S. cerevisiae

J Biol Chem, 1992 Feb 15, 267(5), 3416 - 22
Compartment acidification is required for efficient sorting of proteins to the vacuole in Saccharomyces cerevisiae; Klionsky DJ et al.; The vacuole of the yeast Saccharomyces cerevisiae contains a proton-translocating ATPase that acidifies the vacuolar lumen and generates a pH gradient across the vacuole membrane . We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins . Strains with chromosomal disruptions of the genes encoding the A, B, or c subunit of the vacuolar ATPase are unable to acidify their vacuoles . These vat mutant strains accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A . The kinetics of secretion suggests that missorting occurs in the Golgi complex or in post-Golgi vesicles . The presence of mature forms of the vacuolar proteins within the cell indicates that vat mutations do not cause defects in zymogen processing . Precursor forms of the membrane-associated vacuolar hydrolase alkaline phosphatase are also accumulated in vat mutant cells but to a lesser extent, suggesting that sorting of vacuolar membrane proteins is less sensitive to changes in the lumenal pH . A similar type of missorting defect can be induced in wild-type cells at pH 7.5 . These results indicate that acidification of the vacuolar system is important for efficient sorting of proteins to the vacuole.

Arch Biochem Biophys, 1992 Feb 14, 293(1), 122 - 9
Characterization and partial purification of squalene-2,3-oxide cyclase from Saccharomyces cerevisiae; Balliano G et al.; The membrane nature of squalene oxide cyclase from Saccharomyces cerevisiae was investigated by comparing properties of the enzyme recovered from both microsomes and the soluble fraction of the yeast homogenate . The "apparent soluble" form and microsomal form of the enzyme were both stimulated by the presence of mammalian soluble cytoplasm and corresponded to one another in response to detergents Triton X-100 and Triton X-114 . The observed strong dependence of the enzyme activity on the presence of detergents and the behavior of the enzyme after Triton X-114 phase separation were peculiar to a lipophilic membrane-bound enzyme . A study of the conditions required to extract the enzyme from microsomes confirmed the lipophilic character of the enzyme . Microsomes, exposed to ipotonic conditions to remove peripheral membrane proteins, retained most of the enzyme activity within the integral protein fraction . Quantitative dissociation of the enzyme from membranes occurred only if microsomes were treated with detergents (Triton X-100 or octylglucoside) at concentrations which alter membrane integrity . The squalene oxide cyclase was purified 140 times from yeast microsomes by (a) removal of peripheral proteins, (b) extraction of the enzyme from the integral protein fraction with octylglucoside, and (c) separation of the solubilized proteins by DEAE Bio-Gel A chromatography . Removal of the peripheral proteins seemed to be a key step necessary for obtaining high yields.

Biochim Biophys Acta, 1992 Feb 13, 1119(1), 35 - 8
ATP-dependent Saccharomyces cerevisiae phospho enol pyruvate carboxykinase: isolation and sequence of a peptide containing a highly reactive cysteine; Alvear M et al.; Saccharomyces cerevisiae phospho enol pyruvate carboxykinase (EC 4.1.1.49), inactivated by N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, incorporated 0.95 mol of the fluorescent moiety per mol of enzyme subunit . Reagent incorporation was completely protected by the presence of ADP plus MnCl2 . The labeled protein was digested with trypsin after carboxymethylation . Two labeled peptides were isolated by reverse-phase high-performance liquid chromatography and were sequenced by gas-phase automatic Edman degradation . Both peptides contained overlapping amino acid sequences from Asn-358 to Lys-375, thus identifying Cys-364 as the reactive amino acid residue . The position of the target amino acid residue is immediately preceding a putative phosphoryl-binding sequence proposed for some nucleotide-binding proteins.

Nucleic Acids Res, 1992 Feb 11, 20(3), 449 - 57
The MRE4 gene encodes a novel protein kinase homologue required for meiotic recombination in Saccharomyces cerevisiae; Leem SH et al.; The MRE4 gene was cloned by complementation of the defects of meiotic recombination and haploidization in an mre4-1 mutant . Disruption of MRE4 resulted in reduced meiotic recombination and spore inviability . The mre4 spore lethality can be suppressed by spo13, a mutation that causes cells to bypass the reductional division . Analysis of meiotic DNA extracted from the mre4 mutant cells revealed that double-strand breaks occurred at the two sites of the HIS4-LEU2 recombination hot spot, but at a frequency of about 10-20% of the wild type . Northern blot analysis indicated that the MRE4 gene produces four transcripts of 1.63, 3.2, 4.0 and 6.2 kb . All of these transcripts are absent from mitotic cells and are meiotically induced . The DNA sequence of the MRE4 open reading frame predicts a 497-amino acids protein with a molecular mass of 56.8 kDa . The Mre4 protein contains highly conserved amino acid sequences found specifically in serine-threonine protein kinases . These results suggest that protein phosphorylation is required directly or indirectly for meiotic recombination.

J Biol Chem, 1992 Feb 5, 267(4), 2467 - 73
Regulation by nuclear genes of the mitochondrial synthesis of subunits 6 and 8 of the ATP synthase of Saccharomyces cerevisiae; Pelissier PP et al.; The nuclear mutant AB1-4A/8/100, a respiratory-competent strain altered in the regulation of ATP synthesis, has been shown to be modified in the relative stoichiometry of the mtDNA-encoded proteolipids of the F0 sector of ATP synthase: the ratios mutant/wild type of the proteolipids were equal to 0.4/0.7/1 for Su8/Su6/Su9, respectively . This defect results from the simultaneous presence of two nuclear genes which promote a cryosensitive phenotype on a nonfermentable carbon source . Measurements of mitochondrial protein synthesis carried out "in vivo" and "in organello" evidenced a specific defect in the synthesis of subunits 6 and 8 . Measurements of the steady state levels of mitochondrial mRNA showed that the defect in subunits 6 and 8 was correlated with a modification of the expression of a cotranscript ATP8-ATP6 . This cotranscript is matured at a unique site to give two cotranscripts of 4600 and 5200 bases in length . In mutant mitochondria, the ratio between both cotranscripts, 5200/4600, was lowered . In parallel, expression of the whole mitochondrial transcription unit supporting the genes COXI, ATP8, ATP6, and RF3 was enhanced . However, despite this over expression, the amount of the long cotranscript ATP8-ATP6 remained lower than in wild type mitochondria.

Eur J Biochem, 1992 Feb 1, 203(3), 587 - 92
Primary structure and import pathway of the rotenone-insensitive NADH-ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae; De Vries S et al.; The gene encoding the yeast mitochondrial rotenone-insensitive internal NADH: ubiquinone-6 oxidoreductase has been sequenced . The DNA sequence indicates the presence of an open reading frame of 1539 bp predicted to encode a protein of 513 amino acid residues (57.2 kDa) . The NADH dehydrogenase is synthesized as a precursor protein containing a signal sequence of 26 residues . In vitro import experiments show that the precursor NADH dehydrogenase is cleaved to the mature size by the matrix processing peptidase . Both cleavage and translocation across the mitochondrial membrane(s) are dependent on the membrane potential component of the proton-motive force . Comparison of the protein sequence of the yeast NADH dehydrogenase with the data bank indicates that the enzyme from yeast is homologous to the NADH dehydrogenase of Escherichia coli (22.2% identical residues) . Both NADH dehydrogenases contain in the central part of the protein a sequence predicted to fold into a beta alpha beta structure involved in the binding of NADH or FAD(H2) . Various aspects of the protein structure are discussed.

J Cell Biol, 1992 Feb, 116(3), 597 - 604
Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae; Green N et al.; We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae . The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72 . The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane . Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees . We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER . Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated . Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus.

Curr Genet, 1992 Feb, 21(2), 95 - 9
A novel mutation occurring in the PHO80 gene suppresses the PHO4c mutations of Saccharomyces cerevisiae; Okada H et al.; We have isolated suppressors of a PHO4c (a positive regulator) mutant which normally confers weak constitutivity for acid phosphatase production on the Saccharomyces cell . One dominant suppressor (PHO80-2) was found to be an allele of PHO80 (a negative regulator) that changes G to A, resulting in substitution of isoleucine for methionine 42 of the Pho80 protein . Substitution of valine (PHO80-3) or leucine (PHO80-4) for the same methionine by site-directed mutagenesis also suppressed PHOc . Suppression by PHO80-2) did show some allele specificity . From these results we were able to delimit the region of PHo80 which may interact with the Pho4 protein.

Curr Genet, 1992 Feb, 21(2), 93 - 4
Repair of ultraviolet light damage in Saccharomyces cerevisiae as studied with double- and single-stranded incoming DNAs; Keszenman-Pereyra D et al.; Purified double- and single-stranded DNAs of the autonomously replicating vector M13RK9-T were irradiated with ultraviolet light (UV) in vitro and introduced into competent whole cells of Saccharomyces cerevisiae . Incoming double-stranded DNA was more sensitive to UV in excision repair-deficient rad2-1 cells than in proficient repair RAD+ cells, while single-stranded DNA exhibited high sensitivity in both host cells . The results indicate that in yeast there is no effective rescue of UV-incoming single-stranded DNA by excision repair or other constitutive dark repair processes.

J Gen Microbiol, 1992 Feb, 138 ( Pt 2), 347 - 54
Increased dosage of a transcriptional activator gene enhances iron-limited growth of Saccharomyces cerevisiae; Eide D et al.; We have selected for genes that, when present in multiple copies, enhance growth of wild-type cells of Saccharomyces cerevisiae in an iron-limiting medium . A gene designated FUP1, for 'ferric utilization proficient', was isolated by this approach . Increased dosage of FUP1 reduces the concentration of iron in the medium required for efficient growth and confers elevated levels of iron uptake activity in iron-limited cells . Disruption of the FUP1 locus reduces wild-type iron uptake rates by 2-fold in cells grown on raffinose medium but has no effect on glucose-grown cells . DNA sequencing showed that FUP1 encodes a hydrophilic 43 kDa protein identical to MSN1, a gene encoding a transcriptional activator implicated in carbon source regulation . Our results suggest that FUP1/MSN1 also regulates synthesis of gene products involved in iron uptake.

Yeast, 1992 Feb, 8(2), 95 - 106
The small heat-shock protein Hsp26 of Saccharomyces cerevisiae assembles into a high molecular weight aggregate; Bentley NJ et al.; Hsp26 is one of the major small heat-shock proteins (Hsp) of the yeast Saccharomyces cerevisiae, yet its cellular role remains to be discovered . To examine the cellular consequences of overexpression of Hsp26, the gene encoding this protein (HSP26) was overexpressed from a multicopy plasmid using either its own promoter or by coupling it to the efficient constitutive PGK promoter . The PGK promoter provided the opportunity to overexpress Hsp26 under non-stress conditions and such high level synthesis, prior to a lethal heat shock (50 degrees C), gave a small but reproducible elevation in thermotolerance . In transformed strains overexpressing Hsp26 under either stressed or non-stress conditions, the Hsp26 polypeptide was recovered almost exclusively as a high molecular weight aggregate . This high molecular weight aggregate (or heat-shock granule; HSG) was purified by differential centrifugation and sucrose gradient density centrifugation and shown, by electron microscopic analysis, to be of a uniform size (15-25 nm diameter) . Analysis of the purified HSG demonstrated that it had a molecular weight of 550 kDa, yet contained no other integral polypeptides or other macromolecules.

Yeast, 1992 Feb, 8(2), 147 - 53
Sequence of the sup61-RAD18 region on chromosome III of Saccharomyces cerevisiae; Benit P et al.; A 7965 bp DNA segment from the right arm of chromosome III of Saccharomyces cerevisiae, encompassing the sup61 and RAD18 genes, was sequenced . Four new open reading frames were found in this DNA fragment . One of them, YCR103, is 51% homologous with the G10 gene product of Xenopus laevis.

Yeast, 1992 Feb, 8(2), 133 - 45
Identification of a Saccharomyces cerevisiae homolog of the SNF2 transcriptional regulator in the DNA sequence of an 8.6 kb region in the LTE1-CYS1 interval on the left arm of chromosome I; Clark MW et al.; The DNA sequence of an 8.6 kb region of the left arm of chromosome I has been determined . This region, between the LTE1 and CYS1 loci, is approximately 40 kb from the centromere . There are six potential open-reading frames (ORFs), provisionally named YAL001-006 within this fragment of chromosome I . Four of these ORFs can be aligned with previously identified FUN transcripts: FUN28 with YAL006, FUN29 with YAL004, FUN30 with YAL001 and FUN31 with YAL002 . The YAL001 ORF shows significant homology to the SNF2 transcriptional regulator . A region of the DNA contains an extensive repeat of the bases C-A-T positioned in the 5' terminus of the YAL004 promoter region.

Yeast, 1992 Feb, 8(2), 117 - 20
Thioredoxin genes in Saccharomyces cerevisiae: map positions of TRX1 and TRX2; Muller EG; The two genes encoding thioredoxins in Saccharomyces cerevisiae, TRX1 and TRX2, map to chromosome XII and VII, respectively . From the DNA sequence of the intragenic region TRX1 is 500 bp downstream of PDC1 . Tetrad analysis places TRX2 1.1 cM from ADE3, while a physical map of this region positions TRX2 4.5 kb downstream of ADE3 . The mapping of TRX1 adjacent to PDC1 clarifies previous results (Muller, E . G . D . J . Biol . Chem . 266, 9194-9202, 1991) that suggested a third thioredoxin gene.

Gene, 1992 Feb 1, 111(1), 135 - 9
The K2-type killer toxin- and immunity-encoding region from Saccharomyces cerevisiae: structure and expression in yeast; Meskauskas A et al.; The cDNA copies of M2-1, the larger heat-cleavage product of M2 double-stranded (ds) RNA, have been synthesized, cloned, sequenced and expressed in yeast . This sequence, in combination with the known terminal sequence of M2-1 dsRNA, identifies a translation reading frame for a 362-amino-acid protein of 38.7 kDa, similar in size to the one of several protein species produced from M2-1 dsRNA in vitro translation . The expression of this cDNA clone in yeast confers both killer and immunity phenotypes.

Genetics, 1992 Feb, 130(2), 295 - 304
Analysis of URSG-mediated glucose repression of the GAL1 promoter of Saccharomyces cerevisiae; Flick JS et al.; Repression of GAL1 expression during growth on glucose is mediated in part by cis-acting promoter elements designated URSG . We show that oligonucleotides containing sequences from two regions of URSG confer glucose repression upon a heterologous promoter . Repression caused by URSG is dependent on trans-acting factors of the glucose repression pathway and is independent of orientation or location within a promoter, suggesting that URSG contains binding sites for a glucose-activated repressor protein(s) . Genetic analysis identified three apparently novel genes (URR1, URR3 and URR4) that are specifically required for URSG-mediated repression and may encode such repressor proteins . Mutations in the URR genes suppress the defect in URSG derepression caused by a snf1 mutation.

Genetics, 1992 Feb, 130(2), 263 - 71
A spontaneous chromosomal amplification of the ADH2 gene in Saccharomyces cerevisiae; Paquin CE et al.; A spontaneous antimycin A-resistant mutant carrying approximately four extra copies of ADH2 on chromosome XII was isolated from yeast strain 315-1D which lacks a functional copy of ADH1 and thus is antimycin A-sensitive . The additional copies of the normally glucose-repressed ADH2 are expressed during growth on glucose accounting for the antimycin A resistance . These extra copies are inserted into nonadjacent ribosomal DNA sequences (rDNA) near the recombination stimulating sequence HOT1 . Each extra copy of the ADH2 gene (1548 bp) replaces most of the 37S transcript (approximately 7400 bp) in one of the approximately 200 copies of the rDNA present in the yeast genome . All four extra copies of ADH2 are lost at a rate of approximately 1 x 10(-5) deletions per cell per generation . One of the joints between the rDNA and ADH2 DNA is located 7 nucleotides downstream from 20 adenine residues in the normal copy of ADH2 . This joint occurs at the end of a stretch of 16-29 thymidines in the rDNA which has been expanded to 57-59 thymidines . The other novel joint is located in a short region of sequence similarity between ADH2 and the rDNA . These observations suggest that amplification of ADH2 was a two step process: first the ADH2 gene was inserted into the rDNA, then multiple copies were generated by unequal crossing over or gene conversion within the rDNA.

Mol Gen Genet, 1992 Feb, 231(3), 426 - 32
Negative regulators of the PHO system of Saccharomyces cerevisiae: characterization of PHO80 and PHO85; Uesono Y et al.; Both PHO80 and PHO85 genes are required to establish the repressed state of the PHO system of Saccharomyces cerevisiae . S1 nuclease protection analysis of the PHO85 transcript revealed that the PHO85 gene contains an intron at the 6th codon of the gene . Each of the fusion proteins, LacZ-Pho80 and LacZ-Pho85, was produced into Escherichia coli and used as an antigen to raise antibodies in a rabbit . Using the affinity-purified antibodies in Western blotting experiments, the PHO85 protein was detected as a 36 kDa and the PHO80 protein as a 34 kDa protein . The PHO80 protein was detected only in extracts prepared from an overproducing strain . The immunoprecipitate containing the PHO85 protein showed protein kinase activity suggesting that PHO85 is a protein kinase gene, which is consistent with the observation that the deduced amino acid sequence of the PHO85 protein resembles that of some protein kinases . The PHO80 protein was found to be phosphorylated in the presence of PHO85 protein.

EMBO J, 1992 Feb, 11(2), 733 - 40
DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae; Araki H et al.; Two temperature-sensitive DNA polymerase II mutants (pol2-9 and pol2-18) of the yeast Saccharomyces cerevisiae were isolated by the plasmid shuffling method . DNA polymerase II activity partially purified from both mutants was thermolabile, while DNA polymerase I and III activities remained thermotolerant . At the restrictive temperature, the pol2 mutants were defective in chromosomal DNA replication and exhibited the dumbbell terminal morphology typical of DNA replication mutants . The POL2 transcript accumulated periodically during the cell cycle, peaking at the G1/S boundary in the same manner as the transcripts of more than 10 other DNA replication genes . These results indicate that DNA polymerase II participates in nuclear DNA replication . The similarities in structure and activities between the DNA polymerases of yeast and mammals make it likely that mammalian DNA polymerase epsilon too is required for chromosomal DNA replication.

EMBO J, 1992 Feb, 11(2), 683 - 9
U14 function in Saccharomyces cerevisiae can be provided by large deletion variants of yeast U14 and hybrid mouse-yeast U14 RNAs; Li D et al.; The functional equivalency of yeast and mouse U14 RNAs was examined in Saccharomyces cerevisiae . The test RNAs included mouse U14 and several yeast-mouse bi- and tri-partite hybrid RNAs, all transcribed from yeast U14 gene signals . The ability of the heterologous RNAs to provide essential U14 function was assessed in a test strain containing a single glucose-repressible wild-type U14 gene . Mouse U14 was not functional in yeast . However, wild-type growth was supported by hybrid RNAs that included universal sequence elements from either source, two yeast-specific segments and a 5',3' terminal stem domain . The universal sequences include box C, box D and a sequence complementary to 18S rRNA, all shown previously to be required for function of yeast U14 . Deletion and substitution mapping defined the yeast-specific elements and showed that a major portion of neighboring non-conserved RNA is dispensible . The results are discussed with a view to defining a minimal consensus U14 molecule.

EMBO J, 1992 Feb, 11(2), 423 - 32
The Saccharomyces cerevisiae SEC20 gene encodes a membrane glycoprotein which is sorted by the HDEL retrieval system; Sweet DJ et al.; The SEC20 gene product (Sec20p) is required for endoplasmic reticulum (ER) to Golgi transport in the yeast secretory pathway . We have cloned the SEC20 gene by complementation of the temperature sensitive phenotype of a sec20-1 strain . The DNA sequence predicts a 44 kDa protein with a single membrane-spanning region; Sec20p has an apparent molecular weight of 50 kDa and behaves as an integral membrane protein with carbohydrate modifications that appear to be O-linked . A striking feature of this protein is its C-terminal sequence, which consists of the tetrapeptide HDEL . This signal is known to be required for the retrieval of soluble ER proteins from early Golgi compartments, but has not previously been observed on a membrane protein . The HDEL sequence of Sec20p is not essential for viability but helps to maintain intracellular levels of the protein . Depletion of Sec20p from cells results in the accumulation of an extensive network of ER and clusters of small vesicles . We suggest a possible role for the SEC20 product in the targeting of transport vesicles to the Golgi apparatus.

Yeast, 1992 Feb, 8(2), 107 - 15
Inhibition of glycolysis by 2-deoxygalactose in Saccharomyces cerevisiae; Lagunas R et al.; The enzymatic steps involved in the inhibition of glycolysis by 2-deoxygalactose in Saccharomyces cerevisiae have been investigated . Yeast, incubated with 2-deoxygalactose, accumulates up to 8 mM-2-deoxygalactose, 30 mM-2-deoxygalactose-1-phosphate and 0.25 mM-UDP-2-deoxygalactose and UDP-2-deoxyglucose . An inverse correlation between 2-deoxygalactose-1-phosphate content and rate of glycolysis has been observed . The intracellular concentration of glycolytic intermediates and related metabolites point to the hexokinase and phosphofructokinase steps as the targets for the inhibition of glycolysis by 2-deoxygalactose and rule out all other mechanisms that have been proposed to explain this inhibition.

EMBO J, 1992 Feb, 11(2), 457 - 66
Two different types of lipid moieties are present in glycophosphoinositol-anchored membrane proteins of Saccharomyces cerevisiae; Conzelmann A et al.; Numerous glycoproteins of Saccharomyces cerevisiae are anchored in the lipid bilayer by a glycophosphatidylinositol (GPI) anchor . Mild alkaline hydrolysis reveals that the lipid components of these anchors are heterogeneous in that both base-sensitive and base-resistant lipid moieties can be found on most proteins . The relative abundance of base-resistant lipid moieties is different for different proteins . Strong alkaline or acid hydrolysis of the mild base-resistant lipid component liberates C18-phytosphingosine indicating the presence of a ceramide . Two lines of evidence suggest that proteins are first attached to a base-sensitive GPI anchor, the lipid moiety of which subsequently gets exchanged for a base-resistant ceramide: (i) an early glycolipid intermediate of GPI biosynthesis only contains base-sensitive lipid moieties; (ii) after a pulse with {3H}myo-inositol the relative abundance of base-sensitive GPI anchors decreases significantly during chase . This decrease does not take place if GPI-anchored proteins are retained in the ER.

Can J Microbiol, 1992 Feb, 38(2), 149 - 52
Localization of a 210-kDa microtubule-interacting protein in the yeast Saccharomyces cerevisiae; Hasek J et al.; Using the monoclonal antibody MA-01, which recognizes a 210-kDa protein in cell-free extracts, spindle and cytoplasmic microtubules were visualized in budding yeast, Saccharomyces cerevisiae . In additional, a spot-like staining was found beneath the plasma membrane, revealing in part correlation with F-actin distribution . This pattern was common for cells of all cell-cycle stages . The interaction of the protein recognized by MA-01 with microtubules was confirmed in the double labeling with a polyclonal antitubulin antibody and by the sensitivity of intranuclear structures stained by MA-01 to the microtubule disrupting drug nocodazole.

J Bioenerg Biomembr, 1992 Feb, 24(1), 27 - 31
Determination of the number of polypeptide subunits in a functional VDAC channel from Saccharomyces cerevisiae; Peng S et al.; Genes encoding VDAC proteins containing specific site-directed amino acid alterations were introduced into wild-type Saccharomyces cerevisiae . The mutant VDAC proteins form channels with ion selectivities very different from that of the wild-type channel . Therefore, the resulting yeast strains express two different genes capable of coding for functional, yet distinct, VDAC channels . If VDAC were an oligomeric channel, analysis of VDAC from these strains should have revealed not only the presence of channels with wild-type or mutant selectivity but also channels with intermediate selectivities . While channels with wild-type and mutant selectivities were observed with approximately equal frequency, no channels with intermediate selectivity were observed . Sufficient observations were performed with two different mutant genes K61E.K65E and K19E.K61E) that the likelihood of having missed hybrid channels was less than 1 in 10(7) . These findings favor the hypothesis that each functional VDAC channel is composed of a single 30-kDa polypeptide chain.

Biosci Biotechnol Biochem, 1992 Feb, 56(2), 174 - 9
Cloning of the alpha-amylase cDNA of Aspergillus shirousamii and its expression in Saccharomyces cerevisiae; Shibuya I et al.; alpha-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504 . The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000 . The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3' non-coding regions, respectively, so far determined . The alpha-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1 . The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions . Consequently, active alpha-amylase and glucoamylase were efficiently secreted into the culture medium . The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A . shirousamii.

Biosci Biotechnol Biochem, 1992 Feb, 56(2), 315 - 9
Secretion of mono- and diacylglycerol lipase from Penicillium camembertii U-150 by Saccharomyces cerevisiae and site-directed mutagenesis of the putative catalytic sites of the lipase; Yamaguchi S et al.; Yeast cells carrying intronless mono- and diacylglycerol lipase (MDGL) genes, constructed by recombination of the genomic gene and cDNA, secreted MDGL into the culture supernatant . Most of the yeast MDGL were extensively glycosylated while they had a similar glyceride specificity to that of native MDGL . Site-directed mutagenesis was used to directly confirm the involvements in enzyme activity of the presumptive amino acid residues to form the catalytic center of MDGL . These residues were conserved in the primary structure alignment of a lipase family from filamentous fungi . Mutant lipase proteins in which Ser83, Ser145, or His259 was replaced with glycine were secreted by yeast transformants as inactive proteins . Mutant proteins replacing Asp199 with glycine or asparagine were not detected in the culture supernatant . Replacing other two highly conserved aspartic acids (at positions 232 and 243) with glycine did not render the enzyme inactive . These results indicate that Ser83, Ser145, and His259 in MDGL, are essential to enzyme activity . Asp199 is also likely to be involved.

J Biotechnol, 1992 Feb, 22(3), 329 - 51
The decisive role of the Saccharomyces cerevisiae cell cycle behaviour for dynamic growth characterization; Munch T et al.; The dynamic behaviour of the cell cycle and the physiology of Saccharomyces cerevisiae was monitored in transient experiments . Frequent flow cytometric analyses of the DNA (nuclear phase state) and the cell size enabled us to characterize the proliferation properties of yeast cells under well controlled and undisturbed cultivation conditions . Preliminarily, the correlation between flow cytometric light scattering measurements and the cell size was attested for yeasts . These flow cytometric results are compared with the physiological behaviour of the culture that was detected by high resolution on-line analyses and off-line measurements . The presented results focus on the importance of the yeast cell cycle behaviour for the dynamic growth characterization . Any kind of transients in yeast cultures induced partial synchronization . The characteristics and the time course of the yeast cell cycle were found to be strongly dependent on the physiological environment.

Curr Genet, 1992 Feb, 21(2), 147 - 51
The unusual reversion properties of a mitochondrial mutation in the structural gene of subunit I of cytochrome oxidase of Saccharomyces cerevisiae reveal a probable histidine ligand of the redox center; Netter P et al.; We have analyzed a mutation in the mitochondrial gene oxi3 coding for subunit I of cytochrome-oxidase in the yeast Saccharomyces cerevisiae . This mutation replaces one of the seven invariant histidines of the polypeptide (position 378) by a tyrosine, and leads to a respiratory deficient phenotype . A total of 157 revertants, which have recovered the ability to grow on a respiratory substrate, have been selected from this mutant (tyrosine 378) . The nature of the reversion has been analysed by a rapid screening procedure and 32 of the revertants have been sequenced . They are all true back-mutations reintroducing the histidine in position 378 . This very exceptional situation suggests that this histidine is a ligand of the redox center of cytochrome oxidase.

Gene, 1992 Feb 1, 111(1), 131 - 4
Physical mapping of the Saccharomyces cerevisiae Ap4A phosphorylase I-encoding gene by the Achilles' cleavage method; Garrison PN et al.; LacI-mediated Achilles' cleavage (AC) is a method for selective fragmentation of chromosomes at special lac operator sites introduced by gene targeting methods {Koob and Szybalski, Science 250 (1990) 271-273} . The Saccharomyces cerevisiae APA1 gene, coding for diadenosine 5', 5"'-P1, P4-tetraphosphate phosphorylase I, has previously been shown to be located on chromosome III {Kaushal et al., Gene 95 (1990) 79-84} . We have now used the AC method to map APA1 gene to a site 44 kb from the left terminus of the chromosome, between the HIS4 and HML genes . This location was confirmed by the comparison of restriction maps of the APA1 gene region to published restriction maps of chromosome III.

