional hemoglobin molecule . Purification of the recombinant protein was accomplished by ion exchange chromatography . The N-termini of the alpha and beta chains were correctly processed, and the molecular mass, as determined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier . The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived hemoglobin A0 . The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as previously reported for natural hemoglobin Rainier . Production of recombinant hemoglobin and especially hemoglobin variants like hemoglobin Rainier has the potential to facilitate use of hemoglobin as a blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of hypotension associated with septic shock. Appl Environ Microbiol, 1996 Dec, 62(12), 4441 - 9 Alleviation of glucose repression of maltose metabolism by MIG1 disruption in Saccharomyces cerevisiae; Klein CJ et al.; The MIG1 gene was disrupted in a haploid laboratory strain (B224) and in an industrial polyploid strain (DGI 342) of Saccharomyces cerevisiae . The alleviation of glucose repression of the expression of MAL genes and alleviation of glucose control of maltose metabolism were investigated in batch cultivations on glucose-maltose mixtures . In the MIG1-disrupted haploid strain, glucose repression was partly alleviated; i.e., maltose metabolism was initiated at higher glucose concentrations than in the corresponding wild-type strain . In contrast, the polyploid delta mig1 strain exhibited an even more stringent glucose control of maltose metabolism than the corresponding wild-type strain, which could be explained by a more rigid catabolite inactivation of maltose permease, affecting the uptake of maltose . Growth on the glucose-sucrose mixture showed that the polypoid delta mig1 strain was relieved of glucose repression of the SUC genes . The disruption of MIG1 was shown to bring about pleiotropic effects, manifested in changes in the pattern of secreted metabolites and in the specific growth rate. J Cell Biol, 1996 Dec, 135(5), 1323 - 39 MHP1, an essential gene in Saccharomyces cerevisiae required for microtubule function; Irminger-Finger I et al.; The gene for a microtubule-associated protein (MAP), termed MHP1 (MAP-Homologous Protein 1), was isolated from Saccharomyces cerevisiae by expression cloning using antibodies specific for the Drosophila 205K MAP . MHP1 encodes an essential protein of 1,398 amino acids that contains near its COOH-terminal end a sequence homologous to the microtubule-binding domain of MAP2, MAP4, and tau . While total disruptions are lethal, NH2-terminal deletion mutations of MHP1 are viable, and the expression of the COOH-terminal two-thirds of the protein is sufficient for vegetative growth . Nonviable deletion-disruption mutations of MHP1 can be partially complemented by the expression of the Drosophila 205K MAP . Mhp1p binds to microtubules in vitro, and it is the COOH-terminal region containing the tau-homologous motif that mediates microtubule binding . Antibodies directed against a COOH-terminal peptide of Mhp1p decorate cytoplasmic microtubules and mitotic spindles as revealed by immunofluorescence microscopy . The overexpression of an NH2-terminal deletion mutation of MHP1 results in an accumulation of large-budded cells with short spindles and disturbed nuclear migration . In asynchronously growing cells that overexpress MHP1 from a multicopy plasmid, the length and number of cytoplasmic microtubules is increased and the proportion of mitotic cells is decreased, while haploid cells in which the expression of MHP1 has been silenced exhibit few microtubules . These results suggest that MHP1 is essential for the formation and/or stabilization of microtubules. Mol Cell Biol, 1996 Dec, 16(12), 6794 - 803 Saccharomyces cerevisiae G1 cyclins differ in their intrinsic functional specificities; Levine K et al.; The three budding yeast CLN genes appear to be functionally redundant for cell cycle Start: any single CLN gene is sufficient to promote Start, while the cln1 cln2 cln3 triple mutant is Start defective and inviable . Both quantitative and apparently qualitative differences between CLN genes have been reported, but available data do not in general allow distinction between qualitative functional differences as opposed to simply quantitative differences in expression or function . To determine if there are intrinsic qualitative differences between Cln proteins, we compared CLN2, CLN3, and crippled (but still partially active) CLN2 genes in a range of assays that differentiate genetically between CLN2 and CLN3 . The results suggest that different potencies of Cln2, Cln3, and Cln2 mutants in functional assays cannot be accounted for by a simple quantitative model for their action, since Cln3 is at least as active as Cln2 and much more active than the Cln2 mutants in driving Swi4/Swi6 cell cycle box (SCB)-regulated transcription and cell cycle initiation in cln1 cln2 cln3 bck2 strains, but Cln3 has little or no activity in other assays in which Cln2 and the Cln2 mutants function . Differences in Cln protein abundance are unlikely to account for these results . Cln3-associated kinase is therefore likely to have an intrinsic in vivo substrate specificity distinct from that of Cln2-associated kinase, despite their functional redundancy . Consistent with the idea that Cln3 may be the primary transcriptional activator of CLN1, CLN2, and other genes, the activation of CLN2 transcription was found to be sensitive to the gene dosage of CLN3 but not to the gene dosage of CLN2. Mol Cell Biol, 1996 Dec, 16(12), 6715 - 23 The osmoregulatory pathway represses mating pathway activity in Saccharomyces cerevisiae: isolation of a FUS3 mutant that is insensitive to the repression mechanism; Hall JP et al.; Mitogen-activated protein (MAP) kinase cascades are conserved signal transduction pathways that are required for eukaryotic cells to respond to a variety of stimuli . Multiple MAP kinase pathways can function within a single cell type; therefore, mechanisms that insulate one MAP kinase pathway from adventitious activations by parallel pathways may exist . We have studied interactions between the mating pheromone response and the osmoregulatory (high-osmolarity glycerol response {HOG}) pathways in Saccharomyces cerevisiae which utilize the MAP kinases Fus3p and Hog1p, respectively . Inactivating mutations in HOG pathway kinases cause an increase in the phosphotyrosine content of Fus3p, greater expression of pheromone-responsive genes, and increased sensitivity to growth arrest by pheromone . Therefore, the HOG pathway represses mating pathway activity . In a HOG1+ strain, Fus3p phosphotyrosine increases modestly and transiently following an increase in the extracellular osmolarity; however, it increases to a greater extent and for a sustained duration in a hog1-delta strain . Thus, the HOG-mediated repression of mating pathway activity may insulate the mating pathway from activation by osmotic stress . A FUS3 allele whose gene product is resistant to the HOG-mediated repression of its phosphotyrosine content has been isolated . This mutant encodes an amino acid substitution in the highly conserved DPXDEP motif in subdomain XI . Other investigators have shown that the corresponding amino acid is also mutated in a gain-of-function allele of the MAP kinase encoded by the rolled locus in Drosophila melanogaster . These data suggest that the DPXDEP motif plays a role in the negative regulation of MAP kinases. Mol Cell Biol, 1996 Dec, 16(12), 6617 - 22 Orientation dependence of trinucleotide CAG repeat instability in Saccharomyces cerevisiae; Maurer DJ et al.; To examine the chromosomal stability of repetitions of the trinucleotide CAG, we have cloned CAG repeat tracts onto the 3' end of the Saccharomyces cerevisiae ADE2 gene and placed the appended gene into the ARO2 locus of chromosome VII . Examination of chromosomal DNA from sibling colonies arising from clonal expansion of strains harboring repeat tracts showed that repeat tracts often change in length . Most changes in tract length are decreases, but rare increases also occur . Longer tracts are more unstable than smaller tracts . The most unstable tracts, of 80 to 90 repeats, undergo changes at rates as high as 3 x 10(-2) changes per cell per generation . To examine whether repeat orientation or adjacent sequences alter repeat stability, we constructed strains with repeat tracts in both orientations, either with or without sequences 5' to ADE2 harboring an autonomously replicating sequence (ARS; replication origin) . When CAG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively stable regardless of the orientation of ADE2 . When CTG is in the ADE2 coding strand of strains harboring the ARS, the repeat tract is relatively unstable regardless of the orientation of ADE2 . Removal of the ARS as well as other sequences adjacent to the 5' end of ADE2 alters the orientation dependence such that stability now depends on the orientation of ADE2 in the chromosome . These results suggest that the proximity of an ARS or another sequence has a profound effect on repeat stability. Curr Genet, 1996 Dec, 30(6), 485 - 92 The red/white colony color assay in the yeast Saccharomyces cerevisiae: epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells; Ugolini S et al.; In the yeast Saccharomyces cerevisiae the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment, while epistatic mutations in the same pathway, i.e . ade8, preclude this phenomenon, resulting in normal white colonies . The shift in color from red to white (or vice versa) with a combination of appropriate wild-type and mutant alleles of the adenine-pathway genes has been widely utilized as a non-selective phenotype to visualise and quantify the occurrence of various genetic events such as recombination, conversion and aneuploidy . It has provided an invaluable tool for the study of gene dosage and plasmid stability . In competition experiments between disrupted ade2, ade8-18 transformants carrying either a functional or non-functional episomal ADE8 gene, we verified that white ade8 ade2 cells show a remarkable selective advantage over red ade2 cells, with important implications on the use of this assay for the monitoring of genetic events . The accumulation of the red pigment in ade2 cells is likely to be the cause for impaired growth in these cells. Curr Genet, 1996 Dec, 30(6), 476 - 84 A genetic analysis of the role of calcineurin and calmodulin in Ca++-dependent improvement of NaCl tolerance of Saccharomyces cerevisiae; Danielsson A et al.; Mutants of Saccharomyces cerevisiae lacking activity of the Ca2+/calmodulin-dependent protein phosphatase calcineurin, show sensitivity to high concentrations of sodium that is partly reversed by the external supply of Ca2+ . On long-time exposure to NaCl stress the mutants display an increased intracellular Na+/K+ ratio which is partially corrected by the addition of Ca2+, improving the sodium efflux of not only calcineurin-defective cells but also wild-type cells . We also demonstrate that the NaCl sensitivity of cmd mutants, expressing modified forms of calmodulin that do not bind Ca2+, is strongly reversed by the addition of Ca2+ . This effect is highly dependent on calcineurin, since the NaCl tolerance of a cmd1-3 strain, carrying an additional mutation in calcineurin, is only slightly assisted by Ca2+ . A striking characteristic of the loss of function of calcineurin is a several-fold increased content of intracellular Ca2+, localized mainly in subcellular compartment(s) . If the compartmentalized Ca2+ pool is brought back to normal levels by an additional inactivating mutation of the vacuolar Ca2+-transporting ATPase, such double mutants do not significantly improve their tolerance to NaCl. Curr Genet, 1996 Dec, 30(6), 461 - 8 The repair of DNA methylation damage in Saccharomyces cerevisiae; Xiao W et al.; The major genotoxicity of methyl methanesulfonate (MMS) is due to the production of a lethal 3-methyladenine (3MeA) lesion . An alkylation-specific base-excision repair pathway in yeast is initiated by a Mag1 3MeA DNA glycosylase that removes the damaged base, followed by an Apn1 apurinic/ apyrimidinic endonuclease that cleaves the DNA strand at the abasic site for subsequent repair . MMS is also regarded as a radiomimetic agent, since a number of DNA radiation-repair mutants are also sensitive to MMS . To understand how these radiation-repair genes are involved in DNA methylation repair, we performed an epistatic analysis by combining yeast mag1 and apn1 mutations with mutations involved in each of the RAD3, RAD6 and RAD52 groups . We found that cells carrying rad6, rad18, rad50 and rad52 single mutations are far more sensitive to killing by MMS than the mag1 mutant, that double mutants were much more sensitive than either of the corresponding single mutants, and that the effects of the double mutants were either additive or synergistic, suggesting that post-replication and recombination-repair pathways recognize either the same lesions as MAG1 and APN1, or else some differ- ent lesions produced by MMS treatment . Lesions handled by recombination and post replication repair are not simply 3MeA, since over-expression of the MAG1 gene does not offset the loss of these pathways . Based on the above analyses, we discuss possible mechanisms for the repair of methylation damage by various pathways. Mol Gen Genet, 1996 Nov 27, 253(1-2), 173 - 81 Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae; Gjuracic K et al.; We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid . Plasmid pCW12, containing the ARG4 gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI . Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid . Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants . Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements . Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome. Mol Gen Genet, 1996 Nov 27, 253(1-2), 138 - 48 Cdc20, a beta-transducin homologue, links RAD9-mediated G2/M checkpoint control to mitosis in Saccharomyces cerevisiae; Lim HH et al.; In the budding yeast Saccharomyces cerevisiae, the DNA damage-induced G2 arrest requires the checkpoint control genes RAD9, RAD17, RAD24, MEC1, MEC2 and MEC3 . These genes also prevent entry into mitosis of a temperature-sensitive mutant, cdc13, that accumulates chromosome damage at 37 degrees C . Here we show that a cdc13 mutant overexpressing Cdc20, a beta-transducin homologue, no longer arrests in G2 at the restrictive temperature but instead undergoes nuclear division, exits mitosis and enters a subsequent division cycle, which suggests that the DNA damage-induced G2/M checkpoint control is not functional in these cells . This is consistent with our observation that overexpression of CDC20 in wild-type cells results in increased sensitivity to UV irradiation . Overproduction of Cdc20 does not influence the arrest phenotype of the cdc mutants whose cell cycle block is independent of RAD9-mediated checkpoint control . Therefore, we suggest that the DNA damage-induced checkpoint controls prevent mitosis by inhibiting the nuclear division pathway requiring CDC20 function. Mol Gen Genet, 1996 Nov 27, 253(1-2), 103 - 10 Characterization of a promoter mutation in the CYP3 gene of Saccharomyces cerevisiae which cancels regulation by Cyp1p (Hap1p) without affecting its binding site; Lodi T et al.; Cyp1p (Hap1p) activates, among others, the two structural genes, CYC1 and CYP3 (CYC7) which encode isocytochromes c in Saccharomyces cerevisiae . This activation is believed to occur through the binding of the protein to the dissimilar upstream activation sequences (UASs), UAS1 and UAS', present upstream of CYC1 and CYP3, respectively . In this paper, we describe a novel promoter mutation, CYP3-5, which results from a 39-bp deletion located about 160 bp upstream of the well-characterized CYP3 UAS . This deletion includes a sequence identical to the 3' moiety of the CYC1 UAS1 . Strikingly, a sequence identical to the 5' part of the CYC1 UAS1 is also present 60 bp downstream of the 3' half in the wild-type gene, suggesting that a spatial organization of the promoter might lead to the reconstitution in vivo of an active UAS1-like sequence . Interestingly, we find that in the presence of the CYP3-5 mutation, which disrupts this potential UAS1, the CYP-UAS' complex is importantly diminished and the transcription of CYP3 is insensitive to the wild-type CYP1-activating protein. Nucleic Acids Res, 1996 Nov 15, 24(22), 4543 - 51 Similar upstream regulatory elements of genes that encode the two largest subunits of RNA polymerase II in Saccharomyces cerevisiae; Jansma DB et al.; We have determined the location of cis-acting elements that are important for the expression of RPO21 and RPO22, genes that encode the two largest subunits of RNA polymerase II (RNAPII) in Saccharomyces cerevisiae . A series of 5'-end deletions and nucleotide substitutions in the upstream regions of RPO21 and RPO22 were tested for their effect on the expression of lacZ fusions of these genes . Deletion of sequences from -723 to -693 in RPO21, which disrupted two Reb1p-binding sites and an Abf1p-binding site, resulted in a 10-fold decrease in expression . A T-rich region downstream of these sites was also important for expression . Deletion of sequences from -437 to -392 in the RPO22-upstream, which resulted in a 30-fold decrease in expression, indicated that the Reb1p- and Abf1p-binding sites in this region were important for RPO22 expression, as was a T-rich sequence immediately downstream of these sites . The RPO21 and RPO22 upstream regions were capable of interacting in vitro (gel-mobility-shift assays) with Reb1p and Abf1p . The similarities in the type and organization of elements in the upstream regions of RPO21 and RPO22 suggest that expression of these genes may be regulated coordinately. Nucleic Acids Res, 1996 Nov 15, 24(22), 4450 - 5 Characterization of the basal inhibitor of class II transcription NC2 from Saccharomyces cerevisiae; Goppelt A et al.; Human NC2 utilizes a unique mechanism of repression of transcription by associating with TBP and inhibition of preinitiation complex formation . Here we have cloned two genes from Saccharomyces cerevisiae and functionally characterized them as yeast NC2 . We show that yeast NC2 binds to TBP as a heterodimer and represses RNA polymerase II transcription during assembly of the preinitiation complex . Yeast NC2 is highly homologous to its human counterpart within histone fold domains . C-Terminal regions previously discussed to be important for repression in man are in part not conserved . The human alpha but not the beta subunit efficiently heterodimerizes and represses transcription in combination with the corresponding yeast subunit . Yeast and human NC2 inhibit transcription in the presence of yeast and human TBP . However, repression is optimal within one species . The N-terminus of human TBP supports repression of transcription by human but not by yeast NC2. J Mol Biol, 1996 Nov 15, 263(5), 707 - 14 The anticodon loop is a major identity determinant of Saccharomyces cerevisiae tRNA(Leu); Soma A et al.; The recognition of tRNALeu, one of the class II tRNAs having a long variable arm, by leucyl-tRNA synthetase in Saccharomyces cerevisiae was studied using the T7 transcription system . Exchanging the anticodon arm of tRNALeu but not the D- or T psi C-arm to that of tRNASer seriously affected the leucine accepting activity . Two nucleotides in the anticodon loop, A35 and G37, were found to be important for leucylation . It was also found that the discriminator base, A73, is required for leucylation, and G73 of tRNASer functions as a negative identity determinant for leucyl-tRNA synthetase . Introducing a set of three base substitutions at positions 35, 37 and 73 was sufficient to convert tRNASer into an efficient leucine acceptor . These results indicate that the identity elements of tRNALeu lie at the second position of the anticodon and the 3' adjacent to the anticodon as well as the discriminator position . Such a sequence specific recognition manner is significantly different from that of Escherichia coli, in which not the anticodon but the tertiary structural elements play a key role in discriminating from other class II tRNAs . The leucine system is the first example which shows that the requirement of the anticodon sequence is variable among species. J Mol Biol, 1996 Nov 15, 263(5), 648 - 56 Rat RL23a ribosomal protein efficiently competes with its Saccharomyces cerevisiae L25 homologue for assembly into 60 S subunits; Jeeninga RE et al.; The large subunit protein RL23a from rat liver ribosomes shows 62% sequence identity with the primary rRNA-binding ribosomal protein L25 from Saccharomyces cerevisiae . In vitro binding studies indicated that both r-proteins are able to recognise the L25 binding site on yeast 25 S rRNA and its structural homologue on mammalian 28 S rRNA with equal efficiency . To determine whether the two r-proteins are also functionally equivalent in vivo, a single plasmid-borne copy of either the wild-type L25 gene or the RL23a cDNA, driven by the L25 promoter, was introduced into a yeast strain in which the chromosomal L25 gene is under control of the glucose-repressible GALI-10 promoter . No difference in growth rate could be detected between the two types of transformants when cultured on glucose-based medium . In cells that co-express epitope-tagged versions of L25 and RL23a from single-copy genes, approximately 35% of the 60 S subunits contained the heterologous protein as determined by Western analysis . This value could be increased to 55% by overexpressing RL23a using a multi-copy plasmid . These data demonstrate that rat RL23a can act as a highly efficient substitute for its yeast counterpart in the assembly of functional yeast ribosomes even in the presence of the endogenous L25 protein. Genes Dev, 1996 Nov 15, 10(22), 2831 - 48 Two novel targets of the MAP kinase Kss1 are negative regulators of invasive growth in the yeast Saccharomyces cerevisiae; Cook JG et al.; Haploid cells of budding yeast Saccharomyces cerevisiae respond to mating pheromones by inducing genes required for conjugation, arresting cell cycle progression, and undergoing morphological changes . The same cells respond to nutrient deprivation by altering budding pattern and inducing genes required for invasive growth . Both developmental alternatives to vegetative proliferation require the MAP kinase Kss1 and the transcriptional transactivator Ste12 . Using a two-hybrid screen for gene products that interact with Kss1, two homologous and previously uncharacterized loci (DIG1 and DIG2, for down-regulator of invasive growth) were identified . DIG2 is pheromone-inducible, whereas DIG1 is constitutively expressed . Dig1 colocalizes with Kssl in the nucleus, coimmunoprecipitates with Kss1 from cell extracts in a pheromone-independent manner, and is phosphorylated by Kss1 in immune complexes in a pheromone-stimulated manner . Kss1 binds specifically to a GST-Dig1 fusion in the absence of any other yeast protein . Using the two-hybrid method, both Dig1 and Dig2 also interact with the other MAP kinase of the pheromone response pathway, Fus3 . However, neither dig1 or dig2 single mutants, nor a dig1 dig2 double mutant, have a discernible effect on mating . In contrast, dig1 dig2 cells constitutively invade agar medium, whereas a dig1 dig2 ste12 triple mutant does not, indicating that Dig1 and Dig2 share a role in negatively regulating the invasive growth pathway . High-level expression of Dig1 suppresses invasive growth and also causes cells to appear more resistant to pheromone-imposed cell cycle arrest . Ste12 also binds specifically to GST-Dig1 in the absence of any other yeast protein . Collectively, these findings indicate that Dig1, and most likely Dig2, are physiological substrates of Kssl and suggest that they regulate Ste12 function by direct protein-protein interaction. J Biol Chem, 1996 Nov 15, 271(46), 29295 - 303 Expression of rat aspartyl-tRNA synthetase in Saccharomyces cerevisiae . Role of the NH2-terminal polypeptide extension on enzyme activity and stability; Agou F et al.; Cytoplasmic aspartyl-tRNA synthetase from mammals is one of the components of a multienzyme complex comprising nine synthetase activities . The presence of an amino-terminal extension composed of about 40 residues is a characteristic of the eukaryotic enzyme . We report here the expression in the yeast Saccharomyces cerevisiae of a native form of rat aspartyl-tRNA synthetase and of two truncated derivatives lacking 20 or 36 amino acid residues from their amino-terminal polypeptide extension . The three recombinant enzyme species were purified to homogeneity . They behave as alpha2 dimers and display catalytic parameters in the tRNA aminoacylation reaction identical to those determined for the native, complex-associated form of aspartyl-tRNA synthetase isolated from rat liver . Because the dimer dissociation constant of rat AspRS is much higher than that of its bacterial and yeast counterparts, we could establish a direct correlation between dissociation of the dimer and inactivation of the enzyme . Our results clearly show that the monomer is devoid of amino acid activation and tRNA aminoacylation activities, indicating that dimerization is essential to confer an active conformation on the catalytic site . The two NH2-terminal truncated derivatives were fully active, but proved to be more unstable than the recombinant native enzyme, suggesting that the polypeptide extension fulfills structural rather than catalytic requirements. J Biol Chem, 1996 Nov 15, 271(46), 28953 - 9 The two acetyl-coenzyme A synthetases of Saccharomyces cerevisiae differ with respect to kinetic properties and transcriptional regulation; van den Berg MA et al.; Saccharomyces cerevisiae contains two structural genes, ACS1 and ACS2, each encoding an active acetyl-coenzyme A synthetase . Characterization of enzyme activities in cell-free extracts from strains expressing either of the two genes revealed differences in the catalytic properties of the two enzymes . The Km for acetate of Acs1p was about 30-fold lower than that of Acs2p and Acs1p, but not Acs2p, could use propionate as a substrate . Enzyme activity measurements and mRNA analyses showed that ACS1 and ACS2 were both expressed during carbon-limited growth on glucose, ethanol, and acetate in aerobic chemostat cultures . In anaerobic glucose-limited cultures, only the ACS2 gene was expressed . Based on these facts, the products of the ACS1 and ACS2 genes were identified as the previously described "aerobic" and "non-aerobic" forms of acetyl-coenzyme A synthetase, respectively . Batch and glucose-pulse experiments revealed that transcription of ACS1 is subject to glucose repression . A mutant strain lacking Acs2p was unable to grow on glucose in batch cultures, but grew readily in aerobic glucose-limited chemostat cultures, in which the low residual glucose concentration alleviated glucose repression . Experiments in which ethanol was pulsed to aerobic ethanol-limited chemostat cultures indicated that, in addition to glucose, ethanol also repressed ACS1 transcription, although to a lesser extent . In contrast, transcription of ACS2 was slightly induced by ethanol and glucose . Absence of ACS2 prevented complete glucose repression of ACS1, indicating that ACS2 (in)directly is involved in the transcriptional regulation of ACS1. Science, 1996 Nov 15, 274(5290), 1201 - 4 Identification of subunits of the anaphase-promoting complex of Saccharomyces cerevisiae; Zachariae W et al.; Entry into anaphase and proteolysis of B-type cyclins depend on a complex containing the tetratricopeptide repeat proteins Cdc16p, Cdc23p, and Cdc27p . This particle, called the anaphase-promoting complex (APC) or cyclosome, functions as a cell cycle-regulated ubiquitin-protein ligase . Two additional subunits of the budding yeast APC were identified: The largest subunit, encoded by the APC1 gene, is conserved between fungi and vertebrates and shows similarity to BIMEp from Aspergillus nidulans . A small heat-inducible subunit is encoded by the CDC26 gene . The yeast APC is a 36S particle that contains at least seven different proteins. Gene, 1996 Nov 14, 179(2), 205 - 10 Activation of the human estrogen receptor by estrogenic and antiestrogenic compounds in Saccharomyces cerevisiae: a positive selection system; Shiau SP et al.; The yeast URA3 gene was used as a reporter to investigate the activities of estrogenic and antiestrogenic compounds in yeast Saccharomyces cerevisiae . The control sequences of the wild type (wt) URA3 promoter were replaced with zero, two, or six copies of estrogen-response elements (ERE) . Insertion of two and six copies of ERE rendered the expression of the URA3 gene to be dependent on the presence of the human estrogen receptor (ER) and the hormone 17beta-estradiol (E2) . Two versions of the ER genes were constructed: a full-length wild-type ER (ERa-f) and a truncated ER with domains C, D, and E (ERcde) . Both forms of the ER were able to activate the ERE-URA3 reporter in a hormone-dependent manner . The growth of yeast transformants were hormone-dependent when the reporter constructs were inserted into chromosomes using yeast integrating vectors (YIp) but not with the 2mu-based episomal (high-copy number, YEp) or centromeric (low-copy number, YCp) vectors . The integrated transformants were employed to investigate the effects of estrogenic and antiestrogenic compounds . The estrogenic compounds, E2, diethylstilbestrol (DES), and estrone (EST), activated expression of the reporter genes at 1 nM concentration, which is the same concentration exhibiting activity in mammalian cells . None of the antiestrogens, at concentrations up to 1 microM, including tamoxifen (TAM), raloxifene (RAL), and ICI 164,384 (ICI) antagonized 1 nM of E2 against either form of the ER . In fact, TAM, RAL, and ICI displayed slight agonistic activity at high concentrations of 300 nM or greater to the ERcde . This system can be used to investigate or clone the missing factor(s) that is responsible for the antagonistic activity of the ER in yeast, and is also suitable for screening for the effectors of the ER. Biochim Biophys Acta, 1996 Nov 13, 1285(1), 71 - 8 Characterization of a non-specific lipid transfer protein associated with the peroxisomal membrane of the yeast, Saccharomyces cerevisiae; Ceolotto C et al.; A lipid transfer protein with a broad substrate specificity is associated with the peroxisomal membrane of the yeast Saccharomyces cerevisiae . The protein catalyzes in vitro the transfer of various phospholipids, phosphatidylinositol and phosphatidylserine being translocated at the highest rates . The transfer protein can be released from peroxisomal membranes by treatment with 0.25 M KCl and highly enriched using conventional chromatographic techniques . It is inactivated by heat, detergents, divalent cations and proteinases . During various steps of purification this lipid transfer protein co-fractionated with peroxisomal acyl-CoA oxidase (Pox1p) . In a pox1 disruptant peroxisomal lipid transfer activity was still present, although at a reduced level . The peroxisomal lipid transfer protein from the pox1 mutant exhibited different chromatographic properties as compared to the wild-type strain suggesting that acyl-CoA oxidase and the peroxisomal lipid transfer protein may from a complex. Biochem Biophys Res Commun, 1996 Nov 12, 228(2), 452 - 8 A second gene encoding a putative serine/threonine protein kinase which enhances spermine uptake in Saccharomyces cerevisiae; Nozaki T et al.; We have isolated a new gene (PTK2) which restores spermine uptake of a polyamine uptake-deficient mutant of Saccharomyces cerevisiae (Kakinuma, Y., Maruyama, T., Nozaki, T., Wada, Y., Oshumi, Y., and Igarashi, K., 1995, Biochem, Biophys . Res . Commun . 216, 985-992) . In magnesium-limited medium, the cell growth of a spermine-sensitive polyamine uptake mutant transformed with PTK2 recovered its sensitivity to spermine . The nucleotide sequence of the PTK2 gene indicated that it is identical with the YJR059W open reading frame of chromosome X encoding a putative serine/threonine protein kinase . The deduced amino acid sequence of the PTK2 gene product was 38% identical and 55% similar with that of the PTK1 (POT1) gene product, a putative serine/threonine protein kinase, which was found to enhance spermine uptake of the same mutant . The results indicate that polyamine transport of yeast is regulated by multiple phosphorylation/dephosphorylation pathways. Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 13256 - 61 ORK1, a potassium-selective leak channel with two pore domains cloned from Drosophila melanogaster by expression in Saccharomyces cerevisiae; Goldstein SA et al.; A K+ channel gene has been cloned from Drosophila melanogaster by complementation in Saccharomyces cerevisiae cells defective for K+ uptake . Naturally expressed in the neuromuscular tissues of adult flies, this gene confers K+ transport capacity on yeast cells when heterologously expressed . In Xenopus laevis oocytes, expression yields an ungated K(+)-selective current whose attributes resemble the "leak" conductance thought to mediate the resting potential of vertebrate myelinated neurons but whose molecular nature has long remained elusive . The predicted protein has two pore (P) domains and four membrane-spanning helices and is a member of a newly recognized K+ channel family . Expression of the channel in flies and yeast cells makes feasible studies of structure and in vivo function using genetic approaches that are not possible in higher animals. Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 13054 - 9 Double strand breaks at the HIS2 recombination hot spot in Saccharomyces cerevisiae; Bullard SA et al.; Double strand breaks (DSBs) have been found at several meiotic recombination hot spots in Saccharomyces cerevisiae; more global studies have found that they occur at many places along several yeast chromosomes during meiosis . Indeed, the number of breaks found is consistent with the number of recombination events predicted from the genetic map . We have previously demonstrated that the HIS2 gene is a recombination hot spot, exhibiting a high frequency of gene conversion and associated crossing over . This paper shows that DSBs occur in meiosis at a site in the coding region and at a site downstream of the HIS2 gene and that the DSBs are dependent upon genes required for recombination . The frequency of DSBs at HIS2 increases when the gene conversion frequency is increased by alterations in the DNA around HIS2, and vice versa . A deletion that increases both DSBs and conversion can stimulate both when heterozygous; that is, it is semidominant and acts to stimulate DSBs in trans . These data are consistent with the view that homologous chromosomes associate with each other before the formation of the DSBs. J Biol Chem, 1996 Nov 8, 271(45), 28422 - 9 Structure-function relationships of the Saccharomyces cerevisiae fatty acid synthase . Three-dimensional structure; Kolodziej SJ et al.; The three-dimensional structure of the Saccharomyces cerevisie fatty acid synthase was computed from electron microscopy of stain images . The barrel-shaped structure (point group symmetry 32) has major and minor axes of approximately 245 x 220 A, respectively, and consists of two different subunits organized in an alpha6beta6 complex (Mr = 2.5 x 10(6)) . Two sets of three beta subunits form triangle-shaped caps that enclose the ends of the barrel . The wall of the barrel appears to consist of three N-shaped alpha subunit pairs each with an over and underlying arch-shaped beta subunit . Inside the molecule there are three major interconnected cavities that are tilted approximately 20 degrees with respect to its major axis . An axle-shaped structure extends the length of the cavity on the 3-fold axis and is connected to the two ends of the barrel . The cavities are partially divided on the equator of the molecule by three spokes that extend from the axle on the 2-fold axis to the exterior wall . We propose that these six cavities constitute the six equivalent sites of fatty acid synthesis resulting in an extraordinary structure-function relationship with the 42 catalytic sites involved in fatty acid synthesis inside the molecule . The six cavities each have two funnel-shaped openings ( approximately 20 A in diameter) which may serve to permit the diffusion of substrates and products in and out of these functional units . The subunits appear to be arranged in a manner that affords extensive intermolecular interactions contributing to the stability of this macromolecular complex. Gene, 1996 Nov 7, 179(1), 171 - 7 Regulation of phosphatase synthesis in Saccharomyces cerevisiae--a review; Oshima Y et al.; Transcription of the genes encoding acid and alkaline phosphatases and the inorganic phosphate (Pi) transporter of Saccharomyces cerevisiae are coordinately repressed and derepressed depending on the Pi concentration in the culture medium . This phosphatase system is particularly suited for the study of regulatory mechanisms, because the acid phosphatase activity of each colony on a plate is easily detected by specific staining methods and there is a 500-fold difference between the repressed and derepressed levels of acid phosphatase activity . With these advantages, considerable amounts of genetic and molecular evidence have been accumulated in the past two decades . This article summarizes our current knowledge on this subject. Radiats Biol Radioecol, 1996 Nov-Dec, 36(6), 888 - 94 {The chronic action of gamma radiation on a growing population of Saccharomyces cerevisiae