Nucleic Acids Res, 1992 Jan 25, 20(2), 295 - 301
A new rRNA processing mutant of Saccharomyces cerevisiae; Lindahl L et al.; We have identified from a collection of temperature sensitive yeast mutants strains which fail to process rRNA normally . Characterization of one such mutant is reported here . This strain accumulates increased amounts of the 35S primary transcript, '24S' molecules extending from the transcription start site to the 5.8S region, and two classes of 5.8S rRNA with 5' extensions of 7 and 149 bases, respectively . We show that this pleiotropic change in the rRNA processing pattern is due to a single mutation . Possible models for the function of the mutated gene are discussed.

J Biol Chem, 1992 Jan 25, 267(3), 1648 - 54
Identification and purification of DBF-A, a double-stranded DNA-binding protein from Saccharomyces cerevisiae; Verma R et al.; Using oligonucleotide affinity chromatography with DNase I footprinting as an assay we have looked for proteins that interact with sequence elements within the yeast origin of replication, autonomously replicating sequence 1 (ARS1) . In this work we describe a protein that binds with high affinity to DNA but displays only moderate sequence specificity . It is eluted at 0.7 M salt from an ARS1 oligonucleotide column . Footprinting analysis on ARS1 at a high protein concentration revealed at least three sites of protection flanking element A and its repeats . Element A itself is rendered hypersensitive to DNase I digestion upon protein binding . This pattern is also observed for the H4 and HMR-E ARSs, suggesting that the protein alters the DNA conformation at element A and its repeats . The affinity-purified fraction is also capable of supercoiling a relaxed, covalently closed plasmid in the presence of topoisomerase . Highly purified preparations of the protein are enriched in an 18-kDa polypeptide which can be renatured from a denaturing gel and shown to bind ARS1 DNA . We have designated this protein DBF-A, DNA-binding factor A.

J Biol Chem, 1992 Jan 25, 267(3), 1953 - 61
Hepatitis B virus envelope L protein particles . Synthesis and assembly in Saccharomyces cerevisiae, purification and characterization; Kuroda S et al.; The hepatitis B virus envelope gene encodes three transmembrane proteins in frame; S, the product of S gene; M, the product of M (pre-S2 + S) gene; and L, the product of L (pre-S1 + pre-S2 + S) gene . Unlike the S and M proteins, attempts to efficiently synthesize L proteins and assemble them into L protein particles in various eukaryotic cells have been unsuccessful, probably because of the presence of the pre-S1 peptide with an unknown function which appears to be inhibitory to the host secretory apparatus . To investigate the role of the pre-S1 peptide, we constructed an L gene fused with a synthetic gene for chicken-lysozyme signal peptide (C-SIG) at the 5'-terminal and placed the resultant gene under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter . After the fused-C-SIG peptide was correctly processed by the yeast secretory apparatus, a yeast transformant synthesized a protein with a molecular mass of approximately 52 kDa at a level of 42% of the total soluble protein . Electron micrographic observation showed that the gene products assembled into 23-nm spherical and filamentous particles . The pre-S peptide of the gene product was deposited into the endoplasmic reticulum (ER) lumen and well-glycosylated . It seemed that the gene products were accumulated as particles in certain specific membrane structures of the yeast secretory apparatus . Moreover, both the amount of mRNAs specific for the L gene and the in vivo stability of the synthesized L proteins did not change significantly by the addition of the C-SIG gene . These findings indicated that, if the pre-S1 peptide penetrates the ER membrane efficiently, the L proteins can be synthesized cotranslationally, translocate across the ER membrane with its S region, and then assemble by themselves into the particle form . Therefore, the pre-S1 peptide may involve weak or reduced signal peptide activity for recognition by the secretory apparatus and/or for the transport of the pre-S peptide into the ER lumen.

Biochim Biophys Acta, 1992 Jan 24, 1123(2), 127 - 32
A defect in the sterol:steryl ester interconversion in a mutant of the yeast, Saccharomyces cerevisiae; Keesler GA et al.; A culture cycle dependent interconversion of sterols and steryl esters is disturbed in a mutant of Saccharomyces cerevisiae . Independent extragenic suppressors to this mutant return the mutant's pleiotropic phenotype to that of the parental wild type . Concomitant with the alterations in interconversion, modifications were found in the yeast proteins that antigenically react with antibodies elicited against mammalian apolipoproteins . Suppressor mutations returned the aberrant immunoblot banding pattern of the mutant to that of the wild type in apolipoprotein B.

Biochemistry, 1992 Jan 21, 31(2), 551 - 7
Antagonistic and synergistic peptide analogues of the tridecapeptide mating pheromone of Saccharomyces cerevisiae; Eriotou-Bargiota E et al.; Biologically inactive, truncated analogues of the Saccharomyces cerevisiae alpha-mating factor (WHWLQLKPGQPMY) either antagonized or synergized the activity of the native pheromone . An amino-terminal truncated pheromone {WLQLKPGQP(Nle)Y} had no activity by itself, but the analogue acted as an antagonist by competing with binding and activity of the mating factor . In contrast, a carboxyl-terminal truncated pheromone {WHWLQLKPGQP} was not active by itself nor did the peptide compete with alpha-factor for binding to the alpha-factor receptor, but it acted as a synergist by causing a marked increase in the activity of alpha-factor . The observation that residues near the amino terminus may be involved in signal transduction whereas those near the carboxyl terminus influence binding allows us to separate binding and signal transduction in the yeast pheromone response pathway . If found for other hormone-receptor systems, synergists may have potential as therapeutic compounds.

Biochim Biophys Acta, 1992 Jan 16, 1098(2), 217 - 23
Characterization of a mitochondrial inorganic pyrophosphatase in Saccharomyces cerevisiae; Lundin M et al.; We have studied a mitochondrial inorganic pyrophosphatase (PPase) in the yeast Saccharomyces cerevisiae . The uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and the ionophores valinomycin and nigericin stimulate the PPase activity of repeatedly washed yeast mitochondria 2-3-fold . We have previously cloned a yeast gene, PPA2, encoding the catalytic subunit of a mitochondrial PPase . Uncouplers stimulate the PPase activity several-fold in mitochondria from both cells that overexpress PPA2 from a high copy number plasmid and cells with normal expression . These results indicate that the PPA2 polypeptide functions as an energy linked and membrane associated PPase . The stimulation of mitochondrial PPase activity by FCCP, but not by valinomycin and nigericin, was greatly enhanced by the presence of DTT . The antibiotics Dio-9, equisetin and the F0F1-ATPase inhibitor oligomycin also increase mitochondrial PPase activity several fold . This stimulation is much higher, whereas basal PPase activity is lower, in isotonic than in hypotonic solution, which indicates that intact membranes are a prerequisite for maximal effects.

Mol Microbiol, 1992 Jan, 6(1), 75 - 81
Isolation and characterization of sporulation-specific promoters in the yeast Saccharomyces cerevisiae; Coe JG et al.; A library of random yeast genomic DNA:lacZ fusions has been constructed using an episomal yeast-Escherichia coli shuttle vector (pCS1) . Plasmid pCS1 requires insertion of a promoter and an in frame ATG codon upstream of its resident truncated lacZ gene to regulate expression in yeast . Yeast genomic DNA fragments of 4-6 kb were generated by partial digestion with Sau3A and ligated into the unique BamHI site of plasmid pCS1 to generate a library of 5 x 10(4) individual E . coli transformants . This library was screened to identify promoter-lacZ fusions that were expressed uniquely during sporulation . Of 342 yeast transformants that exhibited beta-galactosidase activity, two were found to express the lacZ gene in a sporulation-specific manner . This paper presents the characterization of two genomic yeast DNA fragments containing promoters that control lacZ expression during the sporulation process . Expression from the promoter present in plasmid pJC18 occurred from 11-21 hours into the sporulation process, while the promoter in plasmid pJC217 was active from 4-14 hours . Staining of nuclear DNA to correlate nuclear morphology with timing of gene expression showed when each of these promoters was active in terms of the morphological stages of sporulation.

Mol Gen Genet, 1992 Jan, 231(2), 329 - 31
The CDC26 gene of Saccharomyces cerevisiae is required for cell growth only at high temperature; Araki H et al.; We have cloned and sequenced the wild-type CDC26 gene and a mutant allele, cdc26-1, of Saccharomyces cerevisiae . Nucleotide sequence analysis revealed that the gene we cloned was the same as SCD26, a dosage-dependent suppressor of cdc26 . However, the cloned gene is in fact the CDC26 gene, because a nucleotide substitution in cdc26-1 was found to be a nonsense mutation in this sequence . Disruption of this gene conferred thermosensitive cell growth and the disrupted cdc26 gene could not complement the cdc26-1 mutant allele . Thus, the CDC26 gene is required for cell growth only at high temperature.

Mol Gen Genet, 1992 Jan, 231(2), 194 - 200
Molecular structure of the DNA cross-link repair gene SNM1 (PSO2) of the yeast Saccharomyces cerevisiae; Richter D et al.; A 3.2 kb yeast DNA fragment containing the DNA interstrand cross-link-specific repair gene SNM1 has been sequenced . Two genes were identified . SNM1 has an open reading frame of 1983 bp and codes for a 661 amino acid protein . Hydrophobic analysis shows that the protein is most probably not directly membrane bound . The second gene, UGX1, has an open reading frame of 573 bp coding for a polypeptide of 191 amino acid residues . The two genes are arranged head to head and share a 192 bp divergent promoter region that contains three TATAAA motives, two for the SNM1 and one for the UGX1 locus . Gene UGX1 has no apparent influence on the sensitivity of the cell to cross-linking nitrogen mustard, as its disruption in wild type does not increase sensitivity to nitrogen mustard and the presence of multiple copies of the gene fails to complement the nitrogen mustard sensitivity phenotype of snm1 disruption mutants . Northern analysis revealed that the expression of SNM1 yields an average of 0.3 copies/cell of a 2.4 kb transcript, while expression of UGX1 yields higher levels of a 0.8 kb poly(A)+ RNA.

Curr Genet, 1992 Jan, 21(1), 85 - 90
The DNA repair gene PSO3 of Saccharomyces cerevisiae belongs to the RAD3 epistasis group; Benfato MS et al.; The mutant allele pso3-1 of Saccharomyces cerevisiae confers sensitivity to treatment with UV365nm (UVA) light-activated mono- and bi-functional psoralens . When pso3-1 is combined in double mutants with selected rad and pso mutant alleles and subjected to 8-MOP + UVA treatment, epistatic interaction with regard to survival is observed with pso1, pso2, and rad3 . With the same treatment the combination of pso3-1 with rad6 and rad52 leads to synergistic interaction . For the monofunctional agent 3-carbethoxypsoralen (3-CPs) the analysis of double mutants yields the same results as with the bifunctional 8-methoxypsoralen (8-MOP) with the exception of the pso1-1pso3-1 double mutant . Here we find an additive interaction, i.e., the sensitivities of both parental strains are summed in the double mutant, which indicates a different substrate specificity of the repair activity encoded by the PSO1 and PSO3 genes.

Biochem J, 1992 Jan 1, 281 ( Pt 1), 67 - 72
Characterization of the structure and conformation of platelet-derived growth factor-BB (PDGF-BB) and proteinase-resistant mutants of PDGF-BB expressed in Saccharomyces cerevisiae; Craig S et al.; A detailed biophysical study of the secondary and tertiary structures of recombinant platelet-derived growth factor (PDGF)-BB produced in yeast has been carried out . The secondary structure of the molecule is composed of 54% beta-sheet with less than 5% ordered helix . The single tryptophan residue has been shown to be solvent-accessible; however, the ability of the side chain to rotate is severely restricted . The fluorescence emission is quenched at pH 7.0 and in the presence of high salt, but dequenched by titration to lower pH with a pK of 5.8 . Two proteinase-resistant mutants of PDGF {( Ser28}- and {Pro32}-PDGF-BB) have also been characterized and shown to have secondary and tertiary structures indistinguishable from wild-type PDGF-BB . These are, therefore, suitable stable background molecules in which to carry out structure-activity-relationship studies on PDGF-BB.

Mol Cell Biol, 1992 Jan, 12(1), 276 - 82
C-terminal sequences can inhibit the insertion of membrane proteins into the endoplasmic reticulum of Saccharomyces cerevisiae; Green N et al.; We have constructed three gene fusions that encode portions of a membrane protein, arginine permease, fused to a reporter domain, the cytoplasmic enzyme histidinol dehydrogenase (HD), located at the C-terminal end . These fusion proteins contain at least one of the internal signal sequences of arginine permease . When the fusion proteins were expressed in Saccharomyces cerevisiae and inserted into the endoplasmic reticulum (ER), two of the fusion proteins placed HD on the luminal side of the ER membrane, but only when a piece of DNA encoding a spacer protein segment was inserted into the fusion joint . The third fusion protein, with or without the spacer included, placed HD on the cytoplasmic side of the membrane . These results suggest that (i) sequences C-terminal to the internal signal sequence can inhibit membrane insertion and (ii) HD requires a preceding spacer segment to be translocated across the ER membrane.

Mol Cell Biol, 1992 Jan, 12(1), 248 - 60
The suil suppressor locus in Saccharomyces cerevisiae encodes a translation factor that functions during tRNA(iMet) recognition of the start codon; Yoon HJ et al.; We initiated a genetic reversion analysis at the HIS4 locus to identify components of the translation initiation complex that are important for ribosomal recognition of an initiator codon . Three unlinked suppressor loci, suil, sui2, and SUI3, that restore expression of both HIS4 and HIS4-lacZ in the absence of an AUG initiator codon were identified . In previous studies, it was demonstrated that the sui2 and SUI3 genes encode mutated forms of the alpha and beta subunits, respectively, of eukaryotic translation initiation factor 2 (eIF-2) . In this report, we describe the molecular and biochemical characterizations of the sui1 suppressor locus . The DNA sequence of the SUI1+ gene shows that it encodes a protein of 108 amino acids with a calculated Mr of 12,300 . The sui1 suppressor genes all contain single base pair changes that alter a single amino acid within this 108-amino-acid sequence . sui1 suppressor strains that are temperature sensitive for growth on enriched medium have altered polysome profiles at the restrictive temperature typical of those caused by alteration of a protein that functions during the translation initiation process . Gene disruption experiments showed that the SUI1+ gene encodes an essential protein, and antibodies directed against the SUI1+ coding region identified a protein with the predicted Mr in a ribosomal salt wash fraction . As observed for sui2 and SUI3 suppression events, protein sequence analysis of His4-beta-galactosidase fusion proteins produced by sui1 suppression events indicated that a UUG codon is used as the site of translation initiation in the absence of an AUG start codon in HIS4 . Changing the penultimate proline codon 3' to UUG at his4 to a Phe codon (UUC) blocks aminopeptidase cleavage of the amino-terminal amino acid of the His4-beta-galactosidase protein, as noted by the appearance of Met in the first cycle of the Edman degradation reaction . The appearance of Met in the first cycle, as noted, in either a sui1 or a SUI3 suppressor strain showed that the mechanism of suppression is the same for both suppressor genes and allows the initiator tRNA to mismatch base pair with the UUG codon . This suggests that the Sui1 gene product performs a function similar to that of the beta subunit of eIF-2 as encoded by the SUI3 gene . However, the Sui1 gene product does not appear to be a required subunit of eIF-2 on the basis of purification schemes designed to identify the GTP-dependent binding activity of eIF-2 for the initiator tRNA . In addition, suppressor mutations in the sui1 gene, in contrast to suppressor mutations in the sui2 or SUI3 gene, do not alter the GTP-dependent binding activity of the eIF-2 . The simplest interpretation of these studies is that the sui1 suppressor gene defines an additional factor that functions in concert with eIF-2 to enable tRNAiMet to establish ribosomal recognition of an AUG initiator codon.

Mol Cell Biol, 1992 Jan, 12(1), 22 - 9
GLC3 and GHA1 of Saccharomyces cerevisiae are allelic and encode the glycogen branching enzyme; Rowen DW et al.; In the yeast Saccharomyces cerevisiae, glycogen serves as a major storage carbohydrate . In a previous study, mutants with altered glycogen metabolism were isolated on the basis of the altered iodine-staining properties of colonies . We found that when glycogen produced by strains carrying the glc-1p (previously called gha1-1) mutation is stained with iodine, the absorption spectrum resembles that of starch rather than that of glycogen, suggesting that this mutation might reduce the level of branching in the glycogen particles . Indeed, glycogen branching activity was undetectable in extracts from a glc3-1p strain but was elevated in strains which expressed GLC3 from a high-copy-number plasmid . These observations suggest that GLC3 encodes the glycogen branching enzyme . In contrast to glc3-1p, the glc3-4 mutation greatly reduces the ability of yeast to accumulate glycogen . These mutations appear to be allelic despite the striking difference in the phenotypes which they produce . The GLC3 clone complemented both glc3-1p and glc3-4 . Deletions and transposon insertions in this clone had parallel effects on its ability to complement glc3-1p and glc3-4 . Finally, a fragment of the cloned gene was able to direct the repair of both glc3-1p and glc3-4 . Disruption of GLC3 yielded the glycogen-deficient phenotype, indicating that glycogen deficiency is the null phenotype . The glc3-1p allele appears to encode a partially functional product, since it is dominant over glc3-4 but recessive to GLC3 . These observations suggest that the ability to introduce branches into glycogen greatly increases the ability of the cell to accumulate that polysaccharide . Northern (RNA) blot analysis identified a single mRNA of 2,300 nucleotides that increased in abundance ca . 20-fold as the culture approached stationary phase . It thus appears that the expression of GLC3 is regulated, probably at the level of transcription.

Mol Cell Biol, 1992 Jan, 12(1), 172 - 82
Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog; Lee KS et al.; The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for yeast cell growth and division . To identify additional components of the pathway in which PKC1 functions, we isolated extragenic suppressors of a pkc1 deletion mutant . All of the suppressor mutations were dominant for suppressor function and defined a single locus, which was designated BCK1 (for bypass of C kinase) . A molecular clone of one suppressor allele, BCK1-20, was isolated on a centromere-containing plasmid through its ability to rescue a conditional pkc1 mutant . The BCK1 gene possesses a 4.4-kb uninterrupted open reading frame predicted to encode a 163-kDa protein kinase . The BCK1 gene product is not closely related to any known protein kinase, sharing only 45% amino acid identity with its closest known relative (the STE11-encoded protein kinase) through a region restricted to its putative C-terminal catalytic domain . Deletion of BCK1 resulted in a temperature-sensitive cell lysis defect, which was suppressed by osmotic stabilizing agents . Because pkc1 mutants also display a cell lysis defect, we suggest that PKC1 and BCK1 may normally function within the same pathway . Suppressor alleles of BCK1 differed from the wild-type gene in a region surrounding a potential PKC phosphorylation site immediately upstream of the predicted catalytic domain . This region may serve as a hinge between domains whose interaction is regulated by PKC1.

J Bacteriol, 1992 Jan, 174(1), 48 - 55
Nitrogen catabolite repression of arginase (CAR1) expression in Saccharomyces cerevisiae is derived from regulated inducer exclusion; Cooper TG et al.; Expression of the Saccharomyces cerevisiae arginase (CAR1) gene is regulated by induction and nitrogen catabolite repression (NCR) . Arginine was demonstrated to be the native inducer . CAR1 sensitivity to NCR has long been accepted to be accomplished through a negative control mechanism, and cis-acting sites for it have been hypothesized . In search of this negatively acting site, we discovered that CAR1 sensitivity to NCR derives from regulated inducer (arginine) exclusion . The route of catabolic entry of arginine into the cell, the general amino acid permease (GAP1), is sensitive to NCR . However, CAR1 expression in the presence of sufficient intracellular arginine is NCR insensitive.

Adv Microb Physiol, 1992, 33, 73 - 144
Secretory pathway function in Saccharomyces cerevisiae; Cleves AE et al.; A genetic analysis of secretory pathway function in yeast was initiated some 12 years ago in the laboratory of Randy Schekman . These mutants held great promise in terms of providing an experimental system with which molecular participants of secretory pathway function could be investigated . This early promise has not failed . For the last five years, analysis of yeast secretory pathway function has been at the cutting edge of our understanding of the mechanisms by which proteins travel between intracellular compartments . In some cases, Sacch . cerevisiae has provided a valuable in vivo corroboration of the concepts derived from biochemical studies of mammalian intercompartmental protein transport in vitro . In other cases, studies conducted in the yeast system have defined previously unanticipated involvements for known catalytic activities in the secretory process . It is clear that yeast will continue to play a major role in setting the pace of research directed towards a detailed molecular understanding of protein secretion . Since it is now apparent that the basic strategies that underlie secretory pathway function have been conserved among eukaryotes, further exploitation of the powerful and complementary yeast and mammalian experimental systems guarantees that the next decade will see even greater progress towards our understanding of protein secretion in eukaryotic cells than did the first.

Yeast, 1992 Jan, 8(1), 71 - 7
A putative serine/threonine protein kinase gene on chromosome III of Saccharomyces cerevisiae; Wilson C et al.; We have sequenced a gene on chromosome III of Saccharomyces cerevisiae which codes for a putative serine/threonine protein kinase of 726 amino acids (calculated molecular weight 82 kDa) . We have called this gene KIN82 . The amino acid sequence of KIN82 is most similar to the cyclic nucleotide-dependent protein kinase subfamily and the protein kinase C subfamily . Gene disruption of KIN82 did not produce any phenotype when tested under a variety of conditions . Reduced stringency hybridizations revealed the presence of another genomic sequence with high homology to the carboxy-terminal catalytic domain of KIN82.

Yeast, 1992 Jan, 8(1), 61 - 70
The complete sequence of a 10.8kb fragment to the right of the chromosome III centromere of Saccharomyces cerevisiae; Biteau N et al.; The complete nucleotide sequence of the D10H fragment (10850 bp) was determined . The D10H fragment is located on the right arm of chromosome III near the centromere and contains the SUF2 gene . Six open reading frames (ORFs) larger than 300 bp were found . One of them is the CIT2 gene encoding the cytoplasmic citrate synthase . The others are new putative genes and show no significant similarity with any known gene . In addition two tRNA genes (Asn and Pro) and a solo delta element were identified . Two ORFs were disrupted; no peculiar phenotype was observed.

Yeast, 1992 Jan, 8(1), 57 - 60
Expression of recombinant platelet-derived endothelial cell growth factor in the yeast Saccharomyces cerevisiae; Finnis C et al.; A platelet-derived endothelial cell growth factor cDNA has been cloned, sequenced and expressed using the Saccharomyces cerevisiae PRB1 promoter . Soluble recombinant platelet-derived endothelial cell growth factor constituted 0.5-1.0% of total soluble protein . Yeast soluble protein extracts containing recombinant platelet-derived endothelial cell growth factor stimulate the growth of calf pulmonary artery endothelial cells in vitro.

Yeast, 1992 Jan, 8(1), 39 - 45
Flow cytometric analysis of Saccharomyces cerevisiae autolytic mutants and protoplasts; de la Fuente JM et al.; Simple methods, based on the technique of flow cytometry, have been developed for the phenotypic characterization of yeast autolytic mutants and for the analysis of the formation and regeneration of the yeast protoplasts . The expression of lytic mutations determined uptake of the fluorescent dye propidium iodide, which could be carefully monitored by flow cytometry . Mixed populations of lysed and viable cells were precisely quantified and sorted, and the technique was also applied to demonstrate protection from lysis of mutant cells with cell wall defects, in the presence of osmotic stabilizers . Protoplast formation and regeneration was monitored by analysing relative cell size; this was facilitated by the preparation of homogeneous protoplast preparations . The technique of flow cytometry proved superior to other conventional methods for these types of study.

J Gen Microbiol, 1992 Jan, 138 ( Pt 1), 97 - 102
Chitinase and chitin synthase 1: counterbalancing activities in cell separation of Saccharomyces cerevisiae; Cabib E et al.; Previous results {E . Cabib, A . Sburlati, B . Bowers & S . J . Silverman (1989) Journal of Cell Biology 108, 1665-1672} strongly suggested that the lysis observed in daughter cells of Saccharomyces cerevisiae defective in chitin synthase 1 (Chs1) was caused by a chitinase that partially degrades the chitin septum in the process of cell separation . Consequently, it was proposed that in wild-type cells, Chs1 acts as a repair enzyme by replenishing chitin during cytokinesis . The chitinase requirement for lysis has been confirmed in two different ways: (a) demethylallosamidin, a more powerful chitinase inhibitor than the previously used allosamidin, is also a much better protector against lysis and (b) disruption of the chitinase gene in chs1 cells eliminates lysis . Reintroduction of a normal chitinase gene, by transformation of those cells with a suitable plasmid, restores lysis . The percentage of lysed cells in strains lacking Chs1 was not increased by elevating the chitinase level with high-copy-number plasmids carrying the hydrolase gene . Furthermore, the degree of lysis varied in different chs1 strains; lysis was abolished in chs1 mutants containing the scs1 suppressor . These results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting.

Folia Microbiol (Praha), 1992, 37(1), 43 - 6
Effect of growth rate on ethanol tolerance of Saccharomyces cerevisiae; Novotny C et al.; delta 5,7-Sterol-accumulating Saccharomyces cerevisiae cells growing in chemostat at a specific growth rate of 0.075/h exhibited higher ethanol tolerance measured as ethanol-induced death and anaerobic growth inhibition than the cells growing at 0.2/h, the difference being dependent on the carbon-to-nitrogen molar proportion in the medium . The observed difference in sensitivity to ethanol of anaerobic growth between the slowly and rapidly-growing cells was completely reversed as a result of a block in sterol synthesis causing a negligible synthesis of delta 5,7-sterols . Two physiological parameters, budding frequency and membrane composition, evidently affected ethanol tolerance . Differences between the delta 5,7-sterol-synthesizing and deficient strains documented a profound effect of the quality of the sterol present on the physiological state of the cell.

Gene Expr, 1992, 2(4), 329 - 37
Interferon regulatory factor-1 (IRF-1) activates the synthetic IRF-1-responsive sequence (GAAAGT)4 in Saccharomyces cerevisiae; Sailer A et al.; In appropriate mammalian cells, interferon regulatory factor-1 (IRF-1) can activate the virus-responsive element of the IFN-beta promoter (VRE beta") or the synthetic oligonucleotide (GAAAGT)4 . The latter contains two copies of the functional equivalent of PRDI, one of the regulatory domains of VRE beta" . We prepared yeast strains containing an IRF-1 expression plasmid under the control of the galactose-inducible Gal1 promoter and a reporter plasmid with either (GAAAGT)4, VRE beta", or other test sequences placed upstream of a minimal promoter linked to the beta-galactosidase coding sequence . Upon induction of IRF-1 expression, the (GAAAGT)4-containing promoter was activated, but VRE beta" and all other sequences tested were inactive . Our results showed that IRF-1 belongs to a class of higher eukaryotic transcription factors that can interact with the yeast transcriptional machinery . Our findings also raised the question why the duplicate PRDI-like sequences in (GAAAGT)4 can be activated by IRF-1 synthesized in yeast, but not VRE beta", which also contains at least two PRDI-like sequences.

Microbios, 1992, 72(290), 27 - 34
Nutritional and physiological factors affecting germination of heterothallic Saccharomyces cerevisiae ascospores; Xu G et al.; Saccharomyces cerevisiae strain AP-3 was examined with respect to those nutritional requirements and physiological conditions which influence its germination rate . It was found that glucose as a carbon source supported the most rapid rate of germination for this heterothallic strain . In contrast, strain AP-3 spore germination was supported the least by the carbon sources potassium acetate and lactose . Of the nitrogen sources tested in culture medium containing glucose, the complex nitrogen sources peptone and casein hydrolysate appeared to be capable of stimulating germination better than a control culture containing ammonium sulphate . None of the amino acids screened were found to stimulate strain AP-3 germination compared with ammonium sulphate . The optimal culture medium pH for ascospore germination was 4.5 although spore germination could still be initiated by glucose between pH 3.0 and pH 7.5 . Germination initiation by glucose was observed over a temperature range from 25 degrees C to 50 degrees C, but the optimal temperature appeared to be 40 degrees C.

Folia Microbiol (Praha), 1992, 37(4), 286 - 8
Ethanol-induced death and lipid composition of Saccharomyces cerevisiae: a comparative study of the role of sterols; Novotny C et al.; Ethanol tolerance of four Saccharomyces cerevisiae strains characterized by different amounts of delta 5,7-sterols was tested . The individual tolerances did not correlate with the strains sterol levels . The highly and medium-accumulating strains exhibited the highest and lowest ethanol tolerances, respectively.

Folia Microbiol (Praha), 1992, 37(4), 273 - 8
Expression of Japanese quail ovalbumin in Saccharomyces cerevisiae; Krizkova L et al.; A cDNA sequence coding for Japanese quail ovalbumin was used for the construction of expression plasmid under the ADH1 promoter of the yeast shuttle vector pVT101-U . The resulting recombinant expression vector pJK2 was used for the transformation of Saccharomyces cerevisiae . Expression of quail ovalbumin in yeast cells was demonstrated by Western blotting followed by immunochemical detection.

Folia Microbiol (Praha), 1992, 37(4), 267 - 72
UV-induced mutability in repair-deficient rad6-1 strains of Saccharomyces cerevisiae is caused by a suppressor gene; Vlckova V et al.; The RAD6 gene is a multifunctional gene required for DNA repair, induced mutagenesis and sporulation . The survival and revertibility of two loci in four rad6-1 mutant strains of different origin after UV irradiation were followed . As expected, all the rad6-1 strains tested were more sensitive to UV radiation in comparison with RAD6 strains . The reversion frequency per survivor in trp1-289 and arg4-17 alleles was significantly higher in all four rad6-1 mutant strains than in wild-type strains after equal doses of UV radiation . On the basis of genetic analysis we suggest that the phenomenon of increased frequency of induced mutagenesis is caused by a suppressor gene.

Annu Rev Microbiol, 1992, 46, 347 - 75
Double-stranded and single-stranded RNA viruses of Saccharomyces cerevisiae; Wickner RB; Yeast RNA viruses include L-A (and its toxin-encoding satellites M1, M2, ...) and L-BC dsRNA viruses and the single-stranded replicons 20S RNA and 23S RNA . L-A has a single-segment 4.6-kb linear genome encoding a major coat protein (gag) and its RNA-dependent RNA polymerase (pol), the latter expressed as a gag-pol fusion protein formed by a -1 ribosomal frameshift . In vitro replication, transcription, and binding systems for L-A have been used to define cis sites necessary for packaging and replication of viral RNA . Cellular functions that promote viral replication include the MAK3-encoded N-acetyltransferase whose modification of the gag N terminus is necessary for L-A virus assembly . The toxins encoded by the M satellite RNAs are processed by enzymes (KEX1 and KEX2, for killer expression) whose study led to discovery of mammalian hormone-processing enzymes . 20S RNA is an apparently naked circular RNA replicon (with a dsRNA form called W) encoding a RNA polymerase-like molecule . Its copy number is induced 10,000-fold in 1% potassium acetate, and it is subject to the same SKI antiviral system that represses L-A, L-BC, and M dsRNA copy number.

Arch Microbiol, 1992, 158(2), 115 - 26
The influence of congo red on the cell wall and (1----3)-beta-D-glucan microfibril biogenesis in Saccharomyces cerevisiae; Kopecka M et al.; Congo red was applied to growing yeast cells and regenerating protoplasts in order to study its effects on wall biogenesis and cell morphogenesis . In the presence of the dye, the whole yeast cells grew and divided to form chains of connected cells showing aberrant wall structures on both sides of the septum . The wall-less protoplasts in solid medium with the dye exhibited an abnormal increase in volume, regeneration of aberrant cell walls and inability to carry out cytokinesis or protoplast reversion to cells . In liquid medium, the protoplasts synthesized glucan nets composed mainly of thin fibrils orientated at random, whereas normally, in the absence of dye, the nets consist of rather thick fibrils, 10 to 20 nm in width, assembled into broad ribbons . These fibrils are known to consist of triple 6/1 helical strands of (1----3)-beta-D-glucan aggregated laterally in crystalline packing . The thin fibrils (c . 4 to 8 nm wide) can contain only a few triple helical strands (c . 1.6 nm wide) and are supposed to be prevented from further aggregation and crystallization by complexing with Congo red on their surfaces . Some loose triple 6/1 helical strands (native elementary fibrils) are also discernible . They represent the first native (1----3)-beta-D-glucan elementary fibrils depicted by electron microscopy . The effects of Congo red on growth and the wall structure in normal cells and regenerating protoplasts in solid medium can be explained by the presence of a complex which the dye forms with (helical) chain parts of the glucan network and which results in a loss of rigidity by a blocked lateral interaction between the helices.

Mutat Res, 1992 Jan, 265(1), 103 - 48
Induction of the cytoplasmic 'petite' mutation by chemical and physical agents in Saccharomyces cerevisiae; Ferguson LR et al.; A range of physical and chemical agents induce the mitochondrial 'petite' mutation in the yeast Saccharomyces cerevisiae . DNA intercalating agents as well as chemicals which can interfere with DNA synthesis induce this mutation, but only in growing cells . Many chemical or physical agents that produce a DNA lesion which is not simply reversed can induce various levels of the petite mutation, and may be more effective in non-growing cells . A limited number of chemicals act like ethidium bromide, inducing a high frequency of petites which is partially reversible with increasing concentration or time . The ability of a specific compound to be transported into mitochondria or its affinity for AT base pairs in DNA may determine whether it acts primarily as a nuclear or mitochondrial mutagen . In mammalian cells, some neoplastic changes occur at the mitochondrial level . Analogies between yeast and mammalian mitochondria suggest that agents which increase petite mutagenesis in yeast may have some carcinogenic potential . Although some types of petite inducer may have potential as antitumour drugs, those which are very effective antimitochondrial agents appear to be too toxic for therapeutic use . A process comparable to early stages in petite mutagensis occurs in human degenerative diseases and it seems possible that a consequence of exposure to petite mutagens could be an increase in the rate of degenerative diseases or of the aging process.

Microbios, 1992, 71(287), 149 - 59
The subcellular distribution of nickel in Ni-sensitive and Ni-resistant strains of Saccharomyces cerevisiae; Joho M et al.; Examination of the subcellular distribution of nickel in a Ni-resistant strain N08 of Saccharomyces cerevisiae showed that 70% of the nickel is distributed in the vascular fraction, which contains large amounts of histidine . The nickel taken up by cells grown in medium containing a high concentration of histidine was preferentially distributed to the vacuole . Arginine and lysine did not affect the intracellular distribution of Ni . In a Ni-sensitive strain 0605-S6, the distribution of nickel into the vacuole was lower than that observed in strain N08 . Strain 0605-S6 exhibited no increase in the histidine content of the vacuolar fraction when grown in a Ni-supplemented medium . The Ni-resistant mechanism appears to involve the sequestration of nickel to the vacuole, and histidine could play an important role in the reduction of free nickel in the vacuole by the formation of histidine-nickel complexes.

Chromosoma, 1992, 102(1 Suppl), S93 - 9
Three new DNA helicases from Saccharomyces cerevisiae; Li X et al.; At least six DNA helicases have been identified during fractionation of extracts from the yeast Saccharomyces cerevisiae . Three of those, DNA helicases B, C, and D, have been further purified and characterized . DNA helicases B and C co-purified with DNA polymerase delta through several chromatographic steps, but were separated from the polymerase by hydrophobic chromatography . DNA helicase D co-purified with Replication Factor C over seven chromatographic steps, and was only separated from it by glycerol gradient centrifugation in the presence of 0.2 M NaCl . All three helicases are DNA dependent ATPases with Km values for ATP of 190 microM, 325 microM, and 60 microM for DNA helicases B, C, and D, respectively . Their DNA helicase activities are comparable . They are 5'-3' helicases and have pH optima of 6.5-7 and Mg2+ optima of 1-2 mM . However, they differ in the nucleotide requirement for helicase action . Whereas all three helicases preferred ATP, dATP, UTP, CTP, and dCTP as cofactors, DNA helicase C also used GTP, but not dTTP . On the other hand, DNA helicase D used dTTP, but not GTP, and DNA helicase B used neither nucleotide as cofactor . These studies allowed us to conclude that DNA helicases B, C, and D are not only distinct enzymes, but also different from two previously identified yeast DNA helicases, the RAD3 protein and ATPase III.

EMBO J, 1992 Jan, 11(1), 87 - 96
GAC1 may encode a regulatory subunit for protein phosphatase type 1 in Saccharomyces cerevisiae; Francois JM et al.; Elevated dosage of the GAC1 gene from the yeast Saccharomyces cerevisiae causes hyperaccumulation of glycogen whereas a gene disruption of GAC1 results in reduced glycogen levels . Glycogen synthase is almost entirely in the active, glucose 6-phosphate-independent, form in cells with increased gene dosage of GAC1 whereas the enzyme is mostly in the inactive form in strains lacking GAC1 . GAC1 encodes an 88 kDa protein that is similar to the regulatory subunit (RG1) of phosphoprotein phosphatase type 1 (PP-1) from skeletal muscle that targets PP-1 to glycogen particles . Taken together, these results suggest that GAC1 encodes a regulatory subunit of PP-1 . As previously shown for glycogen phosphorylase (GPH1), GAC1 RNA accumulates concomitantly with the appearance of glycogen . A strain with a mutation in the regulatory subunit of the cAMP-dependent protein kinase (bcy1) fails to accumulate GPH1 and GAC1 RNA . These results point to coordinate regulation of enzymes involved in glycogen metabolism at the level of RNA accumulation and indicate that at least part of this control is exerted by the RAS-cAMP pathway.

Mol Microbiol, 1992 Jan, 6(1), 15 - 21
Glucose repression in the yeast Saccharomyces cerevisiae; Trumbly RJ; Understanding the mechanism of glucose repression in yeast has proved to be a difficult and challenging problem . A multitude of genes in different pathways are repressed by glucose at the level of transcription . The SUC2 gene, which encodes invertase, is an excellent reporter gene for glucose repression, since its expression is controlled exclusively by this pathway . Genetic analysis has identified numerous regulatory mutations which can either prevent derepression of SUC2 or render its expression insensitive to glucose repression . These mutations allow us to sketch the outlines of a pathway for general glucose repression, which has several key elements: hexokinase PII, encoded by HXK2, which seems to play a role in the sensing of glucose levels; the protein kinase encoded by SNF1, whose activity is required for derepression of many glucose-repressible genes; and the MIG1 repressor protein, which binds to the upstream regions of SUC2 and other glucose-repressible genes . Repression by MIG1 requires the activity of the CYC8 and TUP1 proteins . Glucose repression of other sets of genes seems to be controlled by the general glucose repression pathway acting in concert with other mechanisms . In the cases of the GAL genes and possibly CYC1, regulation is mediated by a cascade in which the general pathway represses expression of a positive transcriptional activator.

Genetics, 1992 Jan, 130(1), 71 - 80
Relationship of the cAMP-dependent protein kinase pathway to the SNF1 protein kinase and invertase expression in Saccharomyces cerevisiae; Hubbard EJ et al.; The SNF1 protein kinase and the associated SNF4 protein are required for release of glucose repression in Saccharomyces cerevisiae . To identify functionally related proteins, we selected genes that in multicopy suppress the raffinose growth defect of snf4 mutants . Among the nine genes recovered were two genes from the cAMP-dependent protein kinase (cAPK) pathway, MSI1 and PDE2 . Increased dosage of these genes partially compensates for defects in nutrient utilization and sporulation in snf1 and snf4 null mutants, but does not restore invertase expression . These results suggest that SNF1 and cAPK affect some of the same cellular responses to nutrients . To examine the role of the cAPK pathway in regulation of invertase, we assayed mutants in which the cAPK is not modulated by cAMP . Expression of invertase was regulated in response to glucose and was dependent on SNF1 function . Thus, a cAMP-responsive cAPK is dispensable for regulation of invertase.

Dev Genet, 1992, 13(6), 498 - 514
Minimal extent of homology required for completion of meiotic recombination in Saccharomyces cerevisiae; Hayden MS et al.; The minimal length of contiguous homology required for successful completion of meiotic recombination was investigated by using heterologous insertions to delimit homologous segments of chromosome III in the yeast Saccharomyces cerevisiae . Constructs created in vitro by insertion of selectable markers into the LEU2 locus were transplaced into haploid strains, which were then mated to create diploids containing pairs of insertion heterologies at various distances . Analysis of the meiotic products from these diploids revealed a gradient in the frequency of both reciprocal and nonreciprocal recombination declining monotonically from the 5' end of LEU2 . Both types of event were found to be restricted by the presence of the insertion heterologies . The spo13 single division meiosis was exploited to develop a plating assay in which LEU2 diploid spores containing reciprocally recombinant strands derived from events occurring completely within the interval flanked by the insertion heterologies were selected by random spore methods . Reciprocal recombination frequencies measured with this assay decreased linearly with extent, extrapolating to a minimal homology requirement of 150-250 nucleotides . When homology was most severely restricted, unexpected flanking marker configurations among reciprocal recombinants within LEU2 demonstrated the occurrence of complex recombination events . In addition to detecting reciprocal recombinants, the system is capable of measuring the probability that a non-reciprocal recombination event will have one end-point between the heterologous inserts and the other lying outside the interval . The minimal length of homology required for this aspect of recombination was found to be 25-60 nucleotides.

Dev Genet, 1992, 13(6), 392 - 402
Characterization of REC104, a gene required for early meiotic recombination in the yeast Saccharomyces cerevisiae; Galbraith AM et al.; The REC104 gene was initially defined by mutations that rescued the inviability of a rad52 spo 13 haploid strain in meiosis . We have observed that rec104 mutant strains undergo essentially no induction of meiotic gene conversion, and we have not been able to detect any meiotic crossing over in such strains . The REC104 gene has no apparent role in mitosis, since mutations have no observable effect on growth, mitotic recombination, or DNA repair . The DNA sequence of REC104 reveals that it is a previously unknown gene with a coding region of 549-bp, and genetic mapping has localized the gene to chromosome VIII near FUR1 . Expression of the REC104 gene is induced in meiosis, and it appears that the gene is not transcribed in mitotic cells . Possible roles for the REC104 gene product in meiosis are discussed.

J Cell Sci Suppl, 1992, 16, 87 - 96
SWI6 is a regulatory subunit of two different cell cycle START-dependent transcription factors in Saccharomyces cerevisiae; Moll T et al.; Most genes involved in DNA replication in the yeast Saccharomyces cerevisiae are transcribed transiently during late G1 as cells undergo START . Their promoters all contain one or more versions of an 8-base pair motif (ACGCGTNA) called the MluI cell cycle box (MCB) . MCBs have been shown to be both necessary and sufficient for the late G1-specific transcription of the TMP1 thymidylate synthase and POLI DNA polymerase genes . A different late G1-specific transcription element called the SCB (CACGAAAA) is bound by a factor containing the SWI4 and SWI6 proteins . We describe here the formation in vitro of complexes on TMP1 MCBs that contain the SWI6 protein and, we suggest, a 120 kDa protein that is distinct from SWI4 . Transcription due to SCBs and MCBs occurs in the absence of SWI6 but it is no longer correctly cell cycle regulated . We suggest that SWI6 is an essential regulatory subunit of two different START-dependent transcription factors . One factor (SBF) contains SWI4 and binds to SCBs whereas the other (MBF) contains p120 and binds MCBs.

Folia Microbiol (Praha), 1992, 37(6), 442 - 9
Effect of nitrogen limitation and sporulation on sterol and lipid formation in Saccharomyces cerevisiae; Behalova B et al.; The content of sterols and lipids was compared in the cells of Saccharomyces cerevisiae cultivated in sporulation and the sterol-induction nitrogen-limited media . After 24 h the measured values in the two cultivations did not significantly differ . However, after subsequent 24 h, further formation of lipid globules and a corresponding increase of lipid and sterol content was detected only in the sterol-induction medium . To demonstrate the similarity of physiological state during the first day of the two cultivations, the combined cultivations were performed . Maximum sporulation, suggesting maximum similarity, of the two processes was achieved when the cells were grown in the sterol-induction medium for 15 h and then transferred to a sporulation medium.

Chromosoma, 1992, 102(1 Suppl), S150 - 6
Use of non-denaturing Southern hybridization and two dimensional agarose gels to detect putative intermediates in telomere replication in Saccharomyces cerevisiae; Wellinger RJ et al.; Telomeres are required for the complete duplication of the ends of linear chromosomes . Saccharomyces telomeres bear approximately 350 bps of C1-3A/TG1-3 sequences . Previous work using non-denaturing Southern blotting has demonstrated the cell cycle controlled appearance of single stranded TG1-3 tails on chromosomal and plasmid telomeres (Wellinger et al . submitted) . Furthermore it was shown that short linear plasmids carrying an origin of replication derived from 2 microns DNA can circularize at the time of telomere replication (Wellinger et al . submitted) . Here we demonstrate that those loci previously shown to acquire single stranded tails are indeed telomeres and that single stranded TG1-3 cannot be observed in non-telomeric C1-3A/TG1-3-tracts . Moreover, we demonstrate that the formation of circular DNA by short linear plasmids is not restricted to plasmids containing a 2 microns origin of replication but can also be detected for plasmids containing ARS1.

Bioseparation, 1992-93, 3(6), 365 - 72
Studies on the extraction of DesPro(2)-Val15-Leu17-aprotinin from the culture broth of a recombinant Saccharomyces cerevisiae; Barthel T et al.; The application of an aqueous two-phase system is described for the extraction of DesPro(2)-Val15-Leu17-aprotinin from yeast culture supernatant, using native chymotrypsin as affinity ligand . The interaction is driven by hydrophobic forces and leads to the accumulation of the aprotinin-chymotrypsin complex in the salt-rich (bottom) phase of a polyethyleglycol/salt system . The complex may be dissociated at low pH values . The separation of the recombinant aprotinin and the protease required chromatographic processes, which proved difficult to interface with the affinity extraction.

J Biol Chem, 1991 Dec 25, 266(36), 24557 - 63
The Saccharomyces cerevisiae TRG1 gene is essential for growth and encodes a lumenal endoplasmic reticulum glycoprotein involved in the maturation of vacuolar carboxypeptidase; Gunther R et al.; We have isolated and mapped to the left end of chromosome III a single-copy gene (TRG1) encoding a 72-kDa glycoprotein, by screening a yeast genomic library with a DNA probe specifying the catalytic center (APWCGHCK) of thioredoxin-related proteins . the TRG1 gene sequence predicts an amino-terminal leader peptide, two thioredoxin-like domains, five N-glycosylation sites and a carboxyl-terminal HDEL retention signal . The TRG1 protein shows about equal sequence similarity to a mammalian multifunctional protein family residing in the lumen of the endoplasmic reticulum (ER), and to a putative cytosolic alpha form of phosphoinositide-specific phospholipase C . Haploid cells do not survive TRG1 gene disruptions, unless an additional wild-type copy is generated by interchromosomal transposition . Antibodies raised against synthetic amino- and carboxyl-terminal epitopes recognize a pair of lumenl ER glycoproteins (gp70/72) and a cytosolic 48-kDa protein . A 1.8-kilobase TRG1 transcript was translated by a reticulocyte lysate into a 60-kDa protein, which was translocated and processed to a 72-kDa glycoprotein in the presence of ER membrane vesicles . The TRG1 gene was placed under the control of the galactose-inducible and glucose-repressible GAL1 promoter, leading to growth arrest in glucose media . Glucose repression of the TRG1 gene caused the disappearance of gp72 and the accumulation of procarboxypeptidase . Our data indicate that the TRG1 gene encodes a growth essential lumenal ER glycoprotein involved the maturation of vacuolar carboxypeptidase.

Gene, 1991 Dec 20, 109(1), 81 - 7
The direct cloning of the yeast genome using the gap-filling method and the complete physical mapping of Saccharomyces cerevisiae chromosome VI; Iwasaki T et al.; The ordered clone library of chromosome VI of Saccharomyces cerevisiae has been constructed by Olson et al . {Proc . Natl . Acad . Sci . USA 83 (1986) 7826-7830, and personal communication} . It is composed of four contiguous stretches from the chromosome, each of 40-70 kb . There remained three gaps of unknown length between these four contigs . We applied the 'gap-repair' method to clone these three gap regions directly from the yeast chromosome . All three gap regions, ranging from 7 to 22 kb, were successfully cloned without any structural changes . Together with these gap regions, a precise physical map of EcoRI and HindIII sites was constructed over the 230-kb fragment which covers most of chromosome VI except for two telomeres.

Gene, 1991 Dec 20, 109(1), 143 - 7
The Saccharomyces cerevisiae ADE1 gene: structure, overexpression and possible regulation by general amino acid control; Myasnikov AN et al.; The ADE1 gene of the yeast Saccharomyces cerevisiae has been cloned by complementation of the ade1 mutation . The nucleotide sequence has been determined for the 918-bp coding region, 240-bp 5'-noncoding region and 292-bp 3'-noncoding region . The sequenced region includes a single large open reading frame coding for a protein of 306 amino acid (aa) residues . The promoter of the ADE1 gene contains a copy of the 5'-TGACTC hexanucleotide, a feature characteristic of promoters under general aa control . Subsequent search of other published purine biosynthesis gene sequences revealed that all of them also contain general aa control signals in their promoter regions . An expression plasmid containing the ADE1 coding region under control of the PHO5 promoter produced N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR) synthetase in yeast cells at a level of 40% of total cellular protein . One-step purification resulted in an almost homogeneous preparation of SAICAR synthetase.

Eur J Biochem, 1991 Dec 18, 202(3), 993 - 1002
Molecular cloning of soluble aminopeptidases from Saccharomyces cerevisiae . Sequence analysis of aminopeptidase yscII, a putative zinc-metallopeptidase; Garcia-Alvarez N et al.; Plasmids capable of complementing lap1, lap2 and lap3 mutations {R.J . Trumbly and G . Bradley (1983) J . Bacteriol . 156, 36-48} were isolated from a yeast YEp13 library by screening for activity against the chromogenic aminopeptidase substrate L-leucine beta-naphthylamide in intact yeast colonies . The genomic inserts were shown to contain the structural genes for aminopeptidases yscII, yscIII and yscIV . Plasmids containing the gene encoding aminopeptidase yscII of Saccharomyces cerevisiae, APE2 (LAP1) were analyzed in detail . APE2 was determined by DNA blot analysis to be a single-copy gene located on chromosome XI . The cloned fragment was used to identify a 2.7-kb mRNA . The cloned APE2 gene was sequenced and found to consist of an open reading frame of 2583 bp encoding a protein of 861 amino acids . The protein sequence contains two putative N-glycosylation sites . A significant amino acid similarity was detected between the APE2 gene product and members of the zinc-dependent metallopeptidase gene family . Chromosomal disruption of the APE2 gene completely abolishes the distinct activity band previously identified as aminopeptidase yscII {H.H . Hirsch, P . Suarez-Rendueles, T . Achstetter and D.H . Wolf (1988) Eur . J . Biochem . 173, 589-598} in crude extracts subjected to non-denaturing polyacrylamide gel electrophoresis and subsequent aminopeptidase activity staining . No vital consequence of aminopeptidase yscII absence on cell growth could be detected.

Proc Natl Acad Sci U S A, 1991 Dec 15, 88(24), 11378 - 82
Mutants of Escherichia coli initiator tRNA that suppress amber codons in Saccharomyces cerevisiae and are aminoacylated with tyrosine by yeast extracts; Lee CP et al.; We recently described mutants of Escherichia coli initiator tRNA that suppress amber termination codons (UAG) in E . coli . These mutants have changes in the anticodon sequence (CAU----CUA) that allow them to read the amber codon and changes in the acceptor stem that allow them to bind to the ribosomal aminoacyl (A) site . We show here that a subset of these mutants suppress amber codons in Saccharomyces cerevisiae and that they are aminoacylated with tyrosine by yeast extracts . Analysis of a number of mutants as substrates for yeast tyrosyl-tRNA synthetase has led to identification of the C1.G72 base pair and the discriminator base A73, conserved in all eukaryotic cytoplasmic and archaebacterial tyrosine tRNAs, as being important for recognition . Our results suggest that the C1.G72 base pair and the discriminator base, in addition to the anticodon nucleotides previously identified {Bare, L.A . & Uhlenbeck, O.C . (1986) Biochemistry 25, 5825-5830} as important in yeast tyrosyl-tRNA synthetase recognition, may comprise the critical identity determinants in yeast tyrosine tRNA.

Gene, 1991 Dec 15, 108(2), 253 - 8
Synthesis of human initiation factor-2 alpha in Saccharomyces cerevisiae; Green SR et al.; A human eIF-2 alpha cDNA (encoding alpha-subunit of the eukaryotic initiation factor-2) was expressed under the control of the galactose-regulated GAL1, 10 promoter, in Saccharomyces cerevisiae, in order to study the possible interactions of human eIF-2 alpha with the yeast protein synthesis apparatus . Isoelectric focusing coupled with Western-blot analysis demonstrated that the human eIF-2 alpha subunit synthesized in yeast under a variety of growth conditions was detected as two bands which co-migrated with the phosphorylated and unphosphorylated forms of rabbit eIF-2 alpha, suggesting covalent modification in vivo . Cell fractionation studies further demonstrated that the synthesised human eIF-2 alpha protein, though present in the cytoplasm, was largely associated with the yeast ribosomes, but could be removed from these by washing with 0.3 M KCl . This possible association of the synthesised human subunit into a three-subunit (alpha, beta and gamma) eIF-2 complex was further examined by partial purification of the yeast eIF-2 complex and estimation of the molecular mass of this complex . Immunoreactive eIF-2 alpha was found in fractions with eIF-2 activity and the estimated molecular mass (130 kDa) corresponded to that predicted for the eIF-2 trimer . These analyses suggest that human eIF-2 alpha subunit synthesised in yeast can become involved with the yeast protein synthetic apparatus, though whether this is a functional incorporation requires further genetic studies.

Gene, 1991 Dec 15, 108(2), 275 - 9
Stable synthesis of viral protein 2 of infectious bursal disease virus in Saccharomyces cerevisiae; Jagadish MN et al.; Viral protein 2 (VP2) from infectious bursal disease virus and its precursor polyprotein (N-VP2-VP4-VP3-C), in the absence of their native N-terminal region (19 amino acids), required fusion of yeast presequences for their stable synthesis in Saccharomyces cerevisiae {Jagadish et al., Gene 95 (1990) 179-186} . Restoration of the missing 19 aa resulted in stable synthesis of VP2, indicating the significance of the N-terminal region in protein stability.

Biochim Biophys Acta, 1991 Dec 3, 1098(1), 79 - 89
Characterisation of proton fluxes across the cytoplasmic membrane of the yeast Saccharomyces cerevisiae; Haworth RS et al.; We have tested the efficacy of fluorescent probes for the measurement of intracellular pH in Saccharomyces cerevisiae . Of the compounds tested (fluorescein, carboxyseminaphthorhodafluor-1 (C.SNARF-1) and 2',7'bis(carboxyethyl)-5(6')-carboxyfluorescein), C.SNARF-1 was found to be the most useful indicator of internal pH . Fluorescence microscopy showed that in Saccharomyces cerevisiae strain DAUL1, C.SNARF-1 and fluorescein had a heterogeneous distribution, with dye throughout the cytoplasm and concentration of the dye to an area close to the cell membrane . This region was also labeled by quinacrine, which is known to accumulate in acidic regions of the cell . Saccharomyces cerevisiae BJ4932, which carries a defect in vacuolar acidification, did not show the same degree of dye concentration, suggesting that the site of C.SNARF-1 and fluorescein localisation in DAUL1 is the acidic vacuole . Changes in intracellular pH could be monitored by measuring changes in the fluorescence intensity of C.SNARF-1 . The addition of glucose caused an initial, rapid decrease in fluorescence intensity, indicating a rise in cellular pH . This was followed by slow acidification . Fluorescence intensity changes were similar in all strains studied, suggesting that the localisation of dye to acidic regions does not affect the measurement of intracellular pH in DAUL1 . The changes in intracellular pH on the addition of glucose correlated well with glucose-induced changes in external pH . Preincubation of cells in the presence of the plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced extracellular acidification and intracellular alkalinisation on the addition of glucose . Both amiloride and 5-(N-ethyl-N-isopropyl)amiloride also inhibited glucose-induced proton fluxes . Phorbol 12-myristate 13-acetate had no effect on the activity of the plasma membrane ATPase.

FEBS Lett, 1991 Dec 9, 294(3), 282 - 4
The sexual inducer of Volvox carteri . Its large-scale production and secretion by Saccharomyces cerevisiae; Haas E et al.; The DNA sequence coding for the sexual inducer glycoprotein of Volvox carteri and its N-terminal signal peptide was placed under the control of the repressible acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a yeast-E . coli shuttle vector . Yeast transformed by this construct synthesized and secreted into the culture medium biologically active inducer in amounts two to three orders of magnitude higher than observed in the Volvox system.

Eur J Biochem, 1991 Dec 5, 202(2), 471 - 7
Efficient expression of bovine alpha-lactalbumin in Saccharomyces cerevisiae; Viaene A et al.; A synthetic gene encoding the mature bovine alpha-lactalbumin fused to the preproregion of the yeast alpha-mating factor has been expressed and secreted at high level in Saccharomyces cerevisiae under the control of the alpha-mating promoter . Growth conditions were found to be critical for the expression: recombinant alpha-lactalbumin could only be detected in the medium provided the culture was grown at neutral pH . The secreted bovine alpha-lactalbumin is enzymatically active and identical to the whey protein, as confirmed by SDS/PAGE, IEF, ultraviolet and CD spectral analysis, and amino-terminal sequence determination.