yeasts at different dose rates}; Ziuzikov NA et al.; Experimental data on the processes of division and death of haploid and diploid yeast Saccharomyces cerevisiae of wild type and of their radiosensitive mutants exposed under optimal for reproduction conditions to chronic action of gamma-radiation at various dose rates are presented . It is shown that the dependence of the integral division/death process on time was exponential for all the studied strains . With dose rate increasing, the duration of the lag period and the probability of cell inactivation increased, while the multiplication rate decreased . These processes, for equal dose rates, were more expressed for the radiosensitive mutants. Appl Microbiol Biotechnol, 1996 Nov, 46(4), 393 - 9 The cytosolic pathway of L-malic acid synthesis in Saccharomyces cerevisiae: the role of fumarase; Pines O et al.; Saccharomyces cerevisiae accumulates L-malic acid but not only minute amounts of fumaric acid . A 13C-nuclear magnetic resonance study following the label from glucose to L-malic acid indicates that the L-malic acid is synthesized from pyruvic acid via oxaloacetic acid . From this, and from previously published studies, we conclude that a cytosolic reductive pathway leading from pyruvic acid via oxaloacetic acid to L-malic acid is responsible for the L-malic acid production in yeast . The non-production of fumaric acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric acid to L-malic but not the reverse . This conclusion is based on the following findings . (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric acid than towards L-malic acid; the Km for L-malic acid is very high indicating that L-malic acid is not an in vivo substrate of the enzyme . (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric acid (but rather more L-malic acid) . (c) According to 13C NMR studies there is no interconversion of cytosolic L-malic and fumaric acids. Electrophoresis, 1996 Nov, 17(11), 1683 - 99 Two-dimensional gel protein database of Saccharomyces cerevisiae; Boucherie H et al.; With the systematic sequencing of the yeast genome, yeast biology has entered a new era where novel challenges have to be faced . One challenge is the identification of the function of the several hundred novel genes discovered by genome sequencing . Another is to understand how all yeast genes act in concert to ensure and maintain cell organization . Two-dimensional (2-D) gel electrophoresis is the technique of choice to take up these challenges because it provides the opportunity of obtaining an overall view of genome expression . In prospect of these studies we have undertaken the construction of a yeast 2-D gel protein database that contains information on polypeptides of the yeast protein map . In this paper we report the information presently contained in this database . The reported information includes the identification of 250 protein spots and the characterization of polypeptides corresponding to N-terminal acetylated proteins, mitochondrial proteins, glucose-repressed proteins, heat shock induced proteins and proteins encoded by intron-containing genes . In all, 600 spots are annotated . These data can be accessed on the Yeast Protein Map server through the World Wide Web network. Biochem Mol Biol Int, 1996 Nov, 40(5), 915 - 21 Isolation and characterization of Saccharomyces cerevisiae SAB2, a suppressor gene for temperature-sensitive phenotype of ARS-binding factor 1 mutant; Shin YH et al.; A high-copy number suppressor of yeast abf1-5 mutant, a temperature-sensitive lethal mutant, was isolated and named SAB2 (suppressor of ABF1) . Hybridization to a yeast chromoblot and to prime clone grid filters revealed that the SAB2 gene was located near the yeast SUP3 on chromosome XV . The suppressor activity was contained in a 2.5 Kbp DNA region of the SAB2 plasmid . The nucleotide sequence of the DNA region contained a long open reading frame, which turned out to encode for yeast tryptophan permease . Four putative ABF1 binding sites were found in the promoter and the structural regions of the tryptophan permease gene . Binding of ABF1 to two of the sites tested in this study was detected . Our results indicate that ABF1 may be involved in the transcriptional control of the yeast tryptophan permease gene. Mol Microbiol, 1996 Nov, 22(4), 739 - 46 Stationary-phase induction of GLR1 expression is mediated by the yAP-1 transcriptional regulatory protein in the yeast Saccharomyces cerevisiae; Grant CM et al.; Glutathione (GSH) is an abundant low-molecular-mass thiol which has been implicated in numerous cellular processes including protection against cytotoxic agents such as xenobiotics, carcinogens and free radicals . Utilization of GSH results in its conversion to the oxidized form (GSSG), and it is recycled to GSH by the action of glutathione reductase (GLR) using the reducing power of NADPH . We show that GLR activity is increased by three- to fourfold during stationary-phase growth compared to exponential phase growth, and that a yeast strain deleted for GLR1, encoding glutathione reductase, shows an elevated sensitivity to H2O2 challenge during stationary phase . These data indicate an increased requirement for GSH as the cell arrests growth and enters stationary phase . The stationary-phase increase in GLR activity is entirely dependent upon the action of the yAP-1 transcriptional regulatory protein, previously implicated in regulating GLR activity in response to oxidative stress . Thus, both oxidant- and growth phase-mediated control of GLR1 expression are regulated by the same transcriptional control mechanism . In addition, strains lacking GLR or yAP-1 do not accumulate GSSG during stationary-phase growth, indicating that the cell possesses alternative means of preventing an accumulation of GSSG during stationary phase. Mol Microbiol, 1996 Nov, 22(4), 681 - 92 Cloning of the ASN1 and ASN2 genes encoding asparagine synthetases in Saccharomyces cerevisiae: differential regulation by the CCAAT-box-binding factor; Dang VD et al.; Two new yeast genes, named ASN1 and ASN2, were isolated by complementation of the growth defect of an asparagine auxotrophic mutant . Genetical analysis indicates that these two genes are allelic to the asnA and asnB loci described previously . Simultaneous disruption of both genes leads to a total asparagine auxotrophy, while disruption of asn1 or asn2 alone has no effect on growth under tested conditions . Nucleotide sequences of ASN1 and ASN2 revealed striking similarities with genes encoding asparagine synthetase (AS) from other organisms . Regulation of ASN1 and ASN2 expression was studied using lacZ fusions and both genes were found to be several times less expressed in the absence of the transcription activator Gcn4p . The HAP complex, another transcription factor that binds to CCAAT-box sequences, was shown to specifically affect ASN1 expression . Hap2p and Hap3p subunits of the HAP complex are required for optimal expression of ASN1, while the Hap4p regulatory subunit, which is required for regulation by the carbon source, plays a minor role in this process . Consistent with the weak effect of Hap4p, the carbon source does not significantly affect expression of ASN1 . Our results show that the role of the HAP complex is not limited to activation of genes required for respiratory metabolism. J Appl Bacteriol, 1996 Nov, 81(5), 481 - 5 Effect of calcium channel blockers on the action of various antitumour agents in the yeast Saccharomyces cerevisiae; Tillgada E et al.; The cytotoxic effects of a 22 h treatment with four antineoplastic agents in the yeast Saccharomyces cerevisiae ATCC 2366 were investigated . Two agents, doxorubicin and 5-fluorouracil (5-FU), were effective in decreasing the colony-forming ability of yeast cells . Following examination under the light microscope, the effect of doxorubicin appeared to be, at least partially, due to killing of yeast cells whereas the effect of 5-FU was rather due to changes in cell structure leading to abnormal bud formation . For amsacrine (AMSA) and melphalan, cytotoxicity was totally absent . In the presence of diltiazem the above described effects were not significantly changed . When verapamil was added in the culture medium the cytotoxic activity of doxorubicin and 5-FU did not change . However, following treatment with AMSA in combination with verapamil, cell survival was significantly decreased whereas the presence of verapamil increased the yeast survival which was observed after melphalan treatment. Nucleic Acids Res, 1996 Nov 1, 24(21), 4222 - 6 Saccharomyces cerevisiae IRE2/HAC1 is involved in IRE1-mediated KAR2 expression; Nikawa J et al.; The Saccharomyces cerevisiae IRE1 gene, encoding a putative receptor-type protein kinase, is known to be required for inositol prototrophy and for the induction of a chaperon molecule, BiP, encoded by KAR2, under stress conditions such as tunicamycin addition . We have characterized a yeast gene, IRE2, which was isolated as a suppressor gene that complements the inositol auxotrophic phenotype of the ire1 mutation . Sequencing analysis revealed that IRE2 is identical to HAC1, which encodes a transcription factor having a basic-leucine zipper motif . Introduction of IRE2/HAC1 into the ire1 mutant clearly restored the expression of KAR2 upon tunicamycin treatment . ire2/hac1-disrupted yeast cells showed not only the inositol auxotrophic phenotype but also the tunicamycin sensitivity, and failed to induce the expression of KAR2 . These results clearly indicate that the IRE2/HAC1 gene product plays a critical role in the induction of KAR2 expression and in the inositol prototrophy mediated by IRE1. Mol Biol Cell, 1996 Nov, 7(11), 1835 - 55 Nuclear transport defects and nuclear envelope alterations are associated with mutation of the Saccharomyces cerevisiae NPL4 gene; DeHoratius C et al.; To identify components involved in nuclear protein import, we used a genetic selection to isolate mutants that mislocalized a nuclear-targeted protein . We identified temperature-sensitive mutants that accumulated several different nuclear proteins in the cytoplasm when shifted to the semipermissive temperature of 30 degrees C; these were termed npl (nuclear protein localization) mutants . We now present the properties of yeast strains bearing mutations in the NPL4 gene and report the cloning of the NPL4 gene and the characterization of the Np14 protein . The npl4-1 mutant was isolated by the previously described selection scheme . The second allele, npl4-2, was identified from an independently derived collection of temperature-sensitive mutants . The npl4-1 and npl4-2 strains accumulate nuclear-targeted proteins in the cytoplasm at the nonpermissive temperature consistent with a defect in nuclear protein import . Using an in vitro nuclear import assay, we show that nuclei prepared from temperature-shifted npl4 mutant cells are unable to import nuclear-targeted proteins, even in the presence of cytosol prepared from wild-type cells . In addition, npl4-2 cells accumulate poly(A)+ RNA in the nucleus at the nonpermissive temperature, consistent with a failure to export mRNA from the nucleus . The npl4-1 and npl4-2 cells also exhibit distinct, temperature-sensitive structural defects: npl4-1 cells project extra nuclear envelope into the cytoplasm, whereas npl4-2 cells from nuclear envelope herniations that appear to be filled with poly(A)+ RNA . The NPL4 gene encodes an essential M(r) 64,000 protein that is located at the nuclear periphery and localizes in a pattern similar to nuclear pore complex proteins . Taken together, these results indicate that this gene encodes a novel nuclear pore complex or nuclear pore complex-associated component required for nuclear membrane integrity and nuclear transport. Curr Genet, 1996 Nov, 30(5), 404 - 9 Cloning and characterization of the first two genes of the non-oxidative part of the Saccharomyces cerevisiae pentose-phosphate pathway; Miosga T et al.; We have cloned and characterized the two remaining unknown genes of the non-oxidative part of the pentose-phosphate pathway of Saccharomyces cerevisiae encoding the enzymes D-ribulose-5-phosphate 3-epimerase (Rpe1p) and D-ribose-5-phosphate ketol-isomerase (Rki1p) . Rpe1p has an unexpected high specific activity of 2148 mU x (mg protein)-1 in crude extracts . Deletion mutants of RPE1 show no enzyme activity and are unable to grow on D-xylulose . Unexpectedly, haploid rki1 deletion mutants are not viable . Functional expression of RKI1 was demonstrated following an increase of gene dosage in the haploid rki1 deletion mutant, which restored viability and specific D-ribose-5-phosphate ketol-isomerase activity . Both enzymes show high similarity to the deduced protein sequences of various open reading frames, expressed sequence tags or cDNAs from different organisms. Curr Genet, 1996 Nov, 30(5), 396 - 403 Molecular analysis of UFE1, a Saccharomyces cerevisiae gene essential for spore formation and vegetative growth; Downing TA et al.; The UFE1 gene of Saccharomyces cerevisiae was cloned, sequenced and characterized . The coding region of UFE1 is separated from the TMP1 gene on chromosome XV by 624 bp . Gene-disruption experiments demonstrated that UFE1 is essential for both the germination of ascospores and for vegetative growth . Translation of the UFE1 coding region generates a protein with significant similarity to cytokeratin and to the coiled-coil region of SED5, USO1 and restin, suggesting that it is involved in the secretory pathway and may also be related to intermediate filament-associated proteins. Arch Microbiol, 1996 Nov, 166(5), 315 - 20 Activity of plasma membrane H+-ATPase and expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cells grown at optimal and low pH; Carmelo V et al.; Cells of Saccharomyces cerevisiae grown in media with an initial pH of 2.5-6.0, acidified with a strong acid (HCl), exhibited the highest plasma membrane H+-ATPase-specific activity at an initial pH of 6.0 . At a lower pH (above pH 2.5) ATPase activity (62-83% of the maximum level) still allowed optimal growth . At pH 2.5, ATPase activity was about 30% of the maximum value and growth was impaired . Quantitative immunoassays showed that the content of ATPase protein in the plasma membrane was similar across the entire pH range tested, although slightly lower at pH 2.5 . The decrease of plasma membrane ATPase activity in cells grown at low pH was partially accounted for by its in vitro stability, which decreased sharply at pH below 5.5, although the reduction of activity was far below the values expected from in vitro measurements . Yeast growth under acid stress changed the pattern of gene expression observed at optimal pH . The level of mRNA from the essential plasma-membrane-ATPase-encoding gene PMA1 was reduced by 50% in cells grown at pH 2.5 as compared with cells grown at the optimal pH 5.0, although the content of ATPase in the plasma membrane was only modestly reduced . As observed in response to other kinds of stress, the PMA2 promoter at the optimal pH was up to eightfold more efficient in cells grown at pH 2.5, although it remained several hundred times less efficient than that of the PMA1 gene. EMBO J, 1996 Nov 1, 15(21), 5752 - 9 The PMT gene family: protein O-glycosylation in Saccharomyces cerevisiae is vital; Gentzsch M et al.; The transfer of mannose to seryl and threonyl residues of secretory proteins is catalyzed by a family of protein mannosyltransferases coded for by seven genes (PMT1-7) . Mannose dolichylphosphate is the sugar donor of the reaction, which is localized at the endoplasmic reticulum . By gene disruption and crosses all single, double and triple mutants of genes PMT1-4 were constructed . Two of the double and three of the triple mutants were not able to grow under normal conditions; three of these mutants could grow, however, when osmotically stabilized . The various mutants were extensively characterized concerning growth, morphology and their sensitivity to killer toxin K1, caffeine and calcofluor white . O-Mannosylation of gp115/Gas1p was affected only in pmt4 mutants, whereas glycosylation of chitinase was mainly affected in pmt1 and pmt2 mutants . The results show that protein O-glycosylation is essential for cell wall rigidity and cell integrity and that this protein modification, therefore, is vital for Saccharomyces cerevisiae. Genetics, 1996 Nov, 144(3), 957 - 66 Regional bivalent-univalent pairing versus trivalent pairing of a trisomic chromosome in Saccharomyces cerevisiae; Koller A et al.; In meiosis I, homologous chromosomes pair, recombine and segregate to opposite poles . These events and subsequent meiosis II ensure that each of the four meiotic products has one complete set of chromosomes . In this study, the meiotic pairing and segregation of a trisomic chromosome in a diploid (2n + 1) yeast strain was examined . We find that trivalent pairing and segregation is the favored arrangement . However, insertions near the centromere in one of the trisomic chromosomes leads to preferential pairing and segregation of the "like" centromeres of the remaining two chromosomes, suggesting that bivalent-univalent pairing and segregation is favored for this region. Genetics, 1996 Nov, 144(3), 923 - 33 Synergy between trehalose and Hsp104 for thermotolerance in Saccharomyces cerevisiae; Elliott B et al.; We isolated a mutant strain unable to acquire heat shock resistance in stationary phase . Two mutations contributed to this phenotype . One mutation was at the TPS2 locus, which encodes trehalose-6-phosphate phosphatase . The mutant fails to make trehalose and accumulates trehalose-6-phosphate . The other mutation was at the HSP104 locus . Gene disruptions showed that tps2 and hsp104 null mutants each produced moderate heat shock sensitivity in stationary phase cells . The two mutations were synergistic and the double mutant had little or no stationary phase-induced heat shock resistance . The same effect was seen in the tps1 (trehalose-6-phosphate synthase) hsp104 double mutant, suggesting that the extreme heat shock sensitivity was due mainly to a lack of trehalose rather than to the presence of trehalose-6-phosphate . However, accumulation of trehalose-6-phosphate did cause some phenotypes in the tps2 mutant, such as temperature sensitivity for growth . Finally, we isolated a high copy number suppressor of the temperature sensitivity of tps2, which we call PMU1, which reduced the levels of trehalose-6-phosphate in tps2 mutants . The encoded protein has a region homologous to the active site of phosphomutases. Genetics, 1996 Nov, 144(3), 905 - 21 Identification and characterization of FAR3, a gene required for pheromone-mediated G1 arrest in Saccharomyces cerevisiae; Horecka J et al.; In haploid Saccharomyces cerevisiae cells, mating pheromones activate a signal transduction pathway that leads to cell cycle arrest in the G1 phase and to transcription induction of genes that promote conjugation . To identify genes that link the signal transduction pathway and the cell cycle machinery, we developed a selection strategy to isolate yeast mutants specifically defective for G1 arrest . Several of these mutants identified previously known genes, including CLN3, FUS3, and FAR1 . In addition, a new gene, FAR3, was identified and characterized . FAR3 encodes a novel protein of 204 amino acid residues that is dispensable for viability . Northern blot experiments indicated that FAR3 expression is constitutive with respect to cell type, pheromone treatment, and cell cycle position . As a first step toward elucidating the mechanism by which Far3 promotes pheromone-mediated G1 arrest, we performed genetic and molecular experiments to test the possibility that Far3 participates in one of the heretofore characterized mechanisms, namely Fus3/Far1-mediated inhibition of Cdc28-Cln kinase activity, G1 cyclin gene repression, and G1 cyclin protein turnover . Our data indicate that Far3 effects G1 arrest by a mechanism distinct from those previously known. Genetics, 1996 Nov, 144(3), 893 - 903 The Saccharomyces cerevisiae RTG2 gene is a regulator of aconitase expression under catabolite repression conditions; Velot C et al.; The ACO1 gene, encoding mitochondrial aconitase of Saccharomyces cerevisiae, is required both for oxidative metabolism and for glutamate prototrophy . This gene is subject to catabolite repression; the ACOI mRNA level is further reduced when glutamate is supplied with glucose . To further explore regulation of ACOI expression, we have screened for mutations that reduce expression of an ACOI-lacZ fusion borne on a multicopy vector . We identified a gene required for wild-type expression of ACOI only under catabolite repression conditions . Sequencing of the corresponding cloned gene revealed that it is identical to RTG2 previously cloned as a pivotal gene in controlling interorganelle retrograde communication . Cells containing either the original rtg2-2 mutation or a null rtg2 allele are not petite but show a residual growth on minimum glucose medium with ammonium sulfate as the sole nitrogen source . This growth defect is partially restored by supplying aspartate or threonine, and fully with glutamate or proline supplement . Surprisingly, this phenotype is not observed on complete medium lacking either of these amino acids . In addition, a genetic analysis revealed an interaction between RTG2 and ASP5 (encoding aspartate amino transferase), thus supporting our hypothesis that RTG2 may be involved in the control of several anaplerotic pathways. J Biol Chem, 1996 Nov 1, 271(44), 27765 - 9 HDF2, the second subunit of the Ku homologue from Saccharomyces cerevisiae; Feldmann H et al.; The high affinity DNA binding factor (HDF) protein of Saccharomyces cerevisiae is composed of two subunits and specifically binds ends of double-stranded DNA . The 70-kDa subunit, HDF1, shows significant homology with the 70-kDa subunit of the human Ku protein . Like the Ku protein, HDF1 has been shown to be involved in recombination and double stranded DNA break repair . We have purified and cloned HDF2, the second subunit of the HDF protein . The amino acid sequence of HDF2 shows a 45.6% homology with the 80-kDa subunit of the Ku protein . HDF1 by itself does not bind DNA, while HDF2 protein on its own seems to displays DNA binding activity . Targeted disruption of the HDF2 gene causes a temperature-sensitive phenotype for growth comparable to the phenotype of hdf1(-) strains . The human Ku protein cannot complement this temperature-sensitive phenotype . hdf2(-) strains are sensitive to bleomycin and methyl methanesulfonate, but this sensitivity is reduced in comparison with hdf1(-) strains. J Biol Chem, 1996 Nov 1, 271(44), 27615 - 22 Role of the cysteine residues in the alpha1,2-mannosidase involved in N-glycan biosynthesis in Saccharomyces cerevisiae . The conserved Cys340 and Cys385 residues form an essential disulfide bond; Lipari F et al.; The Saccharomyces cerevisiae alpha1,2-mannosidase, which removes one specific mannose residue from Man9GlcNAc2 to form Man8GlcNAc2, is a member of a family of alpha1,2-mannosidases with similar amino acid sequences . The yeast alpha1,2-mannosidase contains five cysteine residues, three of which are conserved . Recombinant yeast alpha1, 2-mannosidase, produced as the soluble catalytic domain, was shown to contain two disulfide bonds and one free thiol group using 2-nitro-5-thiosulfobenzoate and 5,5'-dithiobis(2-nitrobenzoate), respectively . Cys485 contains the free thiol group, as demonstrated by sequencing of labeled peptides following modification with {3H}ICH2COOH and by high performance liquid chromatography/mass spectrometry tryptic peptide mapping . A Cys340-Cys385 disulfide was demonstrated by sequencing a purified peptide containing this disulfide and by tryptic peptide mapping . Cys468 and Cys471 were not labeled with {3H}ICH2COOH and a peptide containing these two residues was identified in the tryptic peptide map, showing that Cys468 and Cys471 form the second disulfide bond . The alpha1, 2-mannosidase loses its activity in the presence of dithiothreitol with first order kinetics, suggesting that at least one disulfide bond is essential for activity . Mutagenesis of each cysteine residue to serine showed that Cys340 and Cys385 are essential for production of recombinant enzyme, whereas Cys468, Cys471, and Cys485 are not required for production and enzyme activity . These results indicate that the sensitivity to dithiothreitol is due to reduction of the Cys340-Cys385 disulfide . Since Cys340 and Cys385 are conserved residues, it is likely that this disulfide bond is important to maintain the correct structure in the other members of the alpha1, 2-mannosidase family. J Biol Chem, 1996 Nov 1, 271(44), 27280 - 4 Selective inhibitors of the proteasome-dependent and vacuolar pathways of protein degradation in Saccharomyces cerevisiae; Lee DH et al.; We have studied whether various agents that inhibit purified yeast and mammalian 26 S proteasome can suppress the breakdown of different classes of proteins in Saccharomyces cerevisiae . The degradation of short-lived proteins was inhibited reversibly by peptide