J Biol Chem, 1991 Dec 5, 266(34), 22851 - 7
Activation of the proteinase B precursor of the yeast Saccharomyces cerevisiae by autocatalysis and by an internal sequence; Nebes VL et al.; Proteinase B (PrB) is a subtilisin-like serine protease found in the vacuole of the yeast Saccharomyces cerevisiae . It is first made as a large precursor that consists of a putative signal sequence, a 260-amino acid pro region, the serine protease domain, and two small COOH-terminal post regions (Moehle, C . M., Dixon, C . K., and Jones, E . W . (1989) J . Cell Biol . 108, 309-324) . This precursor is glycosylated and proteolytically processed at least three times before mature enzyme is formed . To determine whether an intact PrB catalytic site is required for proteolytic processing of the precursor, point mutations were generated at the codons for the active site serine or aspartate residues by site-directed mutagenesis . The effect of these mutations on PrB processing suggests that the large pro region may be cleaved by an intramolecular, autocatalytic mechanism . The properties of a prb1 mutant that accumulates a 37-kDa precursor in addition to mature sized mutant PrB antigen suggests that the final proteolytic cleavage step is also autocatalytic . A prb1 deletion that lacks codons for the large pro region was made to test whether this part of the precursor is required for formation of mature PrB . Analysis of this mutant revealed two functions for this region: it prevents N-linked glycosylation of the serine protease domain and it allows the PrB precursor to be processed by proteinase A . The pro region can fulfill this latter function if added as a separate molecule, so long as glycosylation of the catalytic domain is prevented by other means.

Eur J Biochem, 1991 Dec 5, 202(2), 299 - 308
Two lipid-anchored cAMP-binding proteins in the yeast Saccharomyces cerevisiae are unrelated to the R subunit of cytoplasmic protein kinase A; Muller G et al.; We show that the yeast, Saccharomyces cerevisiae, contains two cAMP-binding proteins in addition to the well-characterized regulatory (R) subunit of cytoplasmic cAMP-dependent protein kinase (PKA) . We provide evidence that they comprise a new type of cAMP receptor, membrane-anchored by covalently attached lipid structures . They are genetically not related to the cytoplasmic R subunit . The respective proteins can be detected in sral mutants, in which the gene for the R subunit of PKA has been disrupted and a monoclonal antibody raised against the cytoplasmic R subunit does not cross-react with the two membrane-bound cAMP-binding proteins . In addition, they differ from the cytoplasmic species also with respect to their location and the peptide maps of the photoaffinity-labeled proteins . Although they differ from one another in molecular mass and subcellular location, peptide maps of the cAMP-binding domains resemble each other and both proteins are membrane-anchored by lipid structures, one to the outer surface of the plasma membrane, the other to the outer surface of the inner mitochondrial membrane . Both anchors can be metabolically labeled by Etn, myo-Ins and fatty acids . In addition, the anchor structure of the cAMP receptor from plasma membranes can be radiolabeled by GlcN and Man . After cleavage of the anchor with glycosylphosphatidylinositol-specific phospholipase C from trypanosomes, the solubilized cAMP-binding protein from plasma membranes reacts with antibodies which specifically recognize the cross-reacting determinant from soluble trypanosomal coat protein, suggesting similarity of the anchors . Degradation studies also point to the glycosylphosphatidylinositol nature of the anchor from the plasma membrane, whereas the mitochondrial counterpart is less complex in that it lacks carbohydrates . The plasma membrane cAMP receptor is, in addition, modified by an N-glycosidically linked carbohydrate side chain, responsible mainly for its higher molecular mass.

J Biol Chem, 1991 Dec 5, 266(34), 22893 - 8
Metalloenzymes in DNA repair . Escherichia coli endonuclease IV and Saccharomyces cerevisiae Apn1; Levin JD et al.; Escherichia coli endonuclease IV and its Saccharomyces cerevisiae homologue Apn1, two DNA repair enzymes for free radical damages, were previously shown to be inactivated by metal-chelating agents . In the present study, atomic absorption spectrometry of endonuclease IV revealed the presence of 2.4 zinc and 0.7 manganese atoms, whereas Apn1 contained 3.3 zinc atoms and no significant manganese . EDTA-inactivated endonuclease IV retained 0.7 zinc atom but little detectable manganese . ZnCl2 reactivated 1,10-phenanthroline-treated Apn1, but was ineffective with endonuclease IV treated with either 1,10-phenanthroline or EDTA . In contrast, enzymatic activity was restored to both enzymes after EDTA treatment by incubation with CoCl2 and to a lesser extent by MnCl2 . Endonuclease IV, reactivated with CoCl2 or MnCl2, regained all of the activities characteristic of the native enzyme . MnCl2 was as effective as CoCl2 at restoring activity to the 1,10-phenanthroline-treated enzymes . The results indicate that intrinsic metals play critical roles in both endonuclease IV and Apn1 and that manganese may perform a special function in endonuclease IV . Possible mechanistic roles for the metals in these DNA repair enzymes are discussed.

J Biol Chem, 1991 Dec 5, 266(34), 22832 - 6
Histidine tRNA guanylyltransferase from Saccharomyces cerevisiae . II . Catalytic mechanism; Jahn D et al.; Yeast histidine tRNA guanylyltransferase (TGT) catalyzes in the presence of ATP the addition of GTP to the 5' end of eukaryotic cytoplasmic tRNAHis species . A study of the enzyme mechanism with purified protein showed that during the first step ATP is cleaved to AMP and PPi creating adenylylated TGT . In a second step the activated enzyme forms a stable complex with its cognate tRNA substrate . The 5'-phosphate of the tRNA is adenylylated by nucleotide transfer from the adenylylated guanylyltransferase to form A(5')pp(5')N at the 5'-end of the tRNA . Finally, the 3'-hydroxyl of GTP adds to the activated 5' terminus of the tRNA with the release of AMP . This mechanism of tRNAHis guanylyltransferase is very similar to that of RNA ligases . dATP can substitute for ATP in this reaction . Since among several guanosine compounds active in this reaction GTP is most efficiently added we believe that it is the natural substrate of TGT.

Biochemistry, 1991 Dec 3, 30(48), 11430 - 7
Mouse pulmonary cytochrome P-450 naphthalene hydroxylase: cDNA cloning, sequence, and expression in Saccharomyces cerevisiae; Ritter JK et al.; We have isolated a cDNA clone, Nah-2, encoding the cytochrome P-450Nah (naphthalene hydroxylase) from a mouse lung lambda ZAP cDNA library using anti-cytochrome P-450Nah IgG as a probe . This same antibody selectively blocked {Nagata, K., Martin, B.M., Gillette, J.R., & Sasame, H.A . (1990) Drug Metab . Dispos . 18, 557-564} the cytochrome P-450 in mouse lung microsomes that catalyzed the conversion of naphthalene to (1R,2S)-naphthalene 1,2-oxide, which has been postulated as a causative agent in the naphthalene-induced tissue-specific necrosis of Clara cells in mouse lung . The toxic effect is seen in mouse and not in rat . The cDNA encodes a polypeptide of 491 amino acids with a molecular mass of 50 kDa . Northern blot analysis with an Nah-2-specific probe revealed that the mRNA is expressed in a species- and tissue-specific manner, present only in mouse lung and liver and not in that of rat . The mRNA encoding Nah-2 is constitutively expressed and is not induced by either phenobarbital, pyrazole, pregnenolone 16 alpha-carbonitrile, or 3-methylcholanthrene . Comparative amino acid sequence analyses with other documented members of the P-450 gene superfamily revealed that this encoded protein is in the IIF subfamily . To analyze its substrate specificity, the cDNA was inserted into the vector, pAAH5, and expressed in the Saccharomyces cerevisiae strain, AH22 . The presence of cytochrome P-450Nah in the microsomes isolated from transformed cells and analyzed by Western blot was confirmed by immunocomplexing product with anti-cytochrome P450Nah IgG . Furthermore, activity toward naphthalene in the microsomes from the transformed cells established that this clone encodes a naphthalene hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1991 Dec, 11(12), 6317 - 27
RPD3 encodes a second factor required to achieve maximum positive and negative transcriptional states in Saccharomyces cerevisiae; Vidal M et al.; In Saccharomyces cerevisiae, TRK1 and TRK2 encode the high- and low-affinity K+ transporters, respectively . In cells containing a deletion of TRK1, transcription levels of TRK2 are extremely low and are limiting for growth in media containing low levels of K+ (Trk- phenotype) . Recessive mutations in RPD1 and RPD3 suppress the TRK2, conferring an approximately fourfold increase in transcription . rpd3 mutations confer pleiotropic phenotypes, including (i) mating defects, (ii) hypersensitivity to cycloheximide, (iii) inability to sporulate as homozygous diploids, and (iv) constitutive derepression of acid phosphatase . RPD3 was cloned and is predicted to encode a 48-kDa protein with no extensive similarity to proteins contained in current data bases . Deletion of RPD3 is not lethal but confers phenotypes identical to those caused by spontaneous mutations . RPD3 is required for both full repression and full activation of transcription of target genes including PHO5, STE6, and TY2 . RPD3 is the second gene required for this function, since RPD1 is also required . The effects of mutations in RPD1 and RPD3 are not additive, suggesting that these genes are involved in the same transcriptional regulatory function or pathway.

Mol Cell Biol, 1991 Dec, 11(12), 6205 - 15
Expression of the DAL80 gene, whose product is homologous to the GATA factors and is a negative regulator of multiple nitrogen catabolic genes in Saccharomyces cerevisiae, is sensitive to nitrogen catabolite repression; Cunningham TS et al.; We have cloned the negative regulatory gene (DAL80) of the allantoin catabolic pathway, characterized its structure, and determined the physiological conditions that control DAL80 expression and its influence on the expression of nitrogen catabolic genes . Disruption of the DAL80 gene demonstrated that it regulates multiple nitrogen catabolic pathways . Inducer-independent expression was observed for the allantoin pathway genes DAL7 and DUR1,2, as well as the UGA1 gene required for gamma-aminobutyrate catabolism in the disruption mutant . DAL80 transcription was itself highly sensitive to nitrogen catabolite repression (NCR), and its promoter contained 12 sequences homologous to the NCR-sensitive UASNTR . The deduced DAL80 protein structure contains zinc finger and coiled-coil motifs . The DAL80 zinc finger motif possessed high homology to the transcriptional activator proteins required for expression of NCR-sensitive genes in fungi and the yeast GLN3 gene product required for functioning of the NCR-sensitive DAL UASNTR . It was also homologous to the three GATAA-binding proteins reported to be transcriptional activators in avian and mammalian tissues . The latter correlations raise the possibility that both positive and negative regulators of allantoin pathway transcription may bind to similar sequences.

Mol Cell Biol, 1991 Dec, 11(12), 5910 - 8
Properties of the DNA-binding domain of the Saccharomyces cerevisiae STE12 protein; Yuan YL et al.; The STE12 protein of the yeast Saccharomyces cerevisiae binds to the pheromone response element (PRE) present in the upstream region of genes whose transcription is induced by pheromone . Using DNase I footprinting assays with bacterially made STE12 fragments, we localized the DNA-binding domain to 164 amino acids near the amino terminus . Footprinting of oligonucleotide-derived sequences containing one PRE, or two PREs in head-to-tail or tail-to-tail orientation, showed that the N-terminal 215 amino acids of STE12 has similar binding affinity to either of the dimer sites and a binding affinity 5- to 10-fold lower for the monomer site . This binding cooperativity was also evident on a fragment from the MFA2 gene, which encodes the a-factor pheromone . On this fragment, the 215-amino-acid STE12 fragment protected both a consensus PRE as well as a degenerate PRE containing an additional residue . Mutation of the degenerate site led to a 5- to 10-fold decrease in binding; mutation of the consensus site led to a 25-fold decrease in binding . The ability of PREs to function as pheromone-inducible upstream activation sequences in yeast correlated with their ability to bind the STE12 domain in vitro . The sequence of the STE12 DNA-binding domain contains similarities to the homeodomain, although it is highly diverged from other known examples of this motif . Moreover, the alignment between STE12 and the homeodomain postulates loops after both the putative helix 1 and helix 2 of the STE12 sequence.

Mol Cell Biol, 1991 Dec, 11(12), 5801 - 12
Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae; Preston RA et al.; The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain . Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment . The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein . The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus E1a zinc finger . A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells . Cells bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely small vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37 degrees C, and were incapable of sporulating (as homozygotes) . Fractionation of cells expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cells and is a vacuolar peripheral membrane protein . Sequence identity established that PEP3 and VPS18 (J . S . Robinson, T . R . Graham, and S . D . Emr, Mol . Cell . Biol . 11:5813-5824, 1991) are the same gene.

Mol Cell Biol, 1991 Dec, 11(12), 5813 - 24
A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation; Robinson JS et al.; Saccharomyces cerevisiae strains carrying vps18 mutations are defective in the sorting and transport of vacuolar enzymes . The precursor forms of these proteins are missorted and secreted from the mutant cells . Most vps18 mutants are temperature sensitive for growth and are defective in vacuole biogenesis; no structure resembling a normal vacuole is seen . A plasmid complementing the temperature-sensitive growth defect of strains carrying the vps18-4 allele was isolated from a centromere-based yeast genomic library . Integrative mapping experiments indicated that the 26-kb insert in this plasmid was derived from the VPS18 locus . A 4-kb minimal complementing fragment contains a single long open reading frame predicted to encode a 918-amino-acid hydrophilic protein . Comparison of the VPS18 sequence with the PEP3 sequence reported in the accompanying paper (R . A . Preston, H . F . Manolson, K . Becherer, E . Weidenhammer, D . Kirkpatrick, R . Wright, and E . W . Jones, Mol . Cell . Biol . 11:5801-5812, 1991) shows that the two genes are identical . Disruption of the VPS18/PEP3 gene (vps18 delta 1::TRP1) is not lethal but results in the same vacuolar protein sorting and growth defects exhibited by the original temperature-sensitive vps18 alleles . In addition, vps18 delta 1::TRP1 MAT alpha strains exhibit a defect in the Kex2p-dependent processing of the secreted pheromone alpha-factor . This finding suggests that vps18 mutations alter the function of a late Golgi compartment which contains Kex2p and in which vacuolar proteins are thought to be sorted from proteins destined for the cell surface . The Vps18p sequence contains a cysteine-rich, zinc finger-like motif at the COOH terminus . A mutant in which the first cysteine of this motif was changed to serine results in a temperature-conditional carboxypeptidase Y sorting defect shortly after a shift to nonpermissive conditions . We identified a similar cysteine-rich motif near the COOH terminus of another Vps protein, the Vps11/Pep5/End1 protein . Preston et al . (Mol . Cell . Biol . 11:5801-5812, 1991) present evidence that the Vps18/Pep3 protein colocalizes with the Vps11/Pep5 protein to the cytosolic face of the vacuolar membrane . Together with the similar phenotypes exhibited by both vps11 and vps18 mutants, this finding suggests that they may function at a common step during vacuolar protein sorting and that the integrity of their zinc finger motifs may be required for this function.

Sci China B, 1991 Dec, 34(12), 1478 - 84
Synthesis and secretion of human atrial natriuretic peptide in Saccharomyces cerevisiae; Qin N et al.; A chemically synthesized alpha-hANP gene was inserted into plasmid YFD18, which was an expression-secretion vector of yeast . The recombinant then transformed in the yeast Y33 . The expression level of yeast transformants was about 700 micrograms ANP/L detected by RIA . More than 99% of expression products were secreted in the culture medium . N-terminal analysis of purified product showed that the first 4 amino acid residues of alpha-hANP were deleted.

J Radiat Res (Tokyo), 1991 Dec, 32(4), 366 - 77
Effects of post-treatment incubation on recombinogenesis in incision-proficient and incision-deficient strains of Saccharomyces cerevisiae: II . Recombinogenesis after the photoaddition of furocoumarins; Saeki T et al.; After the photoaddition of mono- and bifunctional furocoumarins to G1 phase cells, most gene conversion and crossing-over occurred without post-irradiation incubation of these cells in incision-proficient strains . In contrast, incision-deficient cells showed marked induction of both recombinational events only after treated cells had been incubated for several hours before selection . These results indicate that when furocoumarins are photoadded to G1 cells, initiation of recombinational events occurs during the same G1 phase in the incision-proficient cells; whereas, it occurs only after post-irradiation DNA replication in incision-deficient cells . The action of the PSO2 gene product specific for the repair of DNA crosslinks in recombination induction is discussed and compared to the actions of the excision repair genes RAD1 and RAD2.

FEMS Microbiol Lett, 1991 Dec 1, 68(3), 245 - 8
A rapid and convenient screening technique for developmental pathway mutants of Saccharomyces cerevisiae; Dickinson JR et al.; A brief exposure to iodine vapour was used to screen for mutants of the yeast Saccharomyces cerevisiae affected in development . Besides obtaining a large number of asporogenous mutants, two novel mutations were identified that permitted germination of spores to occur in conditions (sporulation medium) in which the wild-type would not germinate . These two mutations were named gdr1 and gdr2 for germination derepressed . Both alter nutritional control of germination, but not the kinetics of germination in glucose-containing medium.

Yeast, 1991 Dec, 7(9), 933 - 41
A high-affinity uptake system for branched-chain amino acids in Saccharomyces cerevisiae; Tullin S et al.; In order to isolate mutants with impaired uptake of branched-chain amino acids, mutants were induced which on complex medium were sensitive to an inhibitor of branched-chain amino acid biosynthesis . Eighteen mutants of independent origin were found . Ten of them were assayed for branched-chain amino acid uptake . Three of these were impaired in the uptake of L-valine, L-isoleucine and L-leucine, while the rest were unaffected in uptake of any of the three amino acids . Kinetics of the uptake by one selected mutant and the parental strain S288C were compared to models for one or two systems obeying Michaelis-Menten kinetics . This analysis suggested that a high-affinity system for all three amino acids is absent in the mutant, whereas low-affinity uptake of L-isoleucine and L-leucine by one or more systems remains unaffected . Moreover, medium-affinity uptake components for L-valine and L-leucine, not originally seen in the wild type, were identified in the mutant . In the wild type, 10 mM of any of the amino acids L-alanine, L-cysteine, L-isoleucine, L-leucine, L-tryptophan and L-valine reduce uptake of any of the three branched-chain amino acids . We propose that a permease responsible for high-affinity uptake of the branched-chain amino acids in strain S288C is partially or completely inactive in the mutant . Tetrad analysis shows that the phenotype can be ascribed to a single Mendelian gene . The wild-type allele is denoted BAP1 for branched-chain amino acid permease . The BAP1-dependent system is different from the general amino acid permease.

Yeast, 1991 Dec, 7(9), 913 - 23
The allantoinase (DAL1) gene of Saccharomyces cerevisiae; Buckholz RG et al.; The allantoinase (DAL1) gene from Saccharomyces cerevisiae has been cloned, sequenced, and found to encode a 472 amino acid protein with a Mr of 52,028 . DAL1 is expressed in an inducer-independent manner in strain M970 (sigma 1278b genetic background) and modestly responds to mutation of the dal80 locus . Expression was also sensitive to nitrogen catabolite repression (NCR) . Correlated with these expression characteristics, the upstream region of DAL1 contained five copies of a sequence that is homologous to the DAL UASNTR element previously shown to be required for transcriptional activation and NCR sensitivity of the DAL5 and DAL7 genes . Missing from the DAL1 5' flanking region were any sequences with significant homology to the DAL7 UIS element required for response to inducer . These observations further support the roles of UASNTR and DAL7 UIS in the regulation of allantoin pathway gene expression.

J Interferon Res, 1991 Dec, 11(6), 357 - 64
Cloning and expression in Saccharomyces cerevisiae of a synthetic gene for the type-I trophoblast interferon ovine trophoblast protein-1: purification and antiviral activity; Ott TL et al.; Ovine trophoblast protein-1 (oTP-1) is a unique, Type I, trophoblast interferon (IFN) that possesses potent antiviral activity and is thought to be primarily responsible for maternal recognition of pregnancy in sheep . To provide sufficient amounts of protein for detailed studies, a synthetic gene for oTP-1 was designed and assembled in Escherichia coli, subcloned into a yeast expression plasmid, and used to overproduce recombinant oTP-1 in Saccharomyces cerevisiae . Recombinant oTP-1 was purified from soluble yeast extract using sequential ion-exchange and molecular sieve chromatography . Recombinant oTP-1 purified in this fashion exhibited potent antiviral activity (0.6 x 10(8) U/mg) similar to native oTP-1 . This expression system will enable production of large quantities of soluble, biologically active, and correctly processed recombinant oTP-1 . Furthermore, the synthetic gene construct facilitates introduction of mutations for ongoing structure/function studies of this unique, Type I, trophoblast IFN.

Chromosoma, 1991 Dec, 101(3), 189 - 97
Analysis of centromere function in Saccharomyces cerevisiae using synthetic centromere mutants; Murphy MR et al.; We constructed Saccharomyces cerevisiae centromere DNA mutants by annealing and ligating synthetic oligonucleotides, a novel approach to centromere DNA mutagenesis that allowed us to change only one structural parameter at a time . Using this method, we confirmed that CDE I, II, and III alone are sufficient for centromere function and that A + T-rich sequences in CDE II play important roles in mitosis and meiosis . Analysis of mutants also showed that a bend in the centromere DNA could be important for proper mitotic and meiotic chromosome segregation . In addition we demonstrated that the wild-type orientation of the CDE III sequence, but not the CDE I sequence, is critical for wild-type mitotic segregation . Surprisingly, we found that one mutant centromere affected the segregation of plasmids and chromosomes differently . The implications of these results for centromere function and chromosome structure are discussed.

Mol Gen Mikrobiol Virusol, 1991 Dec, (12), 14 - 9
{Preparation of Saccharomyces cerevisiae strains with regulated MFalpha1 promotor activity}; Brutman AV et al.; Two Saccharomyces cerevisiae strains containing integrated copies of the MAT alpha 1 gene fused to the PHO5 promoter have been obtained by transformation of a MAT alpha 1 mutant . The strains differ in length of 5'-uncoding region of the MAT alpha 1 in the integrated constructions . The mating activity and the ability of these strains to express the yeast MF alpha 1 gene and the hyman alpha-N-interferon gene under the control of MF alpha 1 promoter was shown to be regulated by the exogenous inorganic phosphate . The level of intracellular alpha-N-interferon synthesized in these strains was several fold higher as compared with the wild type alpha mating type strain . At the same time the observed increase in intracellular production is not accompanied by an increase in the level of secreted alpha-N-interferon . On the contrary, one of the strains had a two-fold reduction in the rate of secretion.

Curr Genet, 1991 Dec, 20(6), 471 - 4
Chemical mutagenesis and DNA synthesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae; Baranowska H et al.; Incubation of cdc8 mutants of the yeast Saccharomyces cerevisiae in YPD under permissive conditions, when DNA replication is taking place, prior to transfer to restrictive conditions, strongly stimulates induction of cdc+ colonies of ethyl methane sulphonate (EMS)- and methyl methane sulphonate (MMS)-treated yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1) . After diepoxybutane (DEB) treatment, both the induction of cdc+ colonies and their stimulation after incubation in YPD under permissive conditions is low . The results obtained show that stimulation of induction of cdc+ colonies under permissive conditions occurs not only after UV-treatment, but also after treatment with such mutagens as EMS and MMS.

Biotechnol Appl Biochem, 1991 Dec, 14(3), 391 - 4
Positive effect of GAC gene product on the mRNA level of glyoxalase I gene in Saccharomyces cerevisiae; Inoue Y et al.; A DNA fragment carrying a part of the structural gene for yeast (Saccharomyces cerevisiae) glyoxalase I was cloned from a lambda gt11 expression library using anti-glyoxalase IIgG as a probe . By Northern blotting analysis, the amount of glyoxalase I mRNA was found to increase in yeast cells containing plasmids carrying the GAC gene, which is a positive regulator for yeast glyoxalase I activity . This suggests that the GAC gene product may accelerate the transcription of glyoxalase I gene or may have some positive effects on the accumulation of glyoxalase I mRNA in yeast cells.

Gene, 1991 Dec 1, 108(1), 73 - 80
The CYP2 gene of Saccharomyces cerevisiae encodes a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase with an N-terminal signal sequence; Koser PL et al.; Cells of Saccharomyces cerevisiae contain a major cytosolic cyclophilin (Cyp)-related peptidyl-prolyl cis-trans isomerase (PPIase) which is the target for cyclosporin A (CsA) cytotoxicity and which is encoded by the CYP1 gene {Haendler et al., Gene 83 (1989) 39-46} . We recently identified a second Cyp-related gene in yeast, CYP2 {Koser et al., Nucleic Acids Res . 18 (1990) 1643} which predicts a protein with a hydrophobic leader sequence . A sequence lacking 33 codons from the 5'-end of the CYP2 open reading frame was generated by the polymerase chain reaction and engineered for expression in Escherichia coli . The corresponding recombinant truncated protein was purified and found to exhibit PPIase activity which was inhibited by CsA . The CYP2 gene is genetically unlinked to CYP1 . As with CYP1, genomic disruption of CYP2 had no effect on haploid cell viability . Disruption of all three of the known yeast PPIase-encoding genes {CYP1, CYP2, and RBP1 for rapamycin-binding protein; Koltin et al., Mol . Cell . Biol . 11 (1991) 1718-1723} in the same haploid cell also resulted in no apparent cellular phenotype, suggesting either that none of these enzymes have an essential function or that additional PPIases can compensate for their specific absence . Whereas cells containing a genomic disruption of CYP1 exhibited a CsA-resistant phenotype, genomic disruption of CYP2 had no effect on CsA sensitivity . This suggests that the CYP1 gene product is the primary cellular target for CsA toxicity in yeast . Since both purified Cyps display CsA sensitivity in vitro, our data suggest that Cyp1 and Cyp2 differ in terms of their cellular function and/or localization.

EMBO J, 1991 Dec, 10(13), 4343 - 50
Vaccinia DNA ligase complements Saccharomyces cerevisiae cdc9, localizes in cytoplasmic factories and affects virulence and virus sensitivity to DNA damaging agents; Kerr SM et al.; The functional compatibility of vaccinia virus DNA ligase with eukaryotic counterparts was demonstrated by its ability to complement Saccharomyces cerevisiae cdc9 . The vaccinia DNA ligase is a 63 kDa protein expressed early during infection that is non-essential for virus DNA replication and recombination in cultured cells . This implies complementation by a mammalian DNA ligase, yet no obvious recruitment of host DNA ligase I from the nucleus to the cytoplasm was observed during infection . An antiserum raised against a peptide conserved in eukaryotic DNA ligases identified the virus enzyme in discrete cytoplasmic 'factories', the sites of virus DNA synthesis, demonstrating immunological cross-reactivity between host DNA ligase I and the vaccinia enzyme . DNA ligase was not detected in the factories of a mutant virus lacking the ligase gene . Despite this, no difference in growth between wild-type (WT) and mutant virus was detectable even in Bloom's syndrome cells which have reduced DNA ligase I activity . However, DNA ligase negative virus showed an increased sensitivity to UV or bleomycin in cultured cells, and the importance of DNA ligase for virus virulence in vivo was demonstrated by the attenuated phenotype of the deletion mutant in intranasally infected mice.

EMBO J, 1991 Dec, 10(13), 4081 - 8
Saccharomyces cerevisiae a- and alpha-agglutinin: characterization of their molecular interaction; Cappellaro C et al.; An O-glycosylated protein of approximately 18 kDa responsible for mating type specific agglutination has been isolated from Saccharomyces cerevisiae a cells, purified to homogeneity and via peptide sequences the gene was cloned by PCR . An open reading frame codes for a protein of 69 amino acids . A minimum of five serine and five threonine residues of the mature protein are glycosylated . alpha-Agglutinin is a highly N-glycosylated protein of approximately 250 kDa . Both purified agglutinins form a specific 1:1 complex in vitro . Pretreatment of alpha-agglutinin, but not of alpha-agglutinin, with diethylpyrocarbonate (DEPC) prevents formation of the complex; treatment of alpha-agglutinin in the presence of alpha-agglutinin protects the former from DEPC inactivation . By carboxy terminal shortening of the alpha-agglutinin gene and by replacing three of its eight histidyl residues by arginine, the active region of alpha-agglutinin for interaction with alpha-agglutinin has been defined . Neither the N- nor the O-linked saccharides of the two agglutinins seem to be essential for their interaction.