aldehyde inhibitors of proteasomes, carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132) and carbobenzoxyl-leucinyl-leucinyl-norvalinal (MG115), in a yeast mutant with enhanced permeability, but not in wild-type strains . Lactacystin, an irreversible proteasome inhibitor, had no effect, but the beta-lactone derivative of lactacystin, which directly reacts with proteasomes, inhibited the degradation of short-lived proteins . These inhibitors also blocked the rapid ubiquitin-dependent breakdown of a beta-galactosidase fusion protein and caused accumulation of enzymatically active molecules in cells . The degradation of the bulk of cell proteins, which are long-lived molecules, was not blocked by proteasome inhibitors, but could be blocked by phenylmethylsulfonyl fluoride . This agent, which inhibits multiple vacuolar proteases, did not affect the proteasome or breakdown of short-lived proteins . These two classes of inhibitors can thus be used to distinguish the cytosolic and vacuolar proteolytic pathways and to increase the cellular content of short-lived proteins. RNA, 1996 Nov, 2(11), 1079 - 93 Secondary structure of the yeast Saccharomyces cerevisiae pre-U3A snoRNA and its implication for splicing efficiency; Mougin A et al.; The Saccharomyces cerevisiae U3 snoRNA genes contain long spliceosomal introns with noncanonical branch site sequences . By using chemical and enzymatic methods to probe the RNA secondary structure and site-directed mutagenesis, we established the complete secondary structure of the U3A snoRNA precursor . This is the first determination of the complete secondary structure of an RNA spliced in a spliceosome . The peculiar cruciform structure of the U3A snoRNA 3'-terminal region is formed in the precursor RNA and the conserved Boxes B and C are accessible for binding the U3 snoRNP proteins . The intron forms a highly folded structure with a long central stem-loop structure that brings the 5' box and the branch site together . This is in agreement with the idea that secondary structure interactions are necessary for efficient splicing of long introns in yeast . The 3' splice site is in a bulged loop and the branch site sequence is single-stranded . Surprisingly, the 5' splice site is involved in a 6-base pair interaction . We used in vitro splicing experiments to show that, despite a noncanonical branch site sequence and a base paired 5' splice site, transcripts that mimic the authentic pre-U3A snoRNA are spliced very efficiently in vitro . Sequestering the 5' splice site in a more stable structure had a negative effect on splicing, which was partially compensated by converting the branch site sequence into a canonical sequence . Analysis of spliceosomal complex formation revealed a cumulative negative effect of a base pair interaction at the 5' splice site and of a deviation to the consensus sequence at the branch site on the efficiency of spliceosome formation in vitro. Mol Cell Biol, 1996 Nov, 16(11), 6436 - 43 A novel 66-kilodalton protein complexes with Rrn6, Rrn7, and TATA-binding protein to promote polymerase I transcription initiation in Saccharomyces cerevisiae; Lin CW et al.; We report the cloning of RRN11, a gene coding for a 66-kDa protein essential for transcription initiation by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae . Rrn11 specifically complexes with two previously identified transcription factors, Rrn6 and Rrn7 (D . A . Keys, J . S . Steffan, J . A . Dodd, R . T . Yamamoto, Y . Nogi, and M . Nomura, Genes Dev . 8:2349-2362, 1994) . The Rrn11-Rrn6-Rrn7 complex also binds the TATA-binding protein and is required for transcription by the core domain of the Pol I promoter . Therefore, we have designated the Rrn11-Rrn6-Rrn7-TATA-binding protein complex the yeast Pol I core factor . A two-hybrid assay was used to demonstrate involvement of short leucine heptad repeats on both Rrn11 and Rrn6 in the in vivo association of these two proteins . This assay also verified the previously described strong association between Rrn6 and Rrn7, independent of the Rrn6 leucine repeat. Mol Cell Biol, 1996 Nov, 16(11), 6419 - 26 Rgt1p of Saccharomyces cerevisiae, a key regulator of glucose-induced genes, is both an activator and a repressor of transcription; Ozcan S et al.; The RGT1 gene of Saccharomyces cerevisiae plays a central role in the glucose-induced expression of hexose transporter (HXT) genes . Genetic evidence suggests that it encodes a repressor of the HXT genes whose function is inhibited by glucose . Here, we report the isolation of RGT1 and demonstrate that it encodes a bifunctional transcription factor . Rgt1p displays three different transcriptional modes in response to glucose: (i) in the absence of glucose, it functions as a transcriptional repressor; (ii) high concentrations of glucose cause it to function as a transcriptional activator; and (iii) in cells growing on low levels of glucose, Rgt1p has a neutral role, neither repressing nor activating transcription . Glucose alters Rgt1p function through a pathway that includes two glucose sensors, Snf3p and Rgt2p, and Grr1p . The glucose transporter Snf3p, which appears to be a low-glucose sensor, is required for inhibition of Rgt1p repressor function by low levels of glucose . Rgt2p, a glucose transporter that functions as a high-glucose sensor, is required for conversion of Rgt1p into an activator by high levels of glucose . Grr1p, a component of the glucose signaling pathway, is required both for inactivation of Rgt1p repressor function by low levels of glucose and for conversion of Rgt1p into an activator at high levels of glucose . Thus, signals generated by two different glucose sensors act through Grr1p to determine Rgt1p function. Mol Cell Biol, 1996 Nov, 16(11), 6385 - 97 Spindle pole body separation in Saccharomyces cerevisiae requires dephosphorylation of the tyrosine 19 residue of Cdc28; Lim HH et al.; In eukaryotes, mitosis requires the activation of cdc2 kinase via association with cyclin B and dephosphorylation of the threonine 14 and tyrosine 15 residues . It is known that in the budding yeast Saccharomyces cerevisiae, a homologous kinase, Cdc28, mediates the progression through M phase, but it is not clear what specific mitotic function its activation by the dephosphorylation of an equivalent tyrosine (Tyr-19) serves . We report here that cells expressing cdc28-E19 (in which Tyr-19 is replaced by glutamic acid) perform Start-related functions, complete DNA synthesis, and exhibit high levels of Clb2-associated kinase activity but are unable to form bipolar spindles . The failure of these cells to form mitotic spindles is due to their inability to segregate duplicated spindle pole bodies (SPBs), a phenotype strikingly similar to that exhibited by a previously reported mutant defective in both kinesin-like motor proteins Cin8 and Kip1 . We also find that the overexpression of SWE1, the budding-yeast homolog of wee1, also leads to a failure to segregate SPBs . These results imply that dephosphorylation of Tyr-19 is required for the segregation of SPBs . The requirement of Tyr-19 dephosphorylation for spindle assembly is also observed under conditions in which spindle formation is independent of mitosis, suggesting that the involvement of Cdc28/Clb kinase in SPB separation is direct . On the basis of these results, we propose that one of the roles of Tyr-19 dephosphorylation is to promote SPB separation. Mol Cell Biol, 1996 Nov, 16(11), 6218 - 28 Characterization of rco-1 of Neurospora crassa, a pleiotropic gene affecting growth and development that encodes a homolog of Tup1 of Saccharomyces cerevisiae; Yamashiro CT et al.; The filamentous fungus Neurospora crassa undergoes a well-defined developmental program, conidiation, that culminates in the production of numerous asexual spores, conidia . Several cloned genes, including con-10, are expressed during conidiation but not during mycelial growth . Using a previously described selection strategy, we isolated mutants that express con-10 during mycelial growth . Selection was based on expression of an integrated DNA fragment containing the con-10 promoter-regulatory region followed by the initial segment of the con-10 open reading frame fused in frame with the bacterial hygromycin B phosphotransferase structural gene (con10'-'hph) . Resistance to hygromycin results from mutational alterations that allow mycelial expression of the con-10'-'hph gene fusion . A set of drug-resistant mutants were isolated; several of these had abnormal conidiation phenotypes and were trans-acting, i.e., they allowed mycelial expression of the endogenous con-10 gene . Four of these had alterations at a single locus, designated rco-1 (regulation of conidiation) . Strains with the rco-1 mutant alleles were aconidial, female sterile, had reduced growth rates, and formed hyphae that coiled in a counterclockwise direction, opposite that of the wild type . The four rco-1 mutants had distinct conidiation morphologies, suggesting that conidiation was blocked at different stages . Wild-type rco-1 was cloned by a novel procedure employing heterokaryon-assisted transformation and ligation-mediated PCR . The predicted RCO1 polypeptide is a homolog of Tup1 of Saccharomyces cerevisiae, a multidomain protein that mediates transcriptional repression of genes concerned with a variety of processes . Like tup1 mutants, null mutants of rco-1 are viable and pleiotropic . A promoter element was identified that could be responsible for RCO1-mediated vegetative repression of con-10 and other conidiation genes. Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12428 - 32 Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression; Ozcan S et al.; Glucose is the preferred carbon source for most eukaryotic cells and has profound effects on many cellular functions . How cells sense glucose and transduce a signal into the cell is a fundamental, unanswered question . Here we describe evidence that two unusual glucose transporters in the yeast Saccharomyces cerevisiae serve as glucose sensors that generate an intracellular glucose signal . The Snf3p high-affinity glucose transporter appears to function as a low glucose sensor, since it is required for induction of expression of several hexose transporter (HXT) genes, encoding glucose transporters, by low levels of glucose . We have identified another apparent glucose transporter, Rgt2p, that is strikingly similar to Snf3p and is required for maximal induction of gene expression in response to high levels of glucose . This suggests that Rgt2p is a high glucose-sensing counterpart to Snf3p . We identified a dominant mutation in RGT2 that causes constitutive expression of several HXT genes, even in the absence of the inducer glucose . This same mutation introduced into SNF3 also causes glucose-independent expression of HXT genes . Thus, the Rgt2p and Snf3p glucose transporters appear to act as glucose receptors that generate an intracellular glucose signal, suggesting that glucose signaling in yeast is a receptor-mediated process. Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12245 - 50 Functional cell surface expression of the anion transport domain of human red cell band 3 (AE1) in the yeast Saccharomyces cerevisiae; Groves JD et al.; We expressed the 52-kDa integral membrane domain (B3mem) of the human erythrocyte anion transporter (band 3; AE1) in a protease-deficient strain of the yeast Saccharomyces cerevisiae under the control of the inducible GAL10-CYC1 promoter . Immunoblots of total protein from transformed yeast cells confirmed that the B3mem polypeptide was overexpressed shortly after induction with galactose . Cell surface expression of the functional anion transporter was detected by using a simple transport assay to measure stilbene disulfonate-inhibitable chloride influx into intact yeast cells . The B3mem polypeptide was recycled and degraded by the cells with a half-life of approximately 1-3 hr, which led to a steady-state level of expression in exponentially growing cultures . Our data suggest that 5-10% of total B3mem is functionally active at the cell surface at any one time and that overexpression of this anion transport protein does not interfere with cell growth or survival . This is one of only a few reports of the functional expression of a plasma membrane transport protein in the plasma membrane of yeast cells and to our knowledge is the first report of red cell band 3-mediated anion transport at the plasma membrane of cDNA-transformed cells . The cell surface expression system we describe will provide a simple means for future study of the functional properties of band 3 by using site-directed mutagenesis. Biochemistry, 1996 Oct 29, 35(43), 13788 - 96 Three-dimensional solution structure of Saccharomyces cerevisiae reduced iso-1-cytochrome c; Baistrocchi P et al.; Two-dimensional 1H NMR spectra of Saccharomyces cerevisiae reduced iso-1-cytochrome c have been used to confirm and slightly extend the assignment available in the literature . 1702 NOESY cross-peaks have been assigned, and their intensities have been measured . Through the program DIANA and related protocols (Guntert, 1992), a solution structure has been obtained by using 1442 meaningful NOEs and 13 hydrogen-bond constraints . The RMSD values with respect to the mean structure for the backbone and all heavy atoms for a family of 20 structures are 0.61 +/- 0.09 and 0.98 +/- 0.09 A, the average target function value being as small as 0.57 A2 . The larger number of slowly exchanging amide NHs observed in this system compared to that observed in the cyanide derivative of oxidized Ala 80 cytochrome c suggests that the oxidized form is much more flexible and that the backbone protons are more solvent accessible . Comparison of the present structure with the crystal structures of reduced yeast cytochrome c and of the complex between cytochrome c peroxidase and oxidized yeast cytochrome c reveals substantial similarity among the backbone conformations but differences in the residues located in the region of protein-protein interaction . Interestingly, in solution the peripheral residues involved in the interaction with cytochrome c peroxidase are on average closer to the position found in the crystal structure of the complex than to the solid state structure of the isolated reduced from. Mutat Res, 1996 Oct 28, 358(1), 73 - 80 Utility of a test for chromosomal malsegregation in Saccharomyces cerevisiae strain D61.M for the detection of antianeugens: test of the model combination of chlorophyllin and nocodazole; Verma A et al.; Despite the fact that aneuploidy is a major genetic cause of human morbidity and mortality, antimutagenicity studies have used predominantly short-term tests that detect gene mutations, chromosomal aberrations, and micronuclei . Therefore, the major deficiency in the use of short-term tests for antimutagenicity studies is those that detect chromosomal malsegregation leading to aneuploidy . Thus, we initiated a study on the utility of short-term tests for the detection of antianeugenic activity . We selected strain D61.M of Saccharomyces cerevisiae, nocodazole, and chlorophyllin as a model short-term test, aneugen, and antimutagen, respectively, for our initial study . Chlorophyllin strongly inhibited the aneugenic activity of nocodazole, but had no effect on the endpoints when tested alone, in strain D61.M . To our knowledge, this is the first report of an antianeugen . Furthermore, we conclude that strain D61.M can be used as a relatively simple, inexpensive, and rapid short-term test for the study of antianeugenicity. Mol Gen Genet, 1996 Oct 28, 252(6), 667 - 75 In vitro mutagenesis of the mitochondrial leucyl tRNA synthetase of Saccharomyces cerevisiae shows that the suppressor activity of the mutant proteins is related to the splicing function of the wild-type protein; Li GY et al.; The NAM2 gene of Saccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrial cytb gene (bI4) and the fourth intron of the mitochondrial coxI gene (aI4), as well as for mitochondrial protein synthesis . Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4 . Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein . Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity . Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity . The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity . These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity. J Biol Chem, 1996 Oct 25, 271(43), 27167 - 75 Purification of the Saccharomyces cerevisiae cleavage/polyadenylation factor I . Separation into two components that are required for both cleavage and polyadenylation of mRNA 3' ends; Kessler MM et al.; The cleavage/polyadenylation factor I (CF I) is one of four factors required for mRNA 3' end formation in the yeast Saccharomyces cerevisiae . Here we describe the purification of CF I and its separation into two components, CF IA and CF IB . Both components are needed to reconstitute CF I activity in cleavage and poly(A) addition . CF IA consists of a complex of four polypeptides of 76, 70, 50, and 38 kDa, and CF IB is a single 73-kDa polypeptide . The 76- and 38-kDa subunits of CF IA correspond to the previously identified RNA14 and RNA15 proteins . The RNA14 protein, but not the 70- or 50-kDa proteins, coimmunoprecipitates with the RNA15 protein, indicating that RNA14 and RNA15 proteins exist in a tight complex . RNA15 is the only subunit of CF I that can be cross-linked to pre-mRNA. J Biol Chem, 1996 Oct 25, 271(43), 26713 - 6 Dissociation of import of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria from proteolytic processing of the presequence; Nett JH et al.; The correlation between the import of the Rieske iron-sulfur protein into the mitochondrial matrix and processing of the precursor protein by matrix processing peptidase was investigated using high concentrations of metal chelators and iron-sulfur protein in which the recognition site for the matrix processing peptidase was destroyed by site-directed mutagenesis . High concentrations of EDTA and o-phenanthroline inhibit import of iron-sulfur protein into the matrix . The non-chelating structural isomers m-phenanthroline and p-phenanthroline inhibit import similar to o-phenanthroline, indicating that inhibition of import is mainly independent of the metal chelating ability of the compounds . Iron-sulfur protein in which the recognition site for the matrix processing peptidase had been destroyed by a point mutation was efficiently imported into the matrix space . Import of this mutant iron-sulfur protein was inhibited by the same concentrations of EDTA and o-phenanthroline which inhibit import of the wild-type protein . These results indicate that import of the iron-sulfur protein into the mitochondrial matrix is independent of proteolytic processing of the presequence, and that o-phenanthroline together with EDTA inhibits import of iron-sulfur protein into the matrix space of mitochondria by inhibiting a step other than proteolysis of the presequence. J Biotechnol, 1996 Oct 18, 51(1), 57 - 72 Physiology of Saccharomyces cerevisiae during cell cycle oscillations; Duboc P et al.; Synchronized populations of Saccharomyces cerevisiae CBS 426 are characterized by autonomous oscillations of process variables . CO2 evolution rate, O2 uptake rate and heat production rate varied by a factor of 2 for a continuous culture grown at a dilution rate of 0.10 h-1 . Elemental analysis showed that the carbon mass fraction of biomass did not change . Since the reactor is not at steady state, the elemental and energy balances were calculated on cumulated quantities, i.e . the integral of the reaction rates . It was possible to show that carbon, degree of reduction and energy balances matched . Application of simple mass balance principles for non-steady state systems indicated that oscillations were basically characterized by changes in biomass production rate . In addition, the amount of intermediates, e.g . ethanol or acetate, produced or consumed was negligible . Growth rate was low during the S-phase (0.075 h-1) and high during the G2, M and G1 phases (0.125 h-1) for a constant dilution rate of 0.10 h-1 . However, nitrogen, ash, sulfur and potassium content showed systematic increases during the S-phase (bud initiation) . Cell component analyses showed that changes in cellular fractions during oscillations (storage carbohydrate content decreased during the S-phase) were due to changes in production rates, particularly for protein and carbohydrates . Nevertheless, using the data evaluation techniques for dynamic systems presented here, it was shown that storage carbohydrates are not consumed during the S-phase . Only the synthesis rate of the different cell components changed depending on position in cell cycle . The growth process may be divided into two phenomena: the formation of new cells during mitosis with a low yield, and size increase of new born cells with high yield . Both kinetic and stoichiometric coefficients varied with the position in the oscillation: the results showed that biomass structure changed and that specific growth rate, as well as biomass yield, varied by +/- 25% during the oscillation. J Biol Chem, 1996 Oct 18, 271(42), 26375 - 82 Analysis of the carboxyl-terminal peroxisomal targeting signal 1 in a homologous context in Saccharomyces cerevisiae; Elgersma Y et al.; Most peroxisomal matrix proteins contain a carboxyl-terminal tripeptide that directs them to peroxisomes . Within limits, these amino acids may be varied, without loss of function . The specificity of this peroxisomal targeting signal (PTS1) is remarkable considering its small size and its relaxed consensus sequence . Moreover, several peroxisomal proteins have a PTS1-like signal that does not fit the reported consensus sequence . Because many of these PTS1 variants seem to be functional in a species-dependent or protein context-dependent manner, we investigated the PTS1 requirements in a homologous context, using Saccharomyces cerevisiae and endogenous peroxisomal malate dehydrogenase (MDH3) . Peroxisomal import of the MDH3-PTS1 variants was tested qualitatively by the ability to complement the Deltamdh3 mutant and quantitatively by subcellular fractionation . We observed efficient import of MDH3 into peroxisomes with a large variety of PTS1 tripeptides . Many of these variants do not fit the observed PTS1 requirements for heterologously expressed proteins, which suggests that additional domains in the protein may be of decisive importance whether or not a certain PTS1 variant is recognized by the components of the peroxisomal import machinery . Because we show that dimerization of MDH3 precedes import into the organelle, these domains are most likely conformational domains. Mol Gen Genet, 1996 Oct 16, 252(5), 587 - 96 Gene isolation in Arabidopsis thaliana by conditional overexpression of cDNAs toxic to Saccharomyces cerevisiae: identification of a novel early response zinc-finger gene; Martinez-Garcia M et al.; In an effort to identify novel regulatory plant genes, conditional overexpression of toxic Arabidopsis thaliana gene products in Saccharomyces cerevisiae was evaluated as a genetic selection scheme . The screening method was tested on a fraction of a cDNA expression library and led to the identification of two Arabidopsis cDNA clones that were toxic to yeast; one corresponded to histone H1 and the other to a previously unidentified gene . This new gene, named ATL2, combines a RING-like zinc-binding motif and a putative signal anchor sequence for membrane insertion in the same molecule . Furthermore, inspection of the 3' untranslated region reveals two types of sequences which appear to be key determinants in rapid transcript decay . Indeed, rapid and transient accumulation of transcript occurs in the presence of a protein synthesis inhibitor and of the growth regulator auxin . These features provide evidence that ATL2 is an early-response gene . Thus, ATL2 represents one of the first early-response plant genes to be described which possesses a distinct regulatory domain; the fact that ATL2 mRNA is induced by auxin suggests that it might have a role during the response of plants to this growth regulator. Mol Gen Genet, 1996 Oct 16, 252(5), 552 - 62 Mutations in STS1 suppress the defect in 3' mRNA processing caused by the rna15-2 mutation in Saccharomyces cerevisiae; Amrani N et al.; In a search for proteins associated with Rna15p in processing the 3' ends of messenger RNAs, we have looked for suppressors that correct, even partially, the thermosensitive growth defect of the rna15-2 mutant . Mutations in a single locus that we named SSM5, were able to suppress both the thermosensitivity of cell growth and the mRNA 3' processing defect associated with the rna15-2 mutation, but only slightly alleviated the thermosensitive growth defect of an rna14-1 mutant . The ssm5-1 mutant is sensitive to hydroxyurea at 37 degrees C, a drug that inhibits DNA synthesis . By screening for complementation of the hydroxyurea-sensitive phenotype we cloned the corresponding wild-type gene and found that it corresponds to the essential gene STS1 (also named DBF8) . Sts1p has an apparent molecular weight