Mol Gen Genet, 1991 Dec, 231(1), 7 - 16
Cloning, sequencing and characterization of the Saccharomyces cerevisiae URA7 gene encoding CTP synthetase; Ozier-Kalogeropoulos O et al.; The URA7 gene of Saccharomyces cerevisiae encodes CTP synthetase (EC 6.3.4.2) which catalyses the conversion of uridine 5'-triphosphate to cytidine 5'-triphosphate, the last step of the pyrimidine biosynthetic pathway . We have cloned and sequenced the URA7 gene . The coding region is 1710 bp long and the deduced protein sequence shows a strong degree of homology with bacterial and human CTP synthetases . Gene disruption shows that URA7 is not an essential gene: the level of the intracellular CTP pool is roughly the same in the deleted and the wild-type strains, suggesting that an alternative pathway for CTP synthesis exists in yeast . This could involve either a divergent duplicated gene or a different route beginning with the amination of uridine mono- or diphosphate.

Mol Gen Genet, 1991 Dec, 231(1), 22 - 32
A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter; Bitter GA et al.; The majority of the activation potential of the Saccharomyces cerevisiae TDH3 gene promoter is contained within nucleotides -676 to -381 (relative to the translation initiation codon) . An upstream activation sequence (UAS) in this region has been characterized by in vitro and in vivo assays and demonstrated to be composed of two small, adjacent DNA sequence elements . The essential determinant of this upstream UAS is a general regulatory factor 1 (GRF1) binding site at nucleotides -513 to -501 . A synthetic DNA element comprising this sequence, or an analogue in which two of the degenerate nucleotides of the GRF1 site consensus sequence were altered, activated 5' deleted TDH3 and CYC1 promoters . The second DNA element of the UAS is a 7 bp sequence which is conserved in the promoters of several yeast genes encoding glycolytic enzymes and occurs at positions -486 to -480 of the TDH3 promoter . This DNA sequence represents a novel promoter element: it contains no UAS activity itself, yet potentiates the activity of a GRF1 UAS . The potentiation of the GRF1 UAS by this element occurs when placed upstream from the TATA box of either the TDH3 or CYC1 promoters . The characteristics of this element (termed GPE for GRF1 site potentiator element) indicate that it represents a binding site for a different yeast protein which increases the promoter activation mediated by the GRF1 protein . Site-specific deletion and promoter reconstruction experiments suggest that the entire activation potential of the -676 to -381 region of the TDH3 gene promoter may be accounted for by a combination of the GRF1 site and the GPE.

Biochimie, 1991 Dec, 73(12), 1525 - 32
Identification of nuclear genes which participate to mitochondrial translation in Saccharomyces cerevisiae; Valens M et al.; The mitochondrial protein synthesis presents specific features and uses specific components different from their cytoplasmic counterparts . Since most genes which code for these components are localized in the chromosomes and only a small number are encoded by the mitochondrial DNA, it is important to identify and characterize the nuclear genes involved in this process . In order to do this, we have used a genetic screening which implies the selection and study of nuclear suppressors of mitochondrial mutations (or the reverse situation) which affect the mitochondrial protein synthesis . Three mutations have been used for this purpose . Two of them (ts 1398, cs 909) impair the mitochondrial ribosome; they were used to characterize new interacting components as well as two genes, MBR1 and MBR2, which control the assembly or the regulation of other genes involved in mitochondrial protein synthesis . The third mutation (ts 932), blocks the 3'-end maturation of the mitochondrial aspartyl tRNA . A nuclear suppressor has been obtained which presents all the characteristics of a mutation in the gene encoding the enzyme responsible for this process.

Curr Genet, 1991 Dec, 20(6), 453 - 6
A mutated ARO4 gene for feedback-resistant DAHP synthase which causes both o-fluoro-DL-phenylalanine resistance and beta-phenethyl-alcohol overproduction in Saccharomyces cerevisiae; Fukuda K et al.; o-Fluoro-DL-phenylalanine (OFP)-resistant mutants which overproduce beta-phenethyl-alcohol were isolated from a laboratory strain of Saccharomyces cerevisiae . Cells of one of the mutants accumulated tyrosine and phenylalanine 1.5-3 fold more than did wild-type cells . Its 3-deoxy-D-arabino-hepturosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15), encoded by ARO4, was free from feedback inhibition by tyrosine . Genetic analysis revealed that the mutation was controlled by a single dominant gene, ARO4-OFP, encoding feedback-resistant DAHP synthase by tyrosine, and that this gene caused both the OFP resistance and beta-phenethyl-alcohol overproduction . This was supported by molecular genetic studies using cloned ARO4 both from the wild-type and its mutant strain.

Curr Genet, 1991 Dec, 20(6), 449 - 52
Isolation and genetic study of p-fluoro-DL-phenylalanine-resistant mutants overproducing beta-phenethyl-alcohol in Saccharomyces cerevisiae; Fukuda K et al.; p-Fluoro-DL-phenylalanine (PFP)-resistant mutants which produce a large amount of beta-phenethyl-alcohol, a rose-like flavor component, were isolated from the isogenic strains X2180-1A and X2180-1B of Saccharomyces cerevisiae . Cells of these mutants accumulated phenylalanine and tryptophan more than 3-fold times that of wild-type cells, while they accumulated less than half the tyrosine . The activity of prephenate dehydrogenase (PDG) (EC 1.3.1.12) was markedly decreased while that of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) was increased . Genetic analysis revealed that the mutation occurred at the TYR1 locus, encoding PDG, and that the mutated TYR1 gene, try1-pfp, caused both PFP resistance and beta-phenethyl-alcohol overproduction . This was supported by molecular genetic studies with cloned tyr1-pfp DNA.

Yeast, 1991 Dec, 7(9), 953 - 61
Molecular cloning of the gamma-glutamylcysteine synthetase gene of Saccharomyces cerevisiae; Ohtake Y et al.; The 4.4 kb SphI DNA fragment (GSH1) that complements the gamma-glutamylcysteine synthetase-deficient mutation (gsh1) of Saccharomyces cerevisiae YH1 was cloned into vector plasmid YEp24 . Gene disruption of the cloned fragment confirmed that this segment was the same gene as gsh1 . Mutant strain YH1 with this plasmid not only restored gamma-glutamylcysteine synthetase (GSH-I) activity but the glutathione content and the growth rate . DNA sequence analysis of the SphI fragment showed that the GSH1 structural gene contained 2034 bp and predicted a polypeptide of 678 amino acids . The deduced amino acid sequence had about a 45% homology to that of rat kidney GSH-I, but a very low homology (about 26%) to that of Escherichia coli GSH-I . Northern analysis showed that GSH1 had been transcribed into an approximately 2.7 kb mRNA fragment . Southern analysis showed that GSH1 mapped at chromosome X.

Mol Cell Biol, 1991 Dec, 11(12), 6229 - 47
Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae; Miller SM et al.; We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source . The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium . We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase . Two sites behaved as upstream activation sites (UASs) . The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs . This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.

Mol Cell Biol, 1991 Dec, 11(12), 6216 - 28
Sequence and expression of GLN3, a positive nitrogen regulatory gene of Saccharomyces cerevisiae encoding a protein with a putative zinc finger DNA-binding domain; Minehart PL et al.; The GLN3 gene of Saccharomyces cerevisiae is required for the activation of transcription of a number of genes in response to the replacement of glutamine by glutamate as source of nitrogen . We cloned the GLN3 gene and constructed null alleles by gene disruption . GLN3 is not essential for growth, but increased copies of GLN3 lead to a drastic decrease in growth rate . The complete nucleotide sequence of the GLN3 gene was determined, revealing one open reading frame encoding a polypeptide of 730 amino acids, with a molecular weight of approximately 80,000 . The GLN3 protein contains a single putative Cys2/Cys2 zinc finger which has homology to the Neurospora crassa NIT2 protein, the Aspergillus nidulans AREA protein, and the erythroid-specific transcription factor GATA-1 . Immunoprecipitation experiments indicated that the GLN3 protein binds the nitrogen upstream activation sequence of GLN1, the gene encoding glutamine synthetase . Neither control of transcription nor control of initiation of translation of GLN3 is important for regulation in response to glutamine availability.

New Biol, 1991 Dec, 3(12), 1249 - 59
The products of the SPT10 and SPT21 genes of Saccharomyces cerevisiae increase the amplitude of transcriptional regulation at a large number of unlinked loci; Natsoulis G et al.; The 3' long terminal repeat (LTR) of yeast transposon Ty1 is not normally used as a promoter, although it contains sequences identical to those found in the 5' LTR, which does act as a promoter . We have isolated mutations that fall into two genes, SPT10 and SPT21, that allow the 3' LTRs of Ty1 elements inserted at various positions in the genome of Saccharomyces cerevisiae to act as promoters . We find that mutations in these two genes alter transcriptional regulation of Ty1 LTRs and also of certain non-Ty1-related promoters in two ways: (i) they allow the low-level expression of several genes under repressing conditions, and (ii) they allow transcription from the 5' LTR of Ty1 elements in the absence of a normally required activator, SPT3 . Furthermore, the fully induced levels of transcription of several genes are reduced in these spt mutants . Hence, the products of these two genes increase the amplitude of transcriptional regulation of a wide variety of unlinked loci.

Biochimie, 1991 Dec, 73(12), 1445 - 55
Extragenic suppressors of a translation initiation defect in the cyc1 gene of Saccharomyces cerevisiae; Hampsey M et al.; The cycl-362 allele contains a point mutation that generates an aberrant AUG codon upstream of the normal CYC1 translation initiation codon . Mutants containing this allele express only about 2% of normal iso-1-cytochrome c, presumably due to translation initiation at the upstream AUG, termination at a UAA sequence six codons downstream, and failure to reinitiate at the normal AUG codon two nucleotides later . Both intragenic and extragenic revertants of cycl-362, expressing elevated levels of iso-1-cytochrome c, have been isolated simply by selecting for growth on lactate medium . Here we describe an improved method for isolating and readily distinguishing cis- from trans-acting suppressors of the upstream AUG . Eight different genes, designated sua1-sua8, are represented in our current collection of extragenic suppressors; all are recessive and enhance iso-1-cytochrome c levels to 10-60% of normal . None of the sua genes is allelic to SUI2 or sui3, which encode eIF-2 alpha and eIF-2 beta, respectively, or to SUI1 . Many of the suppressors exhibit pleiotropic phenotypes, including slow growth, cold (16 degrees C) and heat (37 degrees C) sensitivity . These phenotypes have been exploited to clone the SUA5, SUA7 and SUA8 genes, which are presently being characterized . The structure of cyc1-362 and the number of sua genes already uncovered suggest that the SUA genes are likely to encode factors affecting several different cellular processes, including translation initiation, mRNA stability and possibly transcription start site selection.

Genetics, 1991 Dec, 129(4), 1043 - 52
Doubling Ty1 element copy number in Saccharomyces cerevisiae: host genome stability and phenotypic effects; Boeke JD et al.; Haploid yeast strains bearing approximately double the normal number of Ty1 elements have been constructed using marked GAL/Ty1 fusion plasmids . The strains maintain their high transposon copy number and overall genome structure in the absence of selection . The strains bearing extra Ty1 copies are surprisingly similar phenotypically to the parental strain . The results suggest that the limit to transposon copy number, if any, has not been reached . When these strains are crossed by wild-type strains (i.e., bearing the normal complement of Ty1 elements) or by strains of opposite mating type also bearing excess Ty1 elements, normal to very slightly reduced spore viability is observed, indicating that increasing the extent of transposon homology scattered around the genome does not result in significant increases in frequency of ectopic reciprocal recombination . The results suggest that yeast cells have evolved mechanisms for coping with excess transposon copies in the genome.

J Bacteriol, 1991 Dec, 173(24), 7875 - 80
A paradoxical increase of a metabolite upon increased expression of its catabolic enzyme: the case of diadenosine tetraphosphate (Ap4A) and Ap4A phosphorylase I in Saccharomyces cerevisiae; Avila DM et al.; The APA1 gene in Saccharomyces cerevisiae encodes Ap4A phosphorylase I, the catabolic enzyme for diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) . APA1 has been inserted into a multicopy plasmid and into a centromeric plasmid with a GAL1 promoter . Enhanced expression of APA1 via the plasmids resulted in 10- and 90-fold increases in Ap4A phosphorylase activity, respectively, as assayed in vitro . However, the intracellular concentration of Ap4A exhibited increases of 2- and 15-fold, respectively, from the two different plasmids . Intracellular Ap4A increased 3- to 20-fold during growth on galactose of a transformant with APA1 under the control of the GAL1 promoter . Intracellular adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G) also increased in the transformant under these conditions . The chromosomal locus of APA1 has been disrupted in a haploid strain . The Ap4A phosphorylase activity decreased by 80% and the intracellular Ap4A concentration increased by a factor of five in the null mutant . These results with the null mutant agree with previous results reported by Plateau et al . (P . Plateau, M . Fromant, J.-M . Schmitter, J.-M . Buhler, and S . Blancquet, J . Bacteriol . 171:6437-6445, 1989) . The paradoxical increase in Ap4A upon enhanced expression of APA1 indicates that the metabolic consequences of altered gene expression may be more complex than indicated solely by assay of enzymatic activity of the gene product.

Arch Biochem Biophys, 1991 Dec, 291(2), 241 - 6
Saccharomyces cerevisiae nucleoside-diphosphate kinase: purification, characterization, and substrate specificity; Jong AY et al.; Nucleoside-diphosphate kinase is an enzyme which catalyzes the phosphorylation of nucleoside diphosphates into the corresponding triphosphates for nucleic acid biosynthesis . In this communication, we describe the purification and characterization of nucleoside-diphosphate kinase from yeast . The purified protein appears to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel analysis, with a molecular weight of about 17,000-18,000 . An estimate from the fast protein liquid chromatography Superose 12 gel filtration shows a native molecular weight of about 68,000 to 70,000 . The results suggest that yeast nucleoside-diphosphate kinase is composed of four subunits . Substrate specificity studies show that the relative activity of nucleoside diphosphates (NDP) as phosphate acceptors is in the order of dTDP greater than CDP greater than UDP greater than dUDP greater than GDP greater than or equal to dGDP greater than dCDP greater than dADP greater than ADP; and the relative activity of triphosphate donors is in the order of UTP greater than dTTP greater than CTP greater than dCTP greater than dATP greater than ATP greater than or equal to dGTP greater than GTP . The Km and Vm of dTDP, dGDP, dCDP, dUDP, CDP, and UDP have been determined . The rate constant studies indicate that the purified NDP kinase prefers using, to a slight extent, dTDP (approximately 800 min-1) as the substrate rather than other tested deoxyribo- and ribonucleotides (350-450 min-1) . The broad substrate specificity and kinetic data suggest that the enzyme is involved in both DNA and RNA metabolism.

Biotechnology (N Y), 1991 Dec, 9(12), 1382 - 5
Enhanced secretion of human nerve growth factor from Saccharomyces cerevisiae using an advanced delta-integration system; Sakai A et al.; We have designed an advanced delta-integration system (integration of genes into the delta-sequence of yeast retrotransposon Ty) and used it for secretion of human nerve growth factor (hNGF) from Saccharomyces cerevisiae . The expression and secretion of hNGF was directed by the PGK promoter and MF alpha 1 prepro-signal . Using two selectable markers (URA3 and leu2-d), haploid yeast strains were constructed with approximately 20 copies of a delta-integrated hNGF expression cassette on four chromosomes . The strain secreted hNGF at levels 3-4 fold higher than a 2 micron-based plasmid . Northern and Western analyses revealed that the oversecretion was caused by an increased amount of mRNA . We also detected an unusual processing of the MF alpha 1 prepro-hNGF fusion protein that required the pep4 mutation . Application of this system for industrial purposes is discussed.

Nucleic Acids Res, 1991 Nov 25, 19(22), 6255 - 62
Analysis of the interactions of functional domains of a nuclear origin of replication from Saccharomyces cerevisiae; Walker SS et al.; We have determined that ARS121 is an efficient origin of replication on chromosome X of Saccharomyces cerevisiae . This origin is comprised of at least three distinct functional domains . One of these domains is the ARS121 core sequence (approximately 35 bp-long), which is essential for origin activity . This essential core contains an 11 bp sequence resembling (2 bp mismatch) the ARS consensus . Another important domain is an enhancer of DNA replication, which binds the OBF1 protein . The third domain, ATR (A/T-rich, approximately 72 bp), is auxiliary and works in either orientation, but only when located 3' to the essential core . When fused to the ARS121 core both the enhancer and the ATR domain act synergistically to enhance the activity of the origin . Furthermore, when fused to the essential core sequences of heterologous ARSs, ARS1 and ARS307, the auxiliary domains also appeared to stimulate synergistically origin function . These results suggest that (i) in order to elicit maximal origin activity all three domains have to interact and (ii) activation of the essential core sequences at different origins of replication may share a common mechanism.

J Biol Chem, 1991 Nov 25, 266(33), 22698 - 706
Saccharomyces cerevisiae replication factor C . II . Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases delta and epsilon; Burgers PM; Lag times in DNA synthesis by DNA polymerase delta holoenzyme were due to ATP-mediated formation of an initiation complex on the primed DNA by the polymerase with the proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C) . Lag time analysis showed that high affinity binding of RF-C to the primer terminus required PCNA and that this complex was recognized by the polymerase . The formation of stable complexes was investigated through their isolation by Bio-Gel A-5m filtration . A stable complex of RF-C and PCNA on primed single-stranded mp18 DNA was isolated when these factors were preincubated with the DNA and with ATP, or, less efficiently with ATP gamma S . These and additional experiments suggest that ATP binding promotes the formation of a labile complex of RF-C with PCNA at the primer terminus, whereas its hydrolysis is required to form a stable complex . Subsequently, DNA polymerase delta binds to either complex in a replication competent fashion without further energy requirement . DNA polymerase epsilon did not associate stably with RF-C and PCNA onto the DNA, but its transient participation with these cofactors into a holoenzyme-like initiation complex was inferred from its kinetic properties and replication product analysis . The kinetics of the elongation phase at 30 degrees, 110 nucleotides/s by DNA polymerase delta holoenzyme and 50 nucleotides/s by DNA polymerase epsilon holoenzyme, are in agreement with in vivo rates of replication fork movement in yeast . A model for the eukaryotic replication fork involving both DNA polymerase delta and epsilon is proposed.

J Biol Chem, 1991 Nov 25, 266(33), 22689 - 97
Saccharomyces cerevisiae replication factor C . I . Purification and characterization of its ATPase activity; Yoder BL et al.; Saccharomyces cerevisiae replication factor C (RF-C) was purified 25,000-fold from a protease-deficient strain of yeast . RF-C is a complex of 6 subunits of 130, 86, 41, 40, 37, and 27 kDa . None of the subunits are related through proteolysis or differential phosphorylation . The assay for RF-C used as a substrate single-stranded DNA binding protein-coated singly primed single-stranded mp 18 DNA . This DNA was poorly replicated by yeast DNA polymerase delta with or without its cofactor proliferating cell nuclear antigen (PCNA) . In the presence of RF-C, however, replication of the template proceeded efficiently when both ATP and PCNA were present as well . Formation of this replication-proficient complex of DNA polymerase delta required an input of one to two molecules of PCNA per replicated DNA molecule . DNA polymerase epsilon also formed an ATP-dependent complex with PCNA and RF-C . RF-C has a DNA-dependent ATPase activity, equally active on single-stranded and primed single-stranded mp18 DNA . Addition of PCNA stimulated the ATPase of RF-C on primed but not on unprimed DNA, indicating that the increase in ATPase was due to PCNA-enhanced binding of RF-C to the primer terminus . Calf thymus PCNA also stimulated the ATPase activity of yeast RF-C and participated in holoenzyme formation with DNA polymerase delta . These results attest to the structural and functional homology between yeast and mammalian cells for these components of the replication machinery.

J Biol Chem, 1991 Nov 25, 266(33), 22485 - 92
Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae . III . Import, protease processing, and assembly into the cytochrome bc1 complex of iron-sulfur protein lacking the iron-sulfur cluster; Graham LA et al.; We have used site-directed mutagenesis of the Saccharomyces cerevisiae Rieske iron-sulfur protein gene (RIP 1) to convert cysteines 159, 164, 178, and 180 to serines, and to convert histidines 161 and 181 to arginines . These 4 cysteines and 2 histidines are conserved in all Rieske proteins sequenced to date, and 4 of these 6 residues are thought to ligate the iron-sulfur cluster to the apoprotein . We have also converted histidine 184 to arginine . This histidine is conserved only in respiring organisms . The site-directed mutations of the six fully conserved putative iron-sulfur cluster ligands result in an inactive iron-sulfur protein, lacking iron-sulfur cluster, and failure of the yeast to grow on nonfermentable carbon sources . In contrast, when histidine 184 is replaced by arginine, the iron-sulfur cluster is assembled properly and the yeast grow on nonfermentable carbon sources . The site-directed mutations of the 6 fully conserved residues do not prevent post-translational import of iron-sulfur protein precursor into mitochondria, nor do the mutations prevent processing of iron-sulfur protein precursor to mature size protein by mitochondrial proteases . Optical spectra of mitochondria from the six mutants indicate that cytochrome b is normal, in contrast to the deranged spectrum of cytochrome b which results when the iron-sulfur protein gene is deleted . In addition, mature size iron-sulfur apoprotein is associated with cytochrome bc1 complex purified from a site-directed mutant in which iron-sulfur cluster is not inserted . These results indicate that mature size iron-sulfur apoprotein, lacking iron-sulfur cluster, is inserted into the cytochrome bc1 complex, where it interacts with and preserves the optical properties of cytochrome b . Insertion of the iron-sulfur cluster is not an obligatory prerequisite to processing of the protein to its final size . Either the processing protease cannot distinguish between iron-sulfur protein with or without the iron-sulfur cluster, or insertion of the iron-sulfur cluster occurs after the protein is processed to its mature size, possibly after it is assembled in the cytochrome bc1 complex.

FEBS Lett, 1991 Nov 18, 293(1-2), 97 - 100
The promoter of Saccharomyces cerevisiae FBA1 gene contains a single positive upstream regulatory element; Compagno C et al.; The glycolytic enzyme fructose 1,6-bisphosphate aldolase is encoded by the FBA1 gene of Saccharomyces cerevisiae . Transcription of aldolase gene is not regulated by glucose and high levels of expression have been observed also during growth on nonfermentable carbon source . A FBA1::lacZ gene fusion was constructed and a deletion analysis demonstrated the presence of a unique cis-acting positive upstream element (UAS) required for high levels of FBA1 expression . This element is located between positions -550 and -440 upstream of the aldolase open reading frame and it contains sequences known to constitute the binding sites for the multifunctional proteins RAP1 and ABFI and two TTCC motifs.

J Biol Chem, 1991 Nov 15, 266(32), 21474 - 81
Roles of metal ions in the maintenance of the tertiary and quaternary structure of arginase from Saccharomyces cerevisiae; Green SM et al.; Arginase from Saccharomyces cerevisiae has long been known to be a metal ion-requiring enzyme as it requires heating at 45 degrees C in the presence of 10 mM Mn2+ for catalytic activation . Metals are also thought to play a structural role in the enzyme, but the identity of the structural metal and its precise structural role have not been defined . Analysis of the metal ions that bind to yeast arginase by atomic absorption spectroscopy reveals that there is a weakly associated Mn2+ that binds to the trimeric enzyme with a stoichiometry of 1.04 +/- 0.05 mol of Mn2+ bound per subunit and an apparent K'D value of 26 microM at pH 7.0 and 4 degrees C . A more tightly associated Zn2+ ion can only be removed by dialysis against chelating agents . In occasional preparations, this site contained some Mn2+; however, Zn2+ and Mn2+ together bind to high affinity sites with a stoichiometry of 1.14 +/- 0.25/mol of subunit . Both the loosely associated catalytic Mn2+ ion and the more tightly associated structural Zn2+ ion confer stability to the enzyme . Removal of the weakly bound Mn2+ ion results in a 3 degree C decrease in the midpoint of the thermal transition (T 1/2) (from 57 by 54 degrees C) as monitored by UV difference absorption spectroscopy . Removal of the tightly bound Zn2+ ion produces a 19 degrees C decrease in T 1/2 (to 38 degrees C) . Similar results are obtained by circular dichroism measurements . When the Zn2+ ion is removed, the steady-state fluorescence intensity increases 100% as compared to the holoenzyme, with a shift in the emission maximum from 337 to 352 nm . This suggests that in the folded trimeric metalloenzyme, the tryptophan fluorescence is quenched and that upon removal of the structural metal, the quenching is relieved as tryptophan residues become exposed to more polar environments . Equilibrium sedimentation experiments performed after dialysis of the enzyme against EDTA demonstrate that arginase exists in a reversible monomer-trimer equilibrium, in the absence of metal ions, with a KD value of 5.05 x 10(-11) M2 . In contrast, the native enzyme exists as a trimer with no evidence of dissociation when Mn2+ and Zn2+ are present (Eisenstein, E., Duong, L.T., Ornberg, R . L., Osborne, J.C., Jr., and Hensley, P . (1986) J . Biol . Chem . 261, 12814-12819) . In summary, the study presented here demonstrates that binding of a weakly bound Mn2+ ion confers catalytic activity . In contrast, binding of a more tightly associated Zn2+ ion confers substantial stability to the tertiary and quaternary structure of the enzyme.

J Biol Chem, 1991 Nov 15, 266(32), 21347 - 50
Isolation and characterization of a Saccharomyces cerevisiae mutant disrupted for the succinate dehydrogenase flavoprotein subunit; Robinson KM et al.; A partial genomic clone of the flavoprotein subunit of the mitochondrial enzyme, succinate dehydrogenase (EC 1.3.99.1) from Saccharomyces cerevisiae has been isolated . The partial clone was used to construct, by targeted gene disruption, a yeast mutant with a defective flavoprotein subunit gene . Submitochondrial membranes from the mutant are defective in activities requiring a functional succinate dehydrogenase but not in other respiratory chain activities . In addition, the mutant contains significantly lower levels of covalently attached flavin adenine dinucleotide cofactor than does the wild type . Disruption of the flavoprotein subunit gene results in the simultaneous loss of both the iron-sulfur and the flavoprotein subunits from mitochondrial membranes.

Arch Biochem Biophys, 1991 Nov 15, 291(1), 176 - 86
Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae; Kedzie KM et al.; Three mammalian cytochromes P450 from the IIB subfamily, P450IIB11 from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomyces cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gal10 promoter . Cytochromes P450IIB4 and P450IIB5 are closely related proteins, with only 11 amino acid substitutions between them . P450IIB11 is a homologous protein, likely orthologous with IIB4 or IIB5, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the P450IIB5 protein . The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7-ethoxycoumarin, and, in some cases, progesterone . Expressed cytochromes P450IIB4 and P450IIB11 hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins . A striking difference in the androstenedione metabolite profiles of IIB4 and IIB5 was observed, with IIB4 producing almost exclusively the 16 beta-hydroxy metabolite and IIB5 producing the 16 alpha-hydroxy and 15 alpha-hydroxy products . This is the first time that 15 alpha-hydroxylase activity has been associated with IIB4/IIB5 . This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G . These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes.

Mol Cell Biol, 1991 Nov, 11(11), 5639 - 47
The ROX3 gene encodes an essential nuclear protein involved in CYC7 gene expression in Saccharomyces cerevisiae; Rosenblum-Vos LS et al.; The ROX3 gene was identified during a hunt for mutants with increased expression of the heme-regulated CYC7 gene, which encodes the minor species of cytochrome c in the yeast Saccharomyces cerevisiae . The rox3 mutants caused a 10-fold increase in CYC7 expression both in the presence and absence of heme, had slightly increased anaerobic expression of the heme-activated CYC1 gene, and caused decreases in the anaerobic expression of the heme-repressed ANB1 gene and the aerobic expression of its heme-induced homolog . The wild-type ROX3 gene was cloned, and the sequence indicated that it encodes a 220-amino-acid protein . This protein is essential; deletion of the coding sequence was lethal . The coding sequence for beta-galactosidase was fused to the 3' end of the ROX3 coding sequence, and the fusion product was found to be localized in the nucleus, strongly suggesting that the wild-type protein carries out a nuclear function . Mutations in the rox3 gene showed an interesting pattern of intragenic complementation . A deletion of the 5' coding region complemented a nonsense mutation at codon 128 but could not prevent the lethality of the null mutation . These results suggest that the amino-terminal domain is required for an essential function, while the carboxy-terminal domain can be supplied in trans to achieve the wild-type expression of CYC7 . Finally, RNA blots demonstrated that the ROX3 mRNA was expressed at higher levels anaerobically but was not subject to heme repression . The nuclear localization and the lack of viability of null mutants suggest that the ROX3 protein is a general regulatory factor.

Nucleic Acids Res, 1991 Nov 11, 19(21), 5937 - 42
In vitro transcription analysis of the region of Saccharomyces cerevisiae mitochondrial DNA containing the tRNA(fMet) gene; Biswas TK; Prior work has indicated that an octanucleotide {5'TATAAGTA(+1)3'} sequence is used as a promoter in yeast mitochondria . Two such sequences (FP1 and FP2) are present upstream of the tRNA(fMet)-RNAse P RNA -tRNA(Pro) gene cluster but only the FP1 promoter but not the FP2 appears to be active in vivo and in vitro . The results presented in this paper suggest that the downstream ATTAATT sequence close to the initiation site of FP2 causes premature termination of transcription and effectively inhibits transcription from the FP2 octanucleotide sequence . Thus the different levels of RNA synthesis from these tRNA(fMet) promoters might be determined by variable transcriptional initiation and elongation blockage events . Since FP1 is found to be the only active promoter in this gene cluster, these three genes are thought to be transcribed together from the FP1 promoter . In this study, a new promoter (SP) between the tRNA(fMet) and RNase P RNA genes has been identified which may participate in RNase P RNA gene expression . The sequence of the new promoter does not match perfectly to the mitochondrial conserved promoter sequence but does match to the consensus promoter sequence.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9443 - 7
GCR1 of Saccharomyces cerevisiae encodes a DNA binding protein whose binding is abolished by mutations in the CTTCC sequence motif; Baker HV; In Saccharomyces cerevisiae, glycolysis enzymes constitute 30-60% of the soluble protein . GCR1 gene function is required for high-level glycolytic gene expression . In gcr1 mutant strains the levels of most glycolytic enzymes are between 2% and 10% of wild type . Binding sites for the global regulatory protein known as repressor activator protein 1 (RAP1)/general regulatory factor 1 (GRF1)/translation upstream factor (TUF) are found in close proximity to one or more CTTCC sequence motifs in the controlling region of GCR1-dependent genes . RAP1/GRF1/TUF-binding sites are known to be essential elements of upstream activating sequences that control expression of many glycolytic genes . In this report, I demonstrate that GCR1 encodes a DNA binding protein whose ability to bind DNA is dependent on the CTTCC sequence motif . This finding, in addition to the work of others, suggests that the GCR1 gene product and the RAP1/GRF1/TUF gene product act in concert to mediate high-level glycolytic gene expression.

Mutat Res, 1991 Nov, 264(3), 97 - 102
Non-specific incision of DNA due to the presence of 8-methoxypsoralen photoinduced interstrand cross-links in Saccharomyces cerevisiae; Cundari E et al.; The repair of DNA interstrand cross-links (CL) induced by 8-methoxypsoralen (8-MOP) plus UVA irradiation was analyzed by the alkaline step elution technique . A double-exposure protocol was used with 8-MOP, starting with exposure to monochromatic 405-nm radiation inducing only DNA monoadducts (MA), followed, after washing out of unbound 8-MOP molecules, by a second exposure to 365-nm radiation inducing varying relative amounts of CL at a constant level of total photoadducts . In the range of doses used for the second exposure, repair of CL took place; however, in the presence of increased relative amounts of CL induced non-specific incision of DNA occurred . This endonucleolytic cleavage appears to be related to the increased mutagenic and recombinogenic effects observed at increased levels of CL.

Mutat Res, 1991 Nov, 251(1), 123 - 31
Repair of exogenous (plasmid) DNA damaged by photoaddition of 8-methoxypsoralen in the yeast Saccharomyces cerevisiae; Magana-Schwencke N et al.; The contribution of different repair pathways to the repair of 8-methoxypsoralen (8-MOP) plus UVA induced lesions on a centromeric plasmid (YCp50) was investigated in the yeast Saccharomyces cerevisiae using the lithium acetate transformation method . The pathways of excision-resynthesis (RAD1) and recombination (RAD52) were found to be involved in the repair of exogenous as well as of genomic DNA . Mutants in RAD6 and PSO2 genes showed the same transformation efficiency with 8-MOP plus UVA treated plasmid as wild-type cells suggesting that these latter pathways involved in mutagenesis are not operating on plasmid DNA although required for the repair of 8-MOP photoadducts induced in genomic DNA . These results indicate that DNA-repair gene products may be differently involved in the repair of exogenous and endogenous DNA depending on the repair system and the nature of the DNA damage considered.

J Bacteriol, 1991 Nov, 173(22), 7186 - 95
Upstream induction sequence, the cis-acting element required for response to the allantoin pathway inducer and enhancement of operation of the nitrogen-regulated upstream activation sequence in Saccharomyces cerevisiae; van Vuuren HJ et al.; Expression of the DAL2, DAL4, DAL7, DUR1,2, and DUR3 genes in Saccharomyces cerevisiae is induced by the presence of allophanate, the last intermediate of the allantoin degradative pathway . Analysis of the DAL7 5'-flanking region identified an element, designated the DAL upstream induction sequence (DAL UIS), required for response to inducer . The operation of this cis-acting element requires functional DAL81 and DAL82 gene products . We determined the DAL UIS structure by using saturation mutagenesis . A specific dodecanucleotide sequence is the minimum required for response of reporter gene transcription to inducer . There are two copies of the sequence in the 5'-flanking region of the DAL7 gene . There are one or more copies of the sequence upstream of each allantoin pathway gene that responds to inducer . The sequence is also found 5' of the allophanate-inducible CAR2 gene as well . No such sequences were detected upstream of allantoin pathway genes that do not respond to the presence of inducer . We also demonstrated that the presence of a UIS element adjacent to the nitrogen-regulated upstream activation sequence significantly enhances its operation.

J Bacteriol, 1991 Nov, 173(21), 7024 - 8
Fragments of the internal transcribed spacer 1 of pre-rRNA accumulate in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1; Stevens A et al.; The portion of the internal transcribed spacer 1 found on 20S pre-rRNA accumulates in Saccharomyces cerevisiae lacking 5'----3' exoribonuclease 1, showing that an endonucleolytic cleavage at the 3' terminus of 18S rRNA is involved in the 20S pre-rRNA to 18S mature rRNA conversion . Smaller fragments of the spacer sequence are also found . The exoribonuclease may be involved as a cytoplasmic RNase in the hydrolysis of the spacer.

J Bacteriol, 1991 Nov, 173(21), 6709 - 13
Telomere length constancy during aging of Saccharomyces cerevisiae; D'Mello NP et al.; It has been proposed that a decrease in the length of telomeres with the successive rounds of DNA replication that accompany mitotic division could play a causal role in the aging process . To investigate this possibility, telomeres from cells of the budding yeast Saccharomyces cerevisiae that varied in replicative age were examined . No change in the lengths of the telomeres was observed in cells that had completed up to 83% of the mean life span . We conclude that the length of the telomeres is not a contributing factor in the natural aging process in individual yeast cells.

Mol Cell Biol, 1991 Nov, 11(11), 5693 - 700
AAR2, a gene for splicing pre-mRNA of the MATa1 cistron in cell type control of Saccharomyces cerevisiae; Nakazawa N et al.; We have isolated a class of mutants, aar2, showing the alpha mating type due to a defect in a1-alpha 2 repression but with alpha 2 repression activity from a nonmater strain of Saccharomyces cerevisiae expressing both a and alpha mating-type information in duplicate . Cells of the aar2 mutant and the aar2 disruptant also show a growth defect . A DNA fragment complementing the aar2 mutation contains an open reading frame consisting of 355 amino acid codons . Northern hybridization showed that cells of the aar2 mutant and disruptant contained alpha 1 and alpha 2 transcripts of the MAT alpha gene (or HML alpha in sir3 cells), but their a1 transcript of MATa (or HMRa in sir3 cells) migrated more slowly than that of the wild-type cells on gel electrophoresis and gave a diffused band . Primer extension analysis showed that the aar2 mutant and disruptant have a defect in splicing two short introns of the a1 pre-mRNA but not in splicing pre-mRNA of ACT1 . The alpha mating type, but not the slow-growing phenotype, of the aar2 mutant was suppressed by introduction of an intronless MATa1 DNA . Thus, the AAR2 gene is involved in splicing pre-mRNA of the a1 cistron and other genes that are important for cell growth . The AAR2 locus was mapped on chromosome II beside the SSA3 locus, with a 276-bp space, but was not allelic to either PRP5 or PRP6, which are both located on chromosome II and function in splicing pre-mRNA of ACT1.

Mol Cell Biol, 1991 Nov, 11(11), 5648 - 59
A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae; McNally FJ et al.; Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae . The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed . Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus . Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication) . RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer . The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo . Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR . The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer . These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.

Mol Cell Biol, 1991 Nov, 11(11), 5592 - 602
The CDC20 gene product of Saccharomyces cerevisiae, a beta-transducin homolog, is required for a subset of microtubule-dependent cellular processes; Sethi N et al.; Previous analysis of cdc20 mutants of the yeast Saccharomyces cerevisiae suggests that the CDC20 gene product (Cdc20p) is required for two microtubule-dependent processes, nuclear movements prior to anaphase and chromosome separation . Here we report that cdc20 mutants are defective for a third microtubule-mediated event, nuclear fusion during mating of G1 cells, but appear normal for a fourth microtubule-dependent process, nuclear migration after DNA replication . Therefore, Cdc20p is required for a subset of microtubule-dependent processes and functions at multiple stages in the life cycle . Consistent with this interpretation, we find that cdc20 cells arrested by alpha-factor or at the restrictive temperature accumulate anomalous microtubule structures, as detected by indirect immunofluorescence . The anomalous microtubule staining patterns are due to cdc20 because intragenic revertants that revert the temperature sensitivity have normal microtubule morphologies . cdc20 mutants have a sevenfold increase in the intensity of antitubulin fluorescence in intranuclear spindles compared with spindles from wild-type cells, yet the total amount of tubulin is indistinguishable by Western immunoblot analysis . This result suggests that Cdc20p modulates microtubule structure in wild-type cells either by promoting microtubule disassembly or by altering the surface of the microtubules . Finally, we cloned and sequenced CDC20 and show that it encodes a member of a family of proteins that share homology to the beta subunit of transducin.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1991 Nov, 24(4), 311 - 20
Chemical analysis and amino acid content of temperature-sensitive mutants of Saccharomyces cerevisiae; Yang SS et al.; Temperature-sensitive (ts) mutants of Saccharomyces cerevisiae Y-196 were isolated by using UV irradiation and/or EMS treatment . They grew normally at permissive temperature (30 degrees C), but could not grow at restrictive temperature (38 degrees C) . From chemical composition analysis, it was found that ts mutants AMY-159, and AMY-88 had the highest protein and RNA contents, respectively . When the cells were cultivated at 30 degrees C to stationary phase and then shifted to 38 degrees C for 4 hr, protein contents of wild-type and most of the ts mutants increased but RNA contents decreased . Glutamic acid, aspartic acid and lysine were the three major amino acids of protein in all isolates tested at both temperatures . When the ts mutants AMY-136, AMY-161, and AMY-157 were cultured at 38 degrees C for 4 hr, methionine contents increased Amino acid scores of ts mutants AMY-15 and AMY-88 increased when they were shifted to 38 degrees C for 2 to 4 doubling times . The amounts of amino acids produced in some ts mutants were higher than that of wild-type.

FEMS Microbiol Lett, 1991 Nov 1, 68(1), 91 - 5
Confirmation by using mutant strains that the membrane-bound H(+)-ATPase is the major source of non-linear dielectricity in Saccharomyces cerevisiae; Woodward AM et al.; Non-linear dielectric spectroscopy is a novel technique for determining the activity of (predominantly) membranous enzymes as their ability to generate harmonics when excited with a sinusoidal electrical field . In washed suspensions of yeast cells, the ability to generate harmonics is inhibited by low concentrations of sodium vanadate, suggesting that the vanadate-sensitive H(+)-ATPase is the major source of the non-linear dielectricity . This conclusion is greatly strengthened by the demonstration herein that the generation of harmonics by a strain containing a vandate-resistant H(+)-ATPase is also highly resistant to sodium metavanadate.

Mikrobiologiia, 1991 Nov-Dec, 60(6), 26 - 33
{Biochemical basis of varying nystatin resistance of Saccharomyces cerevisiae and Candida maltosa mutants}; Ogorodnikova TE et al.; Six groups of nystatin resistant mutants of C . maltosa and of haploid and diploid Saccharomyces cerevisiae strains were obtained with the help of genetic and biochemical analysis . It has been shown that every group of the mutants was characterized by a specific level of resistance to nystatin . The dependence of the resistance level upon sterol content has been established . It has been shown that the more the structure of the sterol present differed from ergosterol the higher was the resistance level . The results obtained in vivo permit to make conclusions about the role of different functional groups of sterol molecule in the interaction with nystatin.

Z Naturforsch {C}, 1991 Nov-Dec, 46(11-12), 1017 - 23
Studies on porphobilinogen-deaminase from Saccharomyces cerevisiae; Correa Garcia SR et al.; Porphobilinogen-deaminase from Saccharomyces cerevisiae has been isolated and partially purified 80- and 230-fold in the absence or presence of phenylmethylsulphonyl fluoride, respectively . Some properties of the isolated enzyme were studied . Porphyrin formation was linear with time and protein concentration . Optimum pH was about 7.5-7.8 . Molecular mass of the protein was 30,000 +/- 3000 Dalton when the enzyme was purified in the presence of phenylmethylsulphonyl fluoride . A less active and unstable 20,000 Da molecular mass species was obtained when purification was performed in the absence of the protease inhibitor . Porphobilinogen-deaminase exhibited classical Michaelis-Menten kinetics . The apparent Km for uroporphyrinogen formation was 19 microM; Vmax was 3.6 nmol uroporphyrin/h and the Hill coefficient was n = 1 . Also the action of several reagents on the activity was studied . Protective thiol agents had no effect . Heavy metals inhibited both porphyrin formation and porphobilinogen consumption, but known sulphydryl inactivating chemicals inhibit the former without modifying the latter . Ammonium ions had no effect on the activity while hydroxylamine completely inhibited both porphyrin formation and porphobilinogen consumption.

Curr Genet, 1991 Nov, 20(5), 411 - 5
Evolutionarily recent transfer of a group I mitochondrial intron to telomere regions in Saccharomyces cerevisiae; Louis EJ et al.; The junctions between X and Y' subtelomeric repeats in Saccharomyces cerevisiae usually contain a stretch of telomere sequences, (G1-3T)n . Two of three cloned X-Y' junctions from strain YP1 have a replacement of about 200 bp of X, the internal telomere sequence, and 49 bp of Y' by a 292 bp sequence . The first 227 bp of this insertion sequence are 100% identical to the fourth intron of cytochrome b . The rest of the insertion has homology to an unknown dispersed nuclear sequence . Recombination among subtelomeric regions can explain the nuclear distribution of this sequence and why telomeres can trap and maintain sequences that would otherwise be lost.

Curr Genet, 1991 Nov, 20(5), 365 - 72
Isolation and primary structure of the ERG9 gene of Saccharomyces cerevisiae encoding squalene synthetase; Fegueur M et al.; The ERG9 gene of Saccharomyces cerevisiae has been cloned by complementation of the erg9-1 mutation which affects squalene synthetase . From the 5 kb insert isolated, the functional gene has been localized on a DNA fragment of 2.5 kb . The presence of squalene synthetase activity in E . coli bearing the yeast DNA fragment isolated, indicates that the structural gene encoding squalene synthetase has been cloned . The sequence of the 2.5 kb fragment contains an open reading frame which could encode a protein of 444 amino acids with a deduced relative molecular mass of 51,600 . The amino acid sequence reveals one to four potential transmembrane domains with a hydrophobic segment in the C-terminal region . The N-terminus of the deduced protein strongly resembles the signal sequence of yeast invertase suggesting a specific mechanism of integration into the membranes of the endoplasmic reticulum.

Antimicrob Agents Chemother, 1991 Nov, 35(11), 2318 - 21
Petite mutagenesis in Saccharomyces cerevisiae by a series of bis-cationic trypanocidal drugs; Ferguson LR et al.; A group of bis-cationic imidazo{1,2-a}pyridinium salts and related compounds, some of which exhibit in vivo trypanocidal activity, have been investigated for induction of petite mutagenesis in Saccharomyces cerevisiae . All of the compounds which are active trypanocides induce mutagenesis . There appears to be a correlation between trypanocidal activity and mutagenic activity which may have its structural origin in the spatial separation of the cationic centers.

Photochem Photobiol, 1991 Nov, 54(5), 689 - 95
Metabolism of 5-methoxypsoralen by Saccharomyces cerevisiae; Morichetti E et al.; Incubation of methoxypsoralen (5-MOP) in the presence of diploid yeast cells (Saccharomyces cerevisiae) before UV-A exposure leads to an incubation-time dependent decrease of photoinduced genotoxic effects . The reduction in photoinduced genotoxicity is stronger in cells grown in the presence of 20% glucose and containing high levels of cytochrome P-450 than in cells grown in the presence of 0.5% glucose and containing undetectable levels of cytochrome P-450 . Inhibition of P-450 activity by specific inhibitors, such as tetrahydrofuran and metyrapone, strongly affects the observed decrease in 5-MOP genotoxicity, indicating the involvement of P-450 in 5-MOP metabolism . As demonstrated by spectrophotometric and chromatographic (HPLC) analysis during incubation of 5-MOP with P-450 containing yeast cells, 5-MOP gradually disappears from the cell supernatant of the incubation mixture . The reduction in the chromatographic peak corresponding to 5-MOP is accompanied by the appearance of a new peak that probably corresponds to a metabolite . As shown by the use of P-450 specific inhibitors, the metabolite appears to be due to P-450 mediated 5-MOP metabolisation . Its UV absorption spectrum suggests an alteration of the pyrone moiety of the 5-MOP molecule.

Yeast, 1991 Nov, 7(8), 873 - 80
TDH2 is linked to MET3 on chromosome X of Saccharomyces cerevisiae; Mountain HA et al.; The MET3 gene of Saccharomyces cerevisiae was cloned and its restriction map was found to differ in the upstream region from an earlier published map (Cherest et al . Gene 34, 269-281, 1985) and nucleotide sequence (Cherest et al . Mol . Gen . Genet . 210, 307-313, 1987) . Southern blot analysis of genomic DNA from strains S288C and FL100 (the genetic backgrounds from which these different copies of the gene had been cloned) showed that our clone from a S288C-based library had the same restriction map as the chromosomal DNA from both of the strains . Comparison of the nucleotide sequence of the two clones indicated that the earlier published clone probably represented a cloning artifact . In our clone, we found upstream of MET3, the nucleotide sequence of the TDH2 gene (Holland and Holland, J . Biol . Chem . 255, 2596-2605, 1980) . The chromosomal orientation of the two genes was determined to be MET3-TDH2-CEN10.

Yeast, 1991 Nov, 7(8), 867 - 72
The product of the YCR105 gene located on the chromosome III from Saccharomyces cerevisiae presents homologies to ATP-dependent permeases; Purnelle B et al.; During the systematic sequencing of chromosome III from Saccharomyces cerevisiae, carried out by a network of laboratories sponsored by the Commission of the European Community, we have identified the open reading frame YCR105 located on fragment J11D from the circular derivative of chromosome III in strain XJ24-24a (Palzkill et al., 1986) . YCR105 is immediately centromere proximal to the PGK gene (opposite strand) on the right arm of chromosome III about 20 kb from the centromere.

Yeast, 1991 Nov, 7(8), 859 - 65
The PHO80/TUP7 locus in Saccharomyces cerevisiae is on the left arm of chromosome XV: mapping by chromosome engineering; Kawasaki H et al.; The PHO80/TUP7 locus in Saccharomyces cerevisiae is reported to be located on the right arm of chromosome XV close to its centromere . In the present study, the locus has been reassigned to the left arm of the same chromosome by reciprocal recombination between chromosomes V and XV at URA3 (on chromosome V) and PHO80/TUP7 loci by using the site-specific recombination system of the yeast plasmid pSR1.

Yeast, 1991 Nov, 7(8), 843 - 8
Regulation of cystathionine gamma-lyase in Saccharomyces cerevisiae; Ono B et al.; Regulation of the two enzymes in reverse trans-sulfuration was investigated in Saccharomyces cerevisiae . In wild-type strains, cystathionine gamma-lyase, but not cystathionine beta-synthase, was depressed nearly 15-fold if cells were starved for both inorganic and organic sulfur compounds . In a met17 strain which is defective of O-acetylserine and O-acetylhomoserine sulfhydrylase, the same enzyme was derepressed if organic sulfur compounds were limited; the repressive effect was in the order of glutathione greater than methionine greater than cysteine . The repressive effect of methionine was not observed, however, in a cys2 cys4 strain which is deficient of serine O-acetyltransferase and cystathionine beta-synthase, indicating that methionine itself is not the effector . The weak repressive effect of cysteine was attributed to inefficient uptake of this amino acid . Our observations indicate that cystathionine gamma-lyase is the target of regulation in reverse trans-sulfuration and that cysteine is very likely to be the effector of this regulation.

Yeast, 1991 Nov, 7(8), 781 - 803
Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae; Mountain HA et al.; Genes encoding enzymes in the threonine/methionine biosynthetic pathway were cloned and used to investigate their transcriptional response to signals known to affect gene expression on the basis of enzyme specific-activities . Four major responses were evident: strong repression by methionine of MET3, MET5 and MET14, as previously described for MET3, MET2 and MET25; weak repression by methionine of MET6; weak stimulation by methionine but no response to threonine was seen for THR1, HOM2 and HOM3; no response to any of the signals tested, for HOM6 and MES1 . In a BOR3 mutant, THR1, HOM2 and HOM3 mRNA levels were increased slightly . The stimulation of transcription by methionine for HOM2, HOM3 and THR1 is mediated by the GCN4 gene product and hence these genes are under the general amino acid control . In addition to the strong repression by methionine, MET5 is also regulated by the general control.

Appl Environ Microbiol, 1991 Nov, 57(11), 3183 - 6
Role of hydrosulfide ions (HS-) in methylmercury resistance in Saccharomyces cerevisiae; Ono B et al.; Methylmercury-resistant mutants were obtained from Saccharomyces cerevisiae . They were divided into two complementation groups, met2 (homoserine O-acetyltransferase deficiency) and met15 (enzyme deficiency unknown), as reported previously . It was found that met15 was allelic to met17 (O-acetylserine and O-acetylhomoserine sulfhydrylase deficiency) . Methylmercury toxicity was counteracted by exogenously added HS-, and both met2 and met17 (met15) mutants overproduced H2S . On the basis of these results, we conclude that met2 and met17 (met15) cause accumulation of hydrosulfide ions in the cell and that the increased level of hydrosulfide is responsible for detoxification of methylmercury.

Mol Microbiol, 1991 Nov, 5(11), 2845 - 54
A protein kinase gene complements the lytic phenotype of Saccharomyces cerevisiae lyt2 mutants; Torres L et al.; By genetic analysis of a thermosensitive autolytic mutant whose phenotype was complemented by osmotic stabilization with sorbitol, we identified gene LYT2 of Saccharomyces cerevisiae, which is probably involved in cell wall formation . A yeast gene complementing lyt2 strains was cloned and shown to carry an open reading frame coding for a 484-amino-acid protein exhibiting all the characteristic domains of serine/threonine protein kinases and highly homologous to other yeast protein kinases involved in control of the mitotic cycle . Mutants disrupted in the cloned gene also displayed an autolytic phenotype complemented by osmotic stabilization with sorbitol . However, genetic comparison of lyt2 mutants and disruptants of the protein kinase gene revealed that the cloned gene is not the structural gene LYT2 but a suppressor of the lytic phenotype, named gene SLT2, that was mapped to chromosome V . The product of gene SLT2 is the first protein kinase to be described in relation to the yeast cell-wall functions.

New Biol, 1991 Nov, 3(11), 1089 - 96
Description of a baby machine for Saccharomyces cerevisiae; Helmstetter CE; A method for the continuous withdrawal of newly formed daughter cells from a growing population of Saccharomyces cerevisiae is described . An exponential-phase culture of cells was immobilized onto a surface and then flushed continuously with culture medium . Upon division of a cell in the immobilized population, the mother cell remained attached to the surface and the daughter cell was released . The method can be applied to research on the cell cycle, the segregation of components between cells, and cellular aging.

Genetics, 1991 Nov, 129(3), 685 - 96
SUM1-1: a suppressor of silencing defects in Saccharomyces cerevisiae; Laurenson P et al.; The repression of transcription of the silent mating-type locus HMRa in the yeast Saccharomyces cerevisiae requires the four SIR proteins, histone H4 and a flanking site designated HMR-E . The SUM1-1 mutation alleviated the need for many of these components in transcriptional repression . In the absence of each of the SIR proteins, SUM1-1 restored repression in MAT alpha strains; thus, SUM1-1 appeared to bypass the need for the SIR genes in repression of HMRa . Repression was not specific to the genes normally present at HMR, since the TRP1 gene placed at HMR was repressed by SUM1-1 in a sir3 strain . Therefore, like the mechanisms of silencing normally used at HMR, silencing by SUM1-1 was gene-nonspecific . SUM1-1 suppressed point mutations in histone H4, but failed to suppress strongly a deletion mutation in histone H4 . Similarly, SUM1-1 suppressed mutations in the three known elements of HMR-E, but was unable to suppress a deletion of HMR-E . These epistasis analyses implied that the functions required for repression at HMR can be ordered, with the SIR genes and silencer elements acting upstream of SUM1-1 . SUM1-1 itself may function at the level of chromatin in the assembly of inactive DNA at the silent mating-type loci.

Genetics, 1991 Nov, 129(3), 675 - 84
New SNF genes, GAL11 and GRR1 affect SUC2 expression in Saccharomyces cerevisiae; Vallier LG et al.; To identify new genes required for depression of the SUC2 (invertase) gene in Saccharomyces cerevisiae, we have isolated mutants with defects in raffinose utilization . In addition to mutations in SUC2 and previously identified SNF genes, we recovered recessive mutations that define four new complementation groups, designated snf7 through snf10 . These mutations cause defects in the derepression of SUC2 in response to glucose limitation . We also recovered five alleles of gal11 and showed that a gal11 null mutation decreases SUC2 expression to 30% of the wild-type level . Finally, one of the mutants carries a grr1 allele that converts SUC2 from a glucose-inducible gene.

Genetics, 1991 Nov, 129(3), 669 - 73
Seven-base-pair inverted repeats in DNA form stable hairpins in vivo in Saccharomyces cerevisiae; Nag DK et al.; Palindromic sequences in single-stranded DNA and RNA have the potential for intrastrand base pairing, resulting in formation of "hairpin" structures . We previously reported a genetic method for detecting such structures in vivo in the yeast Saccharomyces cerevisiae . Below, we describe evidence indicating that a 14-base-pair palindrome (7 bp per inverted repeat) is sufficient for formation of a hairpin in vivo.

Mol Gen Genet, 1991 Nov, 230(1-2), 45 - 8
Evidence for binding of at least two factors, including T-rich strand-binding factor(s) to the single-stranded ARS1 sequence in Saccharomyces cerevisiae; Kuno K et al.; To study the mechanism of initiation of eukaryotic chromosomal replication, we examined protein factors interacting with the ARS1 region located near the centromere of chromosome IV in Saccharomyces cerevisiae . Using the gel shift assay, we found protein factor(s) which specifically bound to the T-rich strand of the region containing the core consensus and its flanking sequences in ARS1, but not to the opposite strand . We designated this factor ATS (ARS1, T-rich strand-binding factor(s} . Similar specific complexes were also detected with oligonucleotide probes specific for the H4 or C2G1 ARS . As we have previously identified another binding factor, we conclude that at least two factors bind to the single-stranded ARS1 sequence.

Mol Gen Genet, 1991 Nov, 230(1-2), 277 - 87
Nuclear migration in Saccharomyces cerevisiae is controlled by the highly repetitive 313 kDa NUM1 protein; Kormanec J et al.; We have isolated a novel gene (NUM1) with unusual internal periodicity . The NUM1 gene encodes a 313 kDa protein with a potential Ca2+ binding site and a central domain containing 12 almost identical tandem repeats of a 64 amino acid polypeptide . num1-disrupted strains grow normally, but contain many budded cells with two nuclei in the mother cell instead of a single nucleus at the bud neck, while all unbudded cells are uninucleate . This indicates that most G2 nuclei divide in the mother before migrating to the neck, followed by the migration of one of the two daughter nuclei into the bud . Furthermore, haploid num1 strains tend to diploidize during mitosis, and homozygous num1 diploid or tetraploid cells sporulate to form many budded asci with up to eight haploid or diploid spores, respectively, indicating that meiosis starts before nuclear redistribution and cytokinesis . Our data suggest that the NUM1 protein is involved in the interaction of the G2 nucleus with the bud neck.