of 30 kDa and was confirmed to be a cytosolic protein by immunofluorescence analysis . Western blot analysis indicates that the thermosensitive mutant strains rna15-2, rna14-1 and pap1-1 present a very low level of the Rna15p at 37 degrees C . The ssm5-1 mutation restores the level of Rna15p in the rna15-2 ssm5-1 double mutant . Use of the two-hybrid system suggests that Sts1p does not interact directly with Rna15p, but may be active as a homodimer . The present data suggest that Sts1p may play a role in the transport of Rna15p from the cytoplasm to the nucleus. Mol Gen Genet, 1996 Oct 16, 252(5), 530 - 8 Saccharomyces cerevisiae mutants defective in plasmid-chromosome recombination; Elias-Arnanz M et al.; We have studied the recombinational repair of a double-strand break (DSB) in a plasmid-borne ade2::HO-site by an intact ade2 allele following the induction of a galactose-inducible GAL-HO gene . If GAL-HO expression is not attenuated by the presence of a low level of glucose in the galactose medium, deleterious effects are observed . Our comparison of the effects of several rad mutations on the relative efficiencies of DSB repair at both the ade2::HO-site and at the chromosomal MAT locus indicate that the two processes share common functions . Not surprisingly, most of the recombination-defective mutants found using our assay are alleles of genes in the RAD52 epistasis group . The recombination and repair deficiencies vary among the different mutant groups and also among mutants within a group . In general, there is a correlation between the extents of the recombination and repair defects . Our screen also turned up a novel rfa1 allele with a pronounced deficiency in DSB repair and recombination and a srs2 mutation which causes only a mild defect. EMBO J, 1996 Oct 15, 15(20), 5513 - 26 Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH; Morsomme P et al.; In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family . The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5 . In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0 . Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme . Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity . However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells . These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH . Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway. Genes Dev, 1996 Oct 15, 10(20), 2632 - 43 RAD9 and DNA polymerase epsilon form parallel sensory branches for transducing the DNA damage checkpoint signal in Saccharomyces cerevisiae; Navas TA et al.; In response to DNA damage and replication blocks, yeast cells arrest at distinct points in the cell cycle and induce the transcription of genes whose products facilitate DNA repair . Examination of the inducibility of RNR3 in response to UV damage has revealed that the various checkpoint genes can be arranged in a pathway consistent with their requirement to arrest cells at different stages of the cell cycle . While RAD9, RAD24, and MEC3 are required to activate the DNA damage checkpoint when cells are in G1 or G2, POL2 is required to sense UV damage and replication blocks when cells are in S phase . The phosphorylation of the essential central transducer, Rad53p, is dependent on POL2 and RAD9 in response to UV damage, indicating that RAD53 functions downstream of both these genes . Mutants defective for both pathways are severely deficient in Rad53p phosphorylation and RNR3 induction and are significantly more sensitive to DNA damage and replication blocks than single mutants alone . These results show that POL2 and RAD9 function in parallel branches for sensing and transducing the UV DNA damage signal . Each of these pathways subsequently activates the central transducers Mec1p/Esr1p/Sad3p and Rad53p/Mec2p/Sad1p, which are required for both cell-cycle arrest and transcriptional responses. Genes Dev, 1996 Oct 15, 10(20), 2551 - 63 The role of TBP in rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae: TBP is required for upstream activation factor-dependent recruitment of core factor; Steffan JS et al.; Transcription of Saccharomyces cerevisiae rDNA by RNA polymerase I involves at least two transcription factors characterized previously: upstream activation factor (UAF) consisting of Rrn5p, Rrn9p, Rrn10p, and two more uncharacterized proteins; and core factor (CF) consisting of Rrn6p, Rrn7p, and Rrn11p . UAF interacts directly with an upstream element of the promoter and mediates its stimulatory function, and CF subsequently joins a stable preinitiation complex . The TATA-binding protein (TBP) has been known to be involved in transcription by all three nuclear RNA polymerases . We found that TBP interacts specifically with both UAF and CF, the interaction with UAF being stronger than that with CF . Using extracts from a TBP (I143N) mutant, it was shown that TBP is required for stimulation of transcription mediated by the upstream element, but not for basal transcription directed by a template without the upstream element . By template competition experiments, it was shown that TBP is required for UAF-dependent recruitment of CF to the rDNA promoter, explaining the TBP requirement for stimulatory activity of the upstream element . We also studied protein-protein interactions and found specific interactions of TBP with Rrn6p and with Rrn9p both in vitro and in the yeast two-hybrid system in vivo . Thus, these two interactions may be involved in the interactions of TBP with CF and UAF, respectively, contributing to the recruitment of CF to the rDNA promoter . Additionally, we observed an interaction between Rrn9p and Rrn7p both in vitro and in the two-hybrid system; thus, this interaction might also contribute to the recruitment of CF. Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11901 - 6 A Saccharomyces cerevisiae homolog of the human adrenoleukodystrophy transporter is a heterodimer of two half ATP-binding cassette transporters; Shani N et al.; The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half ATP-binding cassette (ABC) transporters in the human peroxisome membrane . ALDP and PMP70 share sequence homology and both are implicated in genetic diseases . PXA1 and YKL741 are Saccharomyces cerevisiae genes that encode homologs of ALDP and PMP70 . Pxa1p, a putative ortholog of ALDP, is involved in peroxisomal beta-oxidation of fatty acids while YKL741 is an open reading frame found by the yeast genome sequencing project . Here we designate YKL741 as PXA2 and show that its protein product, Pxa2p, like Pxa1p, is associated with peroxisomes but not required for their assembly . Yeast strains carrying gene disruption of PXA1, PXA2, or both have similar and, in the case of the latter, nonadditive phenotypes . We also find that the stability of Pxa1p, but not Pxa2p, is markedly reduced in the absence of the other . Finally, we find that Pxa1p and Pxa2p coimmuno-precipitate . These genetic and physical data suggest that Pxa1p and Pxa2p heterodimerize to form a complete peroxisomal ABC transporter involved in fatty acid beta-oxidation . This result predicts the presence of similar heterodimeric ABC transporters in the mammalian peroxisome membrane. Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11569 - 74 The Ku-like protein from Saccharomyces cerevisiae is required in vitro for the assembly of a stable multiprotein complex at a eukaryotic origin of replication; Shakibai N et al.; We have previously shown that three distinct DNA-binding activities, in crude form, are necessary for the ATP-dependent assembly of a specific and stable multiprotein complex at a yeast origin of replication . Here we show the purification of one of these DNA binding activities, referred to as origin binding factor 2 (OBF2) . The purified protein is a heterodimer composed of two polypeptides with molecular mass values of 65 and 80 kDa as determined by SDS/PAGE . Purified OBF2 not only binds DNA but also supports the formation of a protein complex at essential sequences within the ARS121 origin of replication . Interestingly, OBF2 binds tightly and nonspecifically to both duplex DNA and single-stranded DNA . The interaction with duplex DNA occurs at the termini . N-terminal sequencing of the 65-kDa subunit has revealed that this polypeptide is identical to the previously identified HDF1 peptide, a yeast homolog of the small subunit of the mammalian Ku autoantigen . Although the potential involvement of Ku in DNA metabolic events has been proposed, this is the first requirement for a Ku-like protein in the assembly of a protein complex at essential sequences within a eukaryotic origin of replication. J Biol Chem, 1996 Oct 11, 271(41), 25438 - 45 Probing conserved regions of the cytoplasmic LOOP1 segment linking transmembrane segments 2 and 3 of the Saccharomyces cerevisiae plasma membrane H+-ATPase; Wang G et al.; Genetic probing was used to examine conserved amino acid clusters in the first cytoplasmic loop domain (LOOP1) linking transmembrane segments 2 and 3 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae . Deletion of the LOOP1 region in PMA1 resulted in a defective enzyme . Scanning alanine mutagenesis of conserved residues produced lethal cell phenotypes in 14 of 26 amino acids, suggesting major enzyme defects . Most viable mutants showed growth characteristics that were comparable to wild type . Two mutations, I183A and D185A, produced reduced growth rates, hygromycin B resistance, and low pH sensitivity, which are phenotypes associated with defects in the H+-ATPase . However, both mutant enzymes displayed near-normal kinetics for ATP hydrolysis in vitro . Localized random mutagenesis was also performed at sites Glu195, Val196, and Ile210, which all showed lethal phenotypes upon conversion to alanine . Amino acids with polar side groups could substitute for Glu195, while Val196 could not tolerate polar side group moieties . Nine mutations at Ile210 proved lethal, including K, R, E, P, H, N, V, G, and A, while functional enzyme was obtained with S, C, M, and L . Normal rates and extents of pH gradient formation were observed for all mutant enzymes, except I183A and D185A . Detailed analysis of the I183A enzyme indicated that it hydrolyzed ATP like wild type, but it appeared to inefficiently couple ATP hydrolysis to proton transport . In total, these results affirm that conserved amino acids in LOOP1 are important to H+-ATPase function, and purturbations in this region can alter the efficiency of energy coupling. Gene, 1996 Oct 10, 175(1-2), 253 - 60 Probing the limits of expression levels by varying promoter strength and plasmid copy number in Saccharomyces cerevisiae; Nacken V et al.; Heterologous gene expression levels were measured in yeast using the Escherichia coli gusA gene (encoding beta-D-glucuronidase) as a reporter . The influence of two major parameters, promoter activity and plasmid copy number, was studied . (1) Promoters used in this study ranged from the very weak constitutive KEX2, the regulated CYC1 and PGK and the mating type-specific MF alpha 1 to the strong constitutive TEF1 and TDH promoters . Using centromeric vectors, gusA expression levels varied within three orders of magnitude . (2) Plasmid copy number was changed by shifting from a monocopy (centromeric plasmid) over a moderate copy number (2 mu-based plasmid) to a high copy number (2 mu associated with the URA3-d selection marker) . gusA expression levels increased relatively with plasmid copy number in all cases studied, but did not exceed the equivalent of 2% of total soluble yeast proteins . Coupling these variables, a 5-log range in gene expression levels was covered . Taken together, these results provide a framework which allows a comparison of existing and new promoters . This framework will be useful for expressing genes to required levels. J Mol Biol, 1996 Oct 4, 262(4), 473 - 84 The study of methionine uptake in Saccharomyces cerevisiae reveals a new family of amino acid permeases; Isnard AD et al.; The screening of mutants resistant to the oxidized analogues of methionine (methionine sulphoxide and ethionine sulphoxide) allowed the characterisation of a yeast mutant strain lacking the high affinity methionine permease and defining a new locus that was called MUP1 . The study of MUP1 mutants showed that methionine is transported into yeast cells by three different permeases, a high affinity and two low affinity permeases . The MUP1 gene was cloned and was shown to encode an integral membrane protein with 13 putative membrane-spanning regions . Database comparisons revealed that the yeast genome contains an ORF whose produc