Mol Gen Genet, 1991 Nov, 230(1-2), 241 - 50
TFS1: a suppressor of cdc25 mutations in Saccharomyces cerevisiae; Robinson LC et al.; The TFS1 gene of Saccharomyces cerevisiae is a dosage-dependent suppressor of cdc25 mutations . Overexpression of TFS1 does not alleviate defects of temperature-sensitive adenylyl cyclase (cdc35) or ras2 disruption mutations . The ability of TFS1 to suppress cdc25 is allele specific: the temperature-sensitive cdc25-1 mutation is suppressed efficiently but the cdc25-5 mutation and two disruption mutations are only partially suppressed . TFS1 maps to a previously undefined locus on chromosome XII between RDN1 and CDC42 . The DNA sequence of TFS1 contains a single long open reading frame encoding a 219 amino acid polypeptide that is similar in sequence to two mammalian brain proteins . Insertion and deletion mutations in TFS1 are haploviable, indicating that TFS1 is not essential for growth.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9712 - 6
DNA.RNA helicase activity of RAD3 protein of Saccharomyces cerevisiae; Bailly V et al.; The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of UV-damaged DNA and is essential for cell viability . The RAD3 protein exhibits a remarkable degree of sequence homology to the human excision repair protein ERCC2 . The RAD3 protein is a single-stranded DNA-dependent ATPase and a DNA helicase capable of denaturing long regions of duplex DNA . Here, we demonstrate that RAD3 also possesses a potent DNA.RNA helicase activity similar in efficiency to its DNA helicase activity . The rad3 Arg-48 mutant protein, which binds but does not hydrolyze ATP, lacks the DNA.RNA unwinding activity, indicating a dependence on ATP hydrolysis . RAD3 does not show any RNA-dependent NTPase activity and, as expected, does not unwind duplex RNA . This observation suggests that RAD3 translocates on DNA in unwinding DNA.RNA duplexes . That the rad3 Arg-48 mutation inactivates the DNA and DNA.RNA helicase activities and confers a substantial reduction in the incision of UV-damaged DNA suggests a role for these activities in incision . We discuss how RAD3 helicase activities could function in tracking of DNA in search of damage sites and effect enhanced excision repair of actively transcribed genes.

Mutat Res, 1991 Nov, 258(3), 237 - 58
The detection of chemically induced chromosomal malsegregation in Saccharomyces cerevisiae D61.M: a literature survey (1984-1990); Albertini S et al.; Our objective is to summarize the published data obtained with a recently developed tester strain suitable for the detection of chromosomal malsegregation in yeast . Results from 25 papers were reviewed in which numerical data for 111 chemicals tested in Saccharomyces cerevisiae D61.M are reported (a total of 316 independent tests; 279 acceptable, 37 not meeting our criteria) . Of the 111 compounds analyzed 43 compounds are positive for chromosomal malsegregation, 56 compounds are negative and 12 compounds do not meet our criteria for acceptance (inconclusive) . Of the 43 compounds judged positive 5 (acetone, acetonitrile, benzonitrile, ethylacetate and propionitrile) were only positive using a cold interruption protocol . Recommendations are made for standardization of methods and protocols for screening purposes . Finally, a comparison with in vitro tubulin assembly data using mammalian tubulin is presented.

Eur J Biochem, 1991 Nov 1, 201(3), 715 - 21
Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by 1H-NMR and 13C-NMR spectroscopy; Tran-Dinh S et al.; We propose an experimental approach combining 1H-NMR and 13C-NMR spectroscopy to investigate metabolite flux in cells under physiological conditions and present a mathematical model giving the relationships between the following different parameters . 13C fractional enrichment, fluxes in competing pathways, metabolite concentration and experimental time . This model has been used for determining the absolute and/or relative values of five fluxes involving pyruvate, ethanol, acetyl-CoA and glutamate via the Krebs cycle in glucose-grown repressed Saccharomyces cerevisiae cells fed with {1-13C}glucose and/or unlabeled ethanol . The glucose consumption and the production of various compounds such as ethanol, glycerol, trehalose etc . were studied qualitatively and/or quantitatively as a function of time . The 13C fractional enrichment of {2-13C}ethanol was determined by observing the proton resonance of the methyl group . Addition of 25 mM unlabeled ethanol shows no significant effect on the glucose consumption or the production of any metabolites . However unlabeled ethanol exerts a strong influence on the enrichment of glutamate C4, but only induces an insignificant change on glutamate C2 and C3 . Apart from the fact that ethanol is a potential precursor of acetyl-CoA as expected, these results indicate that (a) the probability for citrate and 2-oxoglutarate to make one turn or more in the Krebs cycle is negligible and (b) the scrambling between C4 and C3 via the glyoxylate shunt is virtually absent . The flux of ethanol formation from pyruvate is about three-times and nine-times greater than that of ethanol consumption and acetyl-CoA formation, respectively, from pyruvate via pyruvate dehydrogenase . Without addition of unlabeled ethanol, the ratio of the integrated resonance of glutamate (C2 + C3)/C4 reflecting the activity of pyruvate carboxylase relative to that of citrate synthase, is about 1.1 . By comparing the absolute values of the different fluxes, it was found that 88% of the glucose was used to synthetize ethanol but the observed concentration of ethanol in the supernatant represents only 58% of the glucose consumption . The validity of the present model was supported by the data obtained from similar experiments using unlabeled ethanol and non-NMR techniques.

Genetics, 1991 Nov, 129(3), 659 - 68
Site-directed mutagenesis of a Saccharomyces cerevisiae mitochondrial translation initiation codon; Folley LS et al.; We have used a generally applicable strategy for gene replacement in yeast mitochondria to mutate the translation initiation codon of the COX3 gene from AUG to AUA . The mutation, cox3-1, substantially reduced, but did not eliminate, translation of cytochrome c oxidase subunit III (coxIII) . Strains bearing the mutation exhibited a leaky (partial) nonrespiratory growth phenotype and a reduced incorporation of radiolabeled amino acids into coxIII in vivo in the presence of cycloheximide . Hybridization experiments demonstrated that the mutation had little or no effect on levels of the COX3 mRNA . Residual translation of the cox3-1 mutant mRNA was dependent upon the three nuclearly coded mRNA-specific activators PET494, PET54 and PET122, known from previous studies to work through a site (or sites) upstream of the initiation codon to promote translation of the wild-type mRNA . Furthermore, respiratory growth of cox3-1 mutant strains was sensitive to decreased dosage of genes PET494 and PET122 in heterozygous mutant diploids, unlike the growth of strains carrying wild-type mtDNA . Some residual translation of the cox3-1 mRNA appeared to initiate at the mutant AUA codon, despite the fact that the 610-base 5'-mRNA leader contains numerous AUA triplets . We conclude that, while AUG is an important component of the COX3 translation initiation site, the site probably is also specified by other sequence or structural features.

Mol Gen Genet, 1991 Nov, 230(1-2), 310 - 20
The Saccharomyces cerevisiae SPT14 gene is essential for normal expression of the yeast transposon, Ty, as well as for expression of the HIS4 gene and several genes in the mating pathway; Fassler JS et al.; To investigate the role of the trans-acting transcription factor encoded by the essential SPT14 (SPT = Suppressor of Ty insertion mutations) gene, we have cloned, mapped and sequenced the gene . From the analysis of the effect of spt14 mutations on expression of various genes, we conclude that the SPT14 product has an important role in activation of Ty transcription as well as in the regulation of other genes including HIS4 and several of the a- and alpha-specific mating type genes . Similarities in the phenotypes of spt14 and spt13 mutants (suppression of Ty insertion mutations but not delta insertion mutations), lead to the suggestion that the SPT14 gene and the previously characterized SPT13/GAL11 gene might encode transcriptional regulators with related functions . Our current findings show that in contrast to SPT13/GAL11, which appears negatively to regulate Ty transcription, SPT14 plays a role in the activation of Ty transcription . Thus, despite the similarities in the suppression phenotype exhibited by spt13 and spt14 mutants, SPT13/GAL11 and SPT14 probably differ in their transcriptional roles.

Mol Cell Biol, 1991 Nov, 11(11), 5767 - 80
CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase; Healy AM et al.; Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle . CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII . At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation . A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate . Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies . This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones . An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.

Mol Cell Biol, 1991 Nov, 11(11), 5487 - 96
Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae; Dumont ME et al.; Heme is covalently attached to cytochrome c by the enzyme cytochrome c heme lyase . To test whether heme attachment is required for import of cytochrome c into mitochondria in vivo, antibodies to cytochrome c have been used to assay the distributions of apo- and holocytochromes c in the cytoplasm and mitochondria from various strains of the yeast Saccharomyces cerevisiae . Strains lacking heme lyase accumulate apocytochrome c in the cytoplasm . Similar cytoplasmic accumulation is observed for an altered apocytochrome c in which serine residues were substituted for the two cysteine residues that normally serve as sites of heme attachment, even in the presence of normal levels of heme lyase . However, detectable amounts of this altered apocytochrome c are also found inside mitochondria . The level of internalized altered apocytochrome c is decreased in a strain that completely lacks heme lyase and is greatly increased in a strain that overexpresses heme lyase . Antibodies recognizing heme lyase were used to demonstrate that the enzyme is found on the outer surface of the inner mitochondrial membrane and is not enriched at sites of contact between the inner and outer mitochondrial membranes . These results suggest that apocytochrome c is transported across the outer mitochondrial membrane by a freely reversible process, binds to heme lyase in the intermembrane space, and is then trapped inside mitochondria by an irreversible conversion to holocytochrome c accompanied by folding to the native conformation . Altered apocytochrome c lacking the ability to have heme covalently attached accumulates in mitochondria only to the extent that it remains bound to heme lyase.

J Biotechnol, 1991 Nov, 21(1-2), 63 - 81
Dynamics of activation of a galactose-inducible promoter in Saccharomyces cerevisiae; Eitzman PD et al.; We have investigated the dynamics of accumulation of the Escherichia coli beta-galactosidase (beta-gal) under the control of a promoter containing the galactose-inducible upstream activating sequence (UASG) in single Saccharomyces cerevisiae cells . The accumulation of beta-gal in single cells following the addition of the inducer, galactose, was determined using an in situ combined DNA and immunofluorescent stain in conjunction with flow cytometry . Two strains were studied, D603/2i, which has two copies of the galactose-inducible fusion gene integrated into its genome, and D603/pLGSD5, which carries a 2 microns-based plasmid containing the fusion gene . Flow cytometry results indicate that accumulation of beta-gal within the first three hours following the addition of galactose is dependent on cell cycle position . Two proposed mechanisms explaining this observed behavior are (1) the cell-cycle-dependent synthesis of the fusion protein or (2) the unequal partitioning of the protein at cell division between mother and daughter cells.

Biochem Biophys Res Commun, 1991 Oct 31, 180(2), 1159 - 63
cDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae; Kuno T et al.; We have isolated a cDNA clone encoding a homolog of mammalian calcineurin B (the regulatory subunit of calmodulin-dependent protein phosphatase) by screening a cDNA expression library of Saccharomyces cerevisiae with antiserum against bovine calcineurin B . The yeast calcineurin B homolog (YCNB) is composed of 175 amino acids with a calculated molecular mass of 19,639 daltons and contains four putative Ca(2+)-binding domains . The amino-acid alignment of YCNB with human calcineurin B demonstrates 53% sequence identity and 82% homology . Southern blot analysis indicates that the gene for YCNB is a single-copy gene . Thus, yeast calmodulin-dependent protein phosphatase apparently has a heterodimeric structure similar to that of the enzyme in mammalians.

Biochemistry, 1991 Oct 29, 30(43), 10595 - 600
Crystallization and preliminary X-ray analysis of the cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae; Kuret J et al.; A truncated variant of TPK1, the yeast cAMP-dependent protein kinase catalytic subunit, was overexpressed in an engineered strain of Saccharomyces cerevisiae, purified by liquid chromatography, and crystallized from solutions of 2-propanol and magnesium at alkaline pH . The crystals are hexagonal dipyramids, space group P6(1)22 (P6(5)22), with unit-cell parameters a = b = 61 A, c = 320 A . Large single crystals suitable for diffraction analysis are obtainable by microseeding, and diffract beyond 2.8-A resolution . Crystal density measurements reveal 12 kinase monomers per unit cell with a single kinase monomer per asymmetric unit.

Nucleic Acids Res, 1991 Oct 25, 19(20), 5681 - 7
Induction of multiple plasmid recombination in Saccharomyces cerevisiae by psoralen reaction and double strand breaks; Saffran WA et al.; DNA damage-induced multiple recombination was studied by cotransforming yeast cells with pairs of nonreplicating plasmids carrying different genetic markers . Reaction of one of the plasmids with the interstrand crosslinking agent, psoralen, stimulated cellular transformation by the undamaged plasmid . The cotransformants carried copies of both plasmids cointegrated in tandem arrays at chromosomal sites homologous to either the damaged or the undamaged DNA . Plasmid linearization, by restriction endonuclease digestion, was also found to stimulate the cointegration of unmodified plasmids . Disruption of the RAD1 gene reduced the psoralen damage-induced cotransformation of intact plasmid, but had no effect on the stimulation by double strand breaks . Placement of the double strand breaks within yeast genes produced cointegration only at sequences homologous to the damaged plasmids, while digestion within vector sequences produced integration at chromosomal sites homologous to either the damaged or the undamaged plasmid molecules . These observations suggest a model for multiple recombination events in which an initial exchange occurs between the damaged DNA and homologous sequences on an undamaged molecule . Linked sequences on the undamaged molecule up to 870 base pairs distant from the break site participate in subsequent exchanges with other intact DNA molecules . These events result in recombinants produced by reciprocal exchange between three or more DNA molecules.

J Biol Chem, 1991 Oct 25, 266(30), 19882 - 5
Mitochondrial phosphate transport . The Saccharomyces cerevisiae (threonine 43 to cysteine) mutant protein explicitly identifies transport with genomic sequence; Phelps A et al.; The yeast mitochondrial phosphate transport protein (PTP) has only 38% sequence similarity to the bovine heart protein, and it has recently been postulated to code for a mitochondrial import receptor . Since the reconstitutively active protein is not completely pure, it is important to demonstrate explicitly that the yeast gene codes for PTP . We have replaced Thr43 with Cys (T43C) and show that its unidirectional and pH gradient-dependent inorganic phosphate transport activity becomes highly sensitive to N-ethylmaleimide . This new PTP/T43C catalyzes less than 10% of the wild type transport activity (1 mM {Pi}e, pHe (6.80); 0 mM {Pi}i, pHi (8.07); 30 s {Pi} uptake) suggesting that Thr43 occupies an important position in the PTP.

Nucleic Acids Res, 1991 Oct 25, 19(20), 5731 - 8
A directed DNA sequencing strategy based upon Tn3 transposon mutagenesis: application to the ADE1 locus on Saccharomyces cerevisiae chromosome I; Davies CJ et al.; We have developed a directed DNA sequencing strategy based upon the Escherichia coli transposon Tn3 . This transposon displays little sequence specificity for transposition and is thus well suited to this task . Both mini-Tn3 transposons and sequencing vectors bearing the phage f1 single stranded origin of replication have been constructed . Upon mutagenesis of a target sequence, a population is produced in which each clone has two f1 origins of replication, one of which is at a variable position depending upon the transposon insertion site . When helper phage is added to the mutagenised population, the two f1 origins present on each clone are nicked, dividing the packaged strand into two segments, each of which is packaged into a separate phage particle . One of these segments contains no resistance markers and is lost, whilst the other is recovered as a deleted clone with a single chimeric f1 origin . A unidirectionally, variably-deleted set of sequencing clones is produced, and appropriately sized clones are sequenced using a primer complimentary to the transposon end . In addition to being inexpensive, the method does not require the same degree of technical expertise needed for many in vitro, enzymatically based methods . The strategy has been used to determine 2.6 kilobases of nucleotide sequence in the Saccharomyces cerevisiae ADE 1 locus.

J Biol Chem, 1991 Oct 25, 266(30), 20103 - 9
Vaccinia virus encodes an active thymidylate kinase that complements a cdc8 mutant of Saccharomyces cerevisiae; Hughes SJ et al.; A vaccinia virus open reading frame (ORF) previously predicted to encode thymidylate kinase (TmpK) is shown to encode an active enzyme . A copy of the ORF, generated by polymerase chain reaction, was cloned into an Escherichia coli inducible expression vector . Cell extracts of E . coli expressing the vaccinia gene contained high levels of TmpK activity, whereas extracts of cells without the TmpK gene did not . The vaccinia ORF expressed from a yeast vector complemented a Saccharomyces cerevisiae cdc8 mutant, demonstrating functional compatibility of the vaccinia virus and yeast TmpK enzymes . The gene is shown to be nonessential for the replication of vaccinia virus in cultured cells by the construction of a viable virus mutant that has the coding region of the TmpK gene interrupted by the Ecogpt gene . Synthesis of the vaccinia TmpK protein in infected cells was demonstrated by the use of a polyvalent rabbit antiserum raised against the purified TmpK enzyme expressed in E . coli to immunoprecipitate a 23-kDa early polypeptide from cells infected with wild type vaccinia but not from cells infected with the TmpK mutant . Plasmid vectors that allow the construction of recombinant viruses expressing foreign gene(s) from the nonessential TmpK locus are described.

Biochemistry, 1991 Oct 22, 30(42), 10181 - 90
A cAMP-binding ectoprotein in the yeast Saccharomyces cerevisiae; Muller G et al.; Purified plasma membranes from the yeast Saccharomyces cerevisiae bind about 1.2 pmol of cAMP/mg of protein with high affinity (Kd = 6 nM) . By using photoaffinity labeling with 8-N3-{32P}cAMP, we have identified in plasma membrane vesicles a cAMP-binding protein (Mr = 54,000) that is present also in bcy1 disruption mutants, lacking the cytoplasmic R subunit of protein kinase A (PKA) . This argues that it is genetically unrelated to PKA . Neither high salt, nor alkaline carbonate, nor cAMP extract the protein from the membrane, suggesting that it is not peripherally bound . The observation that (glycosyl)phosphatidylinositol-specific phospholipases (or nitrous acid) release the amphiphilic protein from the membrane, thereby converting it to a hydrophilic form, indicates anchorage by a glycolipidic membrane anchor . Treatment with N-glycanase reduces the Mr to 44,000-46,000 indicative of a modification by N-linked carbohydrate side chain(s) . In addition to the action of a phospholipase, the efficient release from the membrane requires the removal of the carbohydrate side chain(s) or the presence of high salt or methyl alpha-mannopyranoside, suggesting complex interactions with the membrane involving not only the glycolipidic anchor but also the glycan side chain(s) . Topological studies show that the protein is exposed to the periplasmic space, raising intriguing questions for the function of this protein.

FEBS Lett, 1991 Oct 21, 291(2), 303 - 6
Saccharomyces cerevisiae elongation factor 2 is phosphorylated by an endogenous kinase; Donovan MG et al.; Mammalian cells contain a Ca2+/calmodulin-dependent protein kinase that specifically phosphorylates and inactivates elongation factor 2 (EF-2) in response to hormones and other agents which increase intracellular Ca2+ concentrations . Therefore, it has been proposed that the rate of translation in mammals is regulated by EF-2 phosphorylation . In the present study, EF-2 purified from the yeast Saccharomyces cerevisiae is shown to be a substrate for the mammalian EF-2 kinase . Furthermore, evidence was obtained using two-dimensional gel electrophoresis and peptide mapping which suggests that yeast EF-2 is a substrate for an endogenous kinase which phosphorylates the same site as the mammalian EF-2 kinase . Based on these findings, we propose that in yeast as in higher eukaryotes, the protein synthesis elongation cycle is regulated by phosphorylation of EF-2.

Gene, 1991 Oct 15, 106(2), 267 - 71
Secretion of human epidermal growth factor from Saccharomyces cerevisiae using synthetic leader sequences; Clements JM et al.; We have investigated different leader sequences for their ability to direct the efficient secretion of human epidermal growth factor (hEGF) from Saccharomyces cerevisiae . We designed a consensus signal sequence which directs secretion of hEGF from yeast as efficiently as the yeast invertase signal sequence . However, secretion is increased over fivefold by the introduction, after the signal sequence, of a synthetic 19-amino acid (aa) pro-sequence containing a cleavage recognition site for the KEX2 protease . Even in the absence of an Asn-linked glycosylation site, secretion of hEGF using the synthetic prepro-leader was as efficient as that directed by the alpha-factor leader . The role of the KEX2 protease cleavage site was investigated by mutation of the yeast alpha-factor KEX2 site (cleavage after Lys-Arg) . Cleavage was obtained with the following order of efficiency, Lys-Arg greater than Pro-Arg greater than Asp-Arg, although the sequence context was also found to affect efficiency.

Eur J Biochem, 1991 Oct 15, 201(2), 399 - 407
Expression of mammalian tyrosine aminotransferase in Saccharomyces cerevisiae and Escherichia coli . Purification to homogeneity and characterization of the enzyme overproduced in the bacteria; Dietrich JB et al.; Rat liver tyrosine aminotransferase has been expressed in Saccharomyces cerevisiae and Escherichia coli . In yeast, the extent of production is 20-fold higher than that in rat liver after induction by dexamethasone, and reaches 250-fold higher in an E . coli strain carrying the T7 RNA polymerase transcription system . About 250 mg pure and homogeneous enzyme was obtained from 50 g transformed E . coli cells . Determination of Mr and pI, as well as analysis of N- and C-terminal amino acids, suggest that the isolated protein is native . The catalytic properties, similar to those of the enzyme from rat liver, confirm that it is fully active and that post-translational modifications in the mammalian cells are not essential for activity . Pyridoxal 5'-phosphate strongly protects the enzyme against thermal inactivation . After denaturation, 10 thiol groups, out of 16 in the polypeptide chain, react with 5,5'-dithiobis(2-nitrobenzoic acid) whereas only five or six are accessible under native conditions . Two thiols are rapidly modified with concomitant inactivation of the apoenzyme, but pyridoxal 5'-phosphate partially protects them in the holoenzyme . The results are interpreted in the light of the structure/function relationship in this enzyme.

Carbohydr Res, 1991 Oct 14, 219, 203 - 13
A method for the solubilization of a (1----3)-beta-D-glucan isolated from Saccharomyces cerevisiae; Williams DL et al.; This report describes a method for the solubilization of a micro-particulate beta-D-glucan . Insoluble glucan is dissolved in methyl sulfoxide and urea (8M) and partially phosphorylated at 100 degrees . The resulting water-soluble product is called glucan phosphate . The conversion rate is 70%, and the preparation is endotoxin free as determined by the Limulus lysate procedure . Glucan phosphate is composed of 34.66% C, 6.29% H, 42.83% O, and 2.23% P and has a repeating-unit empirical formula of (C6H10O5)7.PO3H2, indicating a phosphate group substitution on every seventh glucose subunit . Molecular-weight averages, polydispersity, and intrinsic viscosity were determined by aqueous high-performance size-exclusion chromatography (s.e.c.) with on-line, multi-angle laser light scattering (m.a.l.l.s.) photometry and differential viscometry (d.v.) . Two polymer peaks were resolved . Peak 1 (Mw = 3.57 x 10(6) daltons), represents approximately 2% of the total polymers, while peak 2 (Mw = 1.10 x 10(5) daltons) comprises approximately 98% of polymers . 13C- and 31P-n.m.r . spectroscopy confirmed the beta-1,3 interchain linkage and the presence of a phosphate group . In solution, glucan phosphate polymers self-associate in a triple-helical arrangement . The ability to prepare a immunologically active, non-toxic, water-soluble beta-D-glucan preparation will greatly enhance the clinical utility of this class of compounds.

FEBS Lett, 1991 Oct 7, 291(1), 45 - 9
Structural and functional analysis of Trichoderma reesei endoglucanase I expressed in yeast Saccharomyces cerevisiae; Aho S; The function of the domains of Trichoderma reesei endoglucanase I (EGI) has been studied . Truncated EGI proteins were expressed from the 3'-end deleted cDNAs in the yeast Saccharomyces cerevisiae under the control of the ADC1 expression cassette . EGI protein was detected by monoclonal antibody EI-2 and EGI activity as cleared zones around growing colonies on agar plates containing hydroxyethylcellulose (HEC) covalently stained with Ostazin brilliant red (OBR) . The results showed that the The-Ser-rich hinge region and the conserved 'tail' are not necessary for the efficient synthesis and secretion of EGI in yeast, but the intact core region is necessary for the enzymatic activity.

J Biol Chem, 1991 Oct 5, 266(28), 18884 - 8
Structural homology among mammalian and Saccharomyces cerevisiae isoprenyl-protein transferases; Kohl NE et al.; Farnesyl-protein transferase (FTase) purified from rat or bovine brain is an alpha/beta heterodimer, comprised of subunits having relative molecular masses of approximately 47 (alpha) and 45 kDa (beta) . In the yeast Saccharomyces cerevisiae, two unlinked genes, RAM1/DPR1 (RAM1) and RAM2, are required for FTase activity . To explore the relationship between the mammalian and yeast enzymes, we initiated cloning and immunological analyses . cDNA clones encoding the 329-amino acid COOH-terminal domain of bovine FTase alpha-subunit were isolated . Comparison of the amino acid sequences deduced from the alpha-subunit cDNA and the RAM2 gene revealed 30% identity and 58% similarity, suggesting that the RAM2 gene product encodes a subunit for the yeast FTase analogous to the bovine FTase alpha-subunit . Antisera raised against the RAM1 gene product reacted specifically with the beta-subunit of bovine FTase, suggesting that the RAM1 gene product is analogous to the bovine FTase beta-subunit . Whereas a ram1 mutation specifically inhibits FTase, mutations in the CDC43 and BET2 genes, both of which are homologous to RAM1, specifically inhibit geranylgeranyl-protein transferase (GGTase) type I and GGTase-II, respectively . In contrast, a ram2 mutation impairs both FTase and GGTase-I, but has little effect on GGTase-II . Antisera that specifically recognized the bovine FTase alpha-subunit precipitated both bovine FTase and GGTase-I activity, but not GGTase-II activity . Together, these results indicate that for both yeast and mammalian cells, FTase, GGTase-I, and GGTase-II are comprised of different but homologous beta-subunits and that the alpha-subunits of FTase and GGTase-I share common features not shared by GGTase-II.

J Mol Biol, 1991 Oct 5, 221(3), 745 - 9
Stable ester conjugate between the Saccharomyces cerevisiae RAD6 protein and ubiquitin has no biological activity; Sung P et al.; The RAD6 gene of Saccharomyces cerevisiae, which encodes a ubiquitin-conjugating enzyme, is required for DNA repair, DNA damage-induced mutagenesis and sporulation . To evaluate the biological relevance of the thioester adduct between RAD6 protein and ubiquitin, formed as an obligatory, transient intermediate during ubiquitin conjugation to substrates, we altered cysteine 88 in RAD6 to serine . Esterification with ubiquitin occurs at serine 88 in the mutant protein, but conjugation of ubiquitin to the test substrate histone H2A is inactivated . Phenotypically, strains harboring the rad6 Ser88 allele are indistinguishable from rad6 deletion (rad6 delta) mutant cells . These findings argue against ligation of ubiquitin at cysteine 88 acting as a functional switch of a cryptic biochemical activity in RAD6.

Proc Natl Acad Sci U S A, 1991 Oct 1, 88(19), 8387 - 91
Evolution of aminoacyl-tRNA synthetase quaternary structure and activity: Saccharomyces cerevisiae mitochondrial phenylalanyl-tRNA synthetase; Sanni A et al.; Phenylalanyl-tRNA synthetases {L-phenylalanine:tRNAPhe ligase (AMP-forming), EC 6.1.1.20} from Escherichia coli, yeast cytoplasm, and mammalian cytoplasm have an unusual conserved alpha 2 beta 2 quaternary structure that is shared by only one other aminoacyl-tRNA synthetase . Both subunits are required for activity . We show here that a single mitochondrial polypeptide from Saccharomyces cerevisiae is an active phenylalanyl-tRNA synthetase . This protein (the MSF1 gene product) is active as a monomer . It has all three characteristic sequence motifs of the class II aminoacyl-tRNA synthetases, and its activity may result from the recruitment of additional sequences into an alpha-subunit-like structure.

Mol Cell Biol, 1991 Oct, 11(10), 5372 - 80
Heteroduplex formation and mismatch repair of the "stuck" mutation during mating-type switching in Saccharomyces cerevisiae; Ray BL et al.; We sequenced two alleles of the MATa locus of Saccharomyces cerevisiae that reduce homothallic switching and confer viability to HO rad52 strains . Both the MATa-stk (J . E . Haber, W . T . Savage, S . M . Raposa, B . Weiffenbach, and L . B . Rowe, Proc . Natl . Acad . Sci . USA 77:2824-2828, 1980) and MATa-survivor (R . E . Malone and D . Hyman, Curr . Genet . 7:439-447, 1983) alleles result from a T----A base change at position Z11 of the MAT locus . These strains also contain identical base substitutions at HMRa, so that the mutation is reintroduced when MAT alpha switches to MATa . Mating-type switching in a MATa-stk strain relative to a MATa Z11T strain is reduced at least 50-fold but can be increased by expression of HO from a galactose-inducible promoter . We confirmed by Southern analysis that the Z11A mutation reduced the efficiency of double-strand break formation compared with the Z11T variant; the reduction was more severe in MAT alpha than in MATa . In MAT alpha, the Z11A mutation also creates a mat alpha 1 (sterile) mutation that distinguishes switches of MATa-stk to either MAT alpha or mat alpha 1-stk . Pedigree analysis of cells induced to switch in G1 showed that MATa-stk switched frequently (23% of the time) to produce one mat alpha 1-stk and one MAT alpha progeny . This postswitching segregation suggests that Z11 was often present in heteroduplex DNA that was not mismatch repaired . When mismatch repair was prevented by deletion of the PMS1 gene, there was an increase in the proportion of mat alpha 1-stk/MAT alpha sectors (59%) and in pairs of switched cells that both retained the stk mutation (27%) . We conclude that at least one strand of DNA only 4 bp from the HO cut site is not degraded in most of the gene conversion events that accompany MAT switching.

Mol Cell Biol, 1991 Oct, 11(10), 5301 - 11
The chromatin structure of Saccharomyces cerevisiae autonomously replicating sequences changes during the cell division cycle; Brown JA et al.; The chromatin structures of two well-characterized autonomously replicating sequence (ARS) elements were examined at their chromosomal sites during the cell division cycle in Saccharomyces cerevisiae . The H4 ARS is located near one of the duplicate nonallelic histone H4 genes, while ARS1 is present near the TRP1 gene . Cells blocked in G1 either by alpha-factor arrest or by nitrogen starvation had two DNase I-hypersensitive sites of about equal intensity in the ARS element . This pattern of DNase I-hypersensitive sites was altered in synchronous cultures allowed to proceed into S phase . In addition to a general increase in DNase I sensitivity around the core consensus sequence, the DNase I-hypersensitive site closest to the core consensus became more nuclease sensitive than the distal site . This change in chromatin structure was restricted to the ARS region and depended on replication since cdc7 cells blocked near the time of replication initiation did not undergo the transition . Subsequent release of arrested cdc7 cells restored entry into S phase and was accompanied by the characteristic change in ARS chromatin structure.

Mol Cell Biol, 1991 Oct, 11(10), 5212 - 21
In vivo analysis of the Saccharomyces cerevisiae centromere CDEIII sequence: requirements for mitotic chromosome segregation; Jehn B et al.; In the yeast Saccharomyces cerevisiae, the complete information needed in cis to specify a fully functional mitotic and meiotic centromere is contained within 120 bp arranged in the three conserved centromeric (CEN) DNA elements CDEI, -II, and -III . The 25-bp CDEIII is most important for faithful chromosome segregation . We have constructed single- and double-base substitutions in all highly conserved residues and one nonconserved residue of this element and analyzed the mitotic in vivo function of the mutated CEN DNAs, using an artificial chromosome . The effects of the mutations on chromosome segregation vary between wild-type-like activity (chromosome loss rate of 4.8 x 10(-4)) and a complete loss of CEN function . Data obtained by saturation mutagenesis of the palindromic core sequence suggest asymmetric involvement of the palindromic half-sites in mitotic CEN function . The poor CEN activity of certain single mutations could be improved by introducing an additional single mutation . These second-site suppressors can be found at conserved and nonconserved positions in CDEIII . Our suppression data are discussed in the context of natural CDEIII sequence variations found in the CEN sequences of different yeast chromosomes.

Mol Cell Biol, 1991 Oct, 11(10), 5181 - 9
Two components of Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) are stereospecifically located upstream of a tRNA gene and interact with the second-largest subunit of TFIIIC; Bartholomew B et al.; A novel photocrosslinking method has been used to identify the components of transcription factor IIIB (TFIIIB) and TFIIIC that associate with DNA upstream of the Saccharomyces cerevisiae SUP4 tRNATyr gene and to map these components to specific positions in DNA . When TFIIIC binds to the tRNA gene, only its second-largest subunit (135 kDa) is accessible for reaction with a photoactive nucleotide, 5-{N-(p-azidobenzoyl)-3-aminoallyl}-dUMP, inserted into DNA upstream of the transcriptional start . Formation of TFIII(C + B)-tRNA gene complexes specifically brings two additional polypeptides (90 and 70 kDa) within reach of upstream photoprobes . A collection of 13 probes has been used to map the locations of these three proteins along a 45-bp segment of DNA upstream of the transcriptional start site . Evidence is presented that the 90- and 70-kDa polypeptides are separate and distinct components of yeast TFIIIB, that they are accessible to crosslinking on opposite sides of the DNA helix in a 6-bp segment centered 35 bp upstream of the tRNATyr gene transcriptional start, and that they interact with the second-largest subunit of TFIIIC.

Mol Cell Biol, 1991 Oct, 11(10), 5101 - 12
GRR1 of Saccharomyces cerevisiae is required for glucose repression and encodes a protein with leucine-rich repeats; Flick JS et al.; Growth of the yeast Saccharomyces cerevisiae on glucose leads to repression of transcription of many genes required for alternative carbohydrate metabolism . The GRR1 gene appears to be of central importance to the glucose repression mechanism, because mutations in GRR1 result in a pleiotropic loss of glucose repression (R . Bailey and A . Woodword, Mol . Gen . Genet . 193:507-512, 1984) . We have isolated the GRR1 gene and determined that null mutants are viable and display a number of growth defects in addition to the loss of glucose repression . Surprisingly, grr1 mutations convert SUC2, normally a glucose-repressed gene, into a glucose-induced gene . GRR1 encodes a protein of 1,151 amino acids that is expressed constitutively at low levels in yeast cells . GRR1 protein contains 12 tandem repeats of a sequence similar to leucine-rich motifs found in other proteins that may mediate protein-protein interactions . Indeed, cell fractionation studies are consistent with this view, suggesting that GRR1 protein is tightly associated with a particulate protein fraction in yeast extracts . The combined genetic and molecular data are consistent with the idea that GRR1 protein is a primary response element in the glucose repression pathway and is required for the generation or interpretation of the signal that induces glucose repression.

Mol Gen Genet, 1991 Oct, 229(2), 307 - 15
DNA sequences in chromosomes II and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains; Stucka R et al.; A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae . We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase . The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere . We localized the previously isolated gene, which we designate PYC1, to chromosome VII . Disruption of either of the genes did not produce marked changes in the phenotype . However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium . This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase . The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level . No appreciable homology was found in the corresponding flanking regions . No differences in the Km values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.

Mol Gen Genet, 1991 Oct, 229(2), 261 - 6
Gene conversions within the Cup1r region from heterologous crosses in Saccharomyces cerevisiae; Welch JW et al.; Meiotic recombination among unselected tetrads was analyzed genetically and physically in a heterologous cross where one parent carried six copies of a 1.6 kb CUP1 repeat while the other parent carried seven copies of a 1.1 kb repeat . In the heterologous cross, 140 unselected, complete tetrads were subjected to Southern analysis and 20% exhibited meiotic copy number alterations at the CUP1 locus . Most events, more than 75%, involved only a single spore of a tetrad, and were largely intrachromosomal or sister chromatid events . However, some conversions and associated crossovers between homologs were also observed . We propose that the high level of heterologies interferes with homologous exchanges and leads to an increase in intrachromosomal events.

J Cell Biol, 1991 Oct, 115(2), 297 - 307
Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway; Wilcox CA et al.; The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues . Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology . Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked mannose in the ER . The earliest detectable species, I1 (approximately 129 kD), undergoes rapid amino-terminal proteolytic removal of a approximately 9-kD pro-segment yielding species I2 (approximately 120 kD) before arrival at the Golgi complex . Transport to the Golgi complex is marked by extensive elaboration of Ser/Thr-linked chains and minor modification of Asn-linked oligosaccharide . During the latter phase of its lifetime, Kex2 protein undergoes a gradual increase in apparent molecular weight . This final modification serves as a marker for association of Kex2 protease with a late compartment of the yeast Golgi complex in which it is concentrated about 27-fold relative to other secretory proteins.

J Bacteriol, 1991 Oct, 173(20), 6432 - 7
Coordinate regulation of phosphatidylserine decarboxylase in Saccharomyces cerevisiae; Lamping E et al.; Regulation of the activity of the mitochondrial enzyme phosphatidylserine decarboxylase (PSD) was measured in vitro by using membrane preparations from wild-type and mutant strains of Saccharomyces cerevisiae . PSD specific activity was not affected by carbon source, and on all carbon sources, the highest specific activity was observed in cells entering the stationary phase of growth . However, PSD activity was found to be regulated in response to soluble precursors of phospholipid biosynthesis . PSD specific activity was reduced to about 63% of the level observed in unsupplemented wild-type cells when the cells were grown in the presence of 75 microM inositol . The presence of 1 mM choline alone had no repressing effect, but the presence of 1 mM choline and 75 microM inositol together led to further repression to a level of about 28% of the derepressed activity . Regulatory mutations known to affect regulation or expression of genes encoding phospholipid-synthesizing enzymes also affected PSD specific activity . opi1 mutants, which are constitutive for a number of phospholipid-biosynthetic enzymes, were found to have high, constitutive levels of PSD . Likewise, in ino2 or ino4 regulatory mutants, PSD activity was found to be at the fully repressed level regardless of growth condition . Regulation of PSD activity was also affected in several structural-gene mutants under conditions of impaired phosphatidylcholine biosynthesis . Together, these data strongly suggest that PSD expression is controlled by the mechanism of general control of phospholipid biosynthesis that regulates many enzymes of phospholipid biosynthesis.

J Bacteriol, 1991 Oct, 173(20), 6332 - 8
Participation of ABF-1 protein in expression of the Saccharomyces cerevisiae CAR1 gene; Kovari LZ et al.; DNA fragments previously shown to be required for expression of the CAR1 (arginase) gene in Saccharomyces cerevisiae and to support transcriptional activation of a reporter gene in a heterologous expression vector were shown to bind purified regulatory protein ABF-1 . Two ABF-1 sites were identified in the CAR1 upstream region, one to which ABF-1 protein bound with high affinity and a second to which it bound much less avidly . The higher-affinity ABF-1 binding site upstream of CAR1 was an effective competitor of the HMRE, ARS1 B domain, and COR2-GFI binding sequences for protein binding . Point mutations in the CAR1 high-affinity ABF-1 binding site resulted in a 12-fold loss of transcriptional activation of a reporter gene compared with the wild-type CAR1 DNA fragment . These data are consistent with the suggestion that ABF-1 protein is one of the transcription factors involved in expression of the CAR1 gene.

EMBO J, 1991 Oct, 10(10), 3033 - 41
Nucleosomes are positioned with base pair precision adjacent to the alpha 2 operator in Saccharomyces cerevisiae; Shimizu M et al.; Analysis of the chromatin structure of minichromosomes containing the binding site for the yeast alpha 2 repressor protein by indirect end-labeling has previously indicated that nucleosomes are stably positioned over sequences adjacent to the alpha 2 operator in the presence of the repressor . Development of a primer extension assay for nucleosome position now allows a more detailed examination of the location of these nucleosomes relative to the operator sequence, and indicates that nucleosomes are precisely and stably positioned both translationally and rotationally over sequences adjoining the operator . In addition, this assay enables analysis of the chromatin structure of single copy, genomic sequences . Chromatin structures determined for two genes regulated by alpha 2, STE6 and BAR1, are consistent with nucleosomes precisely positioned downstream of the operator sequence, incorporating promoter elements, in alpha cells but not in a-cells . The location of these nucleosomes relative to the operator sequence is highly analogous to that observed in the minichromosome . The stability of the nucleosomes adjacent to the operator together with the precision of their location suggests that they may play a role in repression of a specific gene expression by alpha 2 . Further, the primer extension assay allows a comparison of the structure of these positioned nucleosomes formed in vivo to that previously described for core particles reconstituted in vitro.

Curr Opin Genet Dev, 1991 Oct, 1(3), 342 - 50
Budding and cell polarity in Saccharomyces cerevisiae; Chant J et al.; Budding by yeast follows a sequence of three stages . These include selection of a non-random bud-site, organization of that site and establishment of an associated axis of cytoskeletal polarity, and localized growth of the cell surface to produce the bud . Numerous components involved in each stage have been identified . As some of these components have close homologs in other organisms, there may exist common mechanisms involved in the establishment of cell polarity.

Fundam Appl Toxicol, 1991 Oct, 17(3), 627 - 34
False positive result for a peptide drug in the gene conversion assay with Saccharomyces cerevisiae strain D7; DePass LR et al.; A battery of mutagenicity tests was performed with nafarelin, an agonist analogue of luteinizing hormone releasing hormone (LHRH) containing tryptophan (Trp) and histidine (His) . Included were the Ames assay and the gene conversion assay with yeast strain D7 . Both tests were negative without S9 activation, and the Ames test was negative with S9, but the yeast test was positive with S9 activation . Since the yeast test is based on conversion of cells to Trp independence, release of Trp by metabolism of the drug could account for the positive result . The test was repeated using Trp instead of the drug . The result was positive even at the lowest Trp concentration . In another experiment with the drug, amino acid analysis of the incubation mixture revealed the presence of Trp but no detectable His . Since the Ames test is based on mutation to His-independent cells, these data are completely consistent with the negative result in the Ames test and the false positive result in the yeast test . These data suggest the need for caution in interpreting the results from mutagenicity assays with peptide drugs.

Mol Microbiol, 1991 Oct, 5(10), 2417 - 26
Isolation and characterization of osmosensitive vacuolar mutants of Saccharomyces cerevisiae; Latterich M et al.; The yeast vacuole plays an important role in nitrogen metabolism, storage and intracellular macromolecular degradation . Evidence suggests that it is also involved in osmohomeostasis of the cell . We have taken a mutational approach for the analysis of vacuolar function and biogenesis by the isolation of 97 mutants unable to grow if high concentrations of salt are present in the medium . Phenotypic analysis was able to demonstrate that apart from osmosensitivity the mutations also conferred other properties such as altered vacuolar morphology and secretion of the vacuolar enzymes carboxypeptidase Y, proteinase A, proteinase B and alpha-mannosidase . The mutants fall into at least 17 complementation groups, termed ssv for salt-sensitive vacuolar mutants, of which two are identical to complementation groups isolated by others . We conclude that in Saccharomyces cerevisiae correct vacuolar biogenesis and protein targeting is required for osmotolerance as well as other important cellular processes.

Yeast, 1991 Oct, 7(7), 761 - 72
The complete sequence of a 7.5 kb region of chromosome III from Saccharomyces cerevisiae that lies between CRY1 and MAT; Wicksteed BL et al.; We report the sequence of a 7.5 kb region lying between the CRY1 and MAT loci of chromosome III from Saccharomyces cerevisiae . This region lies in the overlap between two major contigs used for the generation of the complete nucleotide sequence of this chromosome . Comparison of this sequence with those reported previously for this overlap {Thierry et al . (1990) Yeast 6, 521; Jia et al . (1991) Yeast 7, 413} reveals 38 nucleotide differences, 45% of which generate changes in the amino acid sequences of the four genes in this region (YCR591, YCR592, YCR521 and YCR522) . These differences appear to reflect true sequence polymorphisms between the two yeast strains used to generate the clones used in the sequencing project . Three of the four genes in this region display weak homologies to proteins in the PIR database . Some properties of YCR521 are analogous to those of ribosomal protein genes . However, the functions of all four genes remain obscure.

Yeast, 1991 Oct, 7(7), 717 - 26
Cell wall glucomannoproteins of Saccharomyces cerevisiae mnn9; Van Rinsum J et al.; Mannoproteins were isolated from Saccharomyces cerevisiae mnn9 mutant cell walls by laminarinase digestion and purified by affinity and anion-exchange chromatography . The purified mannoprotein fraction contained three predominant proteins with molecular masses of 300 kDa, 220 kDa and 160 kDa . These compounds were absent in an SDS extract of cell walls or in a hot-citrate extract of mnn9 cells . The carbohydrate part of the purified mannoproteins consisted of (N-acetyl)glucosamine, mannose and glucose in a molar ratio of 1:53:4 . O-Glycosidically linked chains, containing 70% of the mannose, were released by mild beta-elimination . N-Glycosidically linked chains, representing 80% of the (N-acetyl)glucosamine and 20% of the mannose, were released by peptide N-glycosidase F (PNGase F) digestion . Complete degradation of protein by alkaline hydrolysis released besides the N- and O-glycosidically linked chains, another type of carbohydrate chain containing the residual (N-acetyl)glucosamine, mannose and most of the glucose in a molar ratio of 1:17:18 . Glucose was beta-glycosidically linked . The results indicate that beta-glucose is linked to PNGase F-resistant N-linked chains present on cell wall mannoproteins . We propose that these chains are responsible for the linkage between mannoproteins and glucan in the cell wall.

Yeast, 1991 Oct, 7(7), 693 - 8
The ureidoglycollate hydrolase (DAL3) gene in Saccharomyces cerevisiae; Yoo HS et al.; The DAL3 gene has been sequenced and found to encode a 195 amino acid protein with a molecular weight of 21,727 . The four carboxy-terminal amino acids of DAL3 product (Cys-Ile-Ile-Ile) are homologous to those (CAAX) previously shown to be the primary structural signal for post-translational farnesylation of yeast RAS protein and mating factor . This modification is reported to be responsible for membrane localization of proteins containing it . The upstream region of DAL3 contains six copies of a sequence that is homologous to the positively acting DAL UASNTR reported to be required for transcriptional activation of the DAL5 and DAL7 genes . Missing from the DAL3 upstream region were any sequences related to those shown to be required for a DAL7 response to inducer, the UIS element . This correlates with the previous report that DAL3 expression is independent of the allantoin pathway inducer.

J Gen Microbiol, 1991 Oct, 137 ( Pt 10), 2447 - 54
Vacuolar segregation to the bud of Saccharomyces cerevisiae: an analysis of morphology and timing in the cell cycle; Gomes de Mesquita DS et al.; Vacuoles of Saccharomyces cerevisiae were visualized by phase-contrast microscopy . Visualization was enhanced by adding polyvinylpyrrolidone . Vacuolar segregation during the cell cycle was analysed in 42 individual cells of strain X2180 by time-lapse photomicrography . Within 15 min of bud emergence, more than 80% of the cells contained a vacuolar segregation structure in the form of either a tubule or an alignment of vesicles . The structure emerged from one point of the mother vacuole, then elongated and moved into the bud in a few minutes . The vacuolar segregation structure disappeared, usually within 20 min, before nuclear migration, leaving a separate vacuole in the bud . To test the generality of this observation several strains were grown in the presence of the vacuolar vital dye fluorescein isothiocyanate . The bud size was used to measure progress in the cell cycle . All strains formed vacuolar segregation structures in cells with small buds, although with variations in duration and timing in the cell cycle . In the presence of nocodazole vacuolar segregation occurred normally, thus, microtubules seem not to be essential in this process.

Curr Opin Genet Dev, 1991 Oct, 1(3), 370 - 7
Ras-regulated signaling processes in Saccharomyces cerevisiae; Broach JR; Molecular genetics has proved quite successful in identifying the components of RAS-mediated signal transduction in the yeast Saccharomyces cerevisiae and in defining the nature of their interactions . Recently, the emphasis has shifted to a biochemical approach as the processes of guanine nucleotide exchange, GTPase stimulation activity and posttranslational modification of Ras proteins have all been reproduced in vitro.

Biochem J, 1991 Oct 1, 279 ( Pt 1), 135 - 9
Chemical modification of a functional arginine residue in diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase I from Saccharomyces cerevisiae; Robinson AK et al.; Phenylglyoxal, a reagent with high specificity for arginine residues, inactivated Ap4A phosphorylase I from Saccharomyces cerevisiae in a pseudo-first-order manner . The second-order rate constant was 11.5 +/- 2.5 M-1 min-1 . The loss of activity was a linear function of the incorporation of {7-14C}phenylglyoxal . The incorporation of 1.9 +/- 0.4 mol of phenylglyoxal/mol of enzyme accounted for complete loss of activity . The specificity of inactivation by phenylglyoxal was tested in the presence of ApnA (n = 2-6), ADP, ATP and Pi . The substrates, Ap4A, Ap5A and Pi protected the enzyme against inactivation, but Ap2A, Ap3A and Ap6A did not . Ap4A, Ap5A and Pi reduced the rate of inactivation by about 70%, 60% and 37% respectively . The Ap4A phosphorolysis products, ADP and ATP, also partially protected the enzyme against inactivation by phenylglyoxal . Thus Ap4A phosphorylase I probably contains an arginine residue in the binding site for Ap4A.

Mol Cell Biol, 1991 Oct, 11(10), 5251 - 8
Quantitation of alpha-factor internalization and response during the Saccharomyces cerevisiae cell cycle; Zanolari B et al.; The alpha-factor pheromone binds to specific cell surface receptors on Saccharomyces cerevisiae a cells . The pheromone is then internalized, and cell surface receptors are down-regulated . At the same time, a signal is transmitted that causes changes in gene expression and cell cycle arrest . We show that the ability of cells to internalize alpha-factor is constant throughout the cell cycle, a cells are also able to respond to pheromone throughout the cycle even though there is cell cycle modulation of the expression of two pheromone-inducible genes, FUS1 and STE2 . Both of these genes are expressed less efficiently near or just after the START point of the cell cycle in response to alpha-factor . For STE2, the basal level of expression is modulated in the same manner.

Mol Cell Biol, 1991 Oct, 11(10), 4876 - 84
Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis; Ronne H et al.; We have cloned three genes for protein phosphatases in the yeast Saccharomyces cerevisiae . Two of the genes, PPH21 and PPH22, encode highly similar proteins that are homologs of the mammalian protein phosphatase 2A (PP2A), while the third gene, PPH3, encodes a new PP2A-related protein . Disruptions of either PPH21 or PPH22 had no effects, but spores disrupted for both genes produced very small colonies with few surviving cells . We conclude that PP2A performs an important function in yeast cells . A disruption of the third gene, PPH3, did not in itself affect growth, but it completely prevented growth of spores disrupted for both PPH21 and PPH22 . Thus, PPH3 provides some PP2A-complementing activity which allows for a limited growth of PP2A-deficient cells . Strains were constructed in which we could study the phenotypes caused by either excess PP2A or total PP2A depletion . We found that the level of PP2A activity has dramatic effects on cell shape . PP2A-depleted cells develop an abnormal pear-shaped morphology which is particularly pronounced in the growing bud . In contrast, overexpression of PP2A produces more elongated cells, and high-level overexpression causes a balloonlike phenotype with huge swollen cells filled by large vacuoles.

J Bacteriol, 1991 Oct, 173(19), 6124 - 31
Regulation of phosphatidylglycerolphosphate synthase in Saccharomyces cerevisiae by factors affecting mitochondrial development; Gaynor PM et al.; Phosphatidylglycerolphosphate synthase (PGPS; CDP-diacylglycerol glycerol 3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the first step in the synthesis of cardiolipin, an acidic phospholipid found in the mitochondrial inner membrane . In the yeast Saccharomyces cerevisiae, PGPS expression is coordinately regulated with general phospholipid synthesis and is repressed when cells are grown in the presence of the phospholipid precursor inositol (M . L . Greenberg, S . Hubbell, and C . Lam, Mol . Cell . Biol . 8:4773-4779, 1988) . In this study, we examined the regulation of PGPS in growth conditions affecting mitochondrial development (carbon source, growth stage, and oxygen availability) and in strains with genetic lesions affecting mitochondrial function . PGPS derepressed two- to threefold when cells were grown in a nonfermentable carbon source (glycerol-ethanol), and this derepression was independent of the presence of inositol . PGPS derepressed two- to fourfold as cells entered the stationary phase of growth . Stationary-phase derepression occurred in both glucose- and glycerol-ethanol-grown cells and was slightly greater in cells grown in the presence of inositol and choline . PGPS expression in mitochondria was not affected when cells were grown in the absence of oxygen . In mutants lacking mitochondrial DNA {( rho0} mutants), PGPS activity was 30 to 70% less than in isogenic {rho+} strains . PGPS activity in {rho0} strains was subject to inositol-mediated repression . PGPS activity in {rho0} cell extracts was derepressed twofold as the {rho0} cells entered the stationary phase of growth . No growth phase derepression was observed in mitochondrial extracts of the {rho0} cells . Relative cardiolipin content increased in glycerol-ethanol-grown cells but was not affected by growth stage or by growth in the presence of the phospholipid precursors inositol and choline . These results demonstrate that (i) PGPS expression is regulated by factors affecting mitochondrial development; (ii) regulation of PGPS by these factors is independent of cross-pathway control; and (iii) PGPS expression is never fully repressed, even during anaerobic growth.

J Biotechnol, 1991 Oct, 20(3), 291 - 300
Production of functional IgM Fab fragments by Saccharomyces cerevisiae; Edqvist J et al.; The aim of this study was to express and secrete functional mouse IgM fragments in yeast . The heavy chain cDNA was truncated at two different sites, yielding genes coding for the complete VH domain . In one of the truncated genes, the CH1 domain is complete, while in the other gene 18 bp are missing from the 3' terminus of the CH1 region . Both shortened genes were coexpressed in Saccharomyces cerevisiae with a cDNA gene encoding a full length mouse Ig light chain . We show that only the longer form of the truncated heavy chain together with the light chain produced and secreted functional IgM Fab fragments.

Gene, 1991 Sep 30, 106(1), 35 - 42
An androgen-inducible expression system for Saccharomyces cerevisiae; Purvis IJ et al.; A novel controllable expression system for Saccharomyces cerevisiae has been developed . Expression of the gene encoding the human androgen receptor, from a strong yeast promoter, results in transactivation of a hybrid promoter carrying androgen-responsive sequences such that a target gene may be expressed in an androgen-dependent manner . By selection of an appropriate combination of androgen receptor level, target-gene copy number and concentration of the androgenic ligand, dihydrotestosterone, the expression level can be set within a 1400-fold range with no detectable effect on normal cell growth.

Nucleic Acids Res, 1991 Sep 25, 19(18), 5059 - 64
A temperature sensitive mutant of Saccharomyces cerevisiae defective in pre-rRNA processing; Shuai K et al.; A recessive temperature sensitive mutant has been isolated that is defective in ribosomal RNA processing . By Northern analysis, this mutant was found to accumulate three novel rRNA species: 23S', 18S' and 7S', each of which contains sequences from the spacer region between 25S and 18S rRNA . 35S pre-rRNA accumulates, while the level of the 20S and 27S rRNA processing intermediates is depressed . Pulse-chase analysis demonstrates that the processing of 35S pre-rRNA is slowed . The defect in the mutant appears to be at the first processing step, which generates 20S and 27S rRNA . 7S' RNA is a form of 5.8S RNA whose 5' end is extended by 149 nucleotides to a position just 5 nucleotides downstream of the normal cleavage site that produces 20S and 27S rRNA . 7S' RNA can assemble into 60S ribosomal subunits, but such subunits are relatively ineffective in joining polyribosomes . A single lesion is responsible for the pre-rRNA processing defect and the temperature sensitivity . The affected gene is designated RRP2.

Nucleic Acids Res, 1991 Sep 25, 19(18), 4867 - 72
Cloning DPB3, the gene encoding the third subunit of DNA polymerase II of Saccharomyces cerevisiae; Araki H et al.; DNA polymerase II purified from Saccharomyces cerevisiae contains polypeptides with apparent molecular masses of greater than 200, 80, 34, 30 and 29 kDa, the two largest of which (subunits A and B) are encoded by the essential genes POL2 and DPB2 . By probing a lambda gt11 expression library of yeast DNA with antiserum against DNA polymerase II, we isolated a single gene, DPB3, that encodes both the 34- and 30-kDa polypeptides (subunit C and C') . The nucleotide sequence of DPB3 contained an open reading frame encoding a 23-kDa protein, significantly smaller than the observed molecular masses, 34- or 30-kDa, which might represent post-translationally modified forms of the DPB3 product . The predicted amino acid sequence contained a possible NTP-binding motif and a glutamate-rich region . NTP-binding motif and a glutamate-rich region . A dpb3 deletion mutant (dpb3 delta) was viable and yielded a DNA polymerase II lacking the 34- and 30-kDa polypeptides . dpb3 delta strains exhibited an increased spontaneous mutation rate, suggesting that the DPB3 product is required to maintain fidelity of chromosomal replication . Since a fifth, 29-kDa polypeptide was present in DNA polymerase II preparations from wild-type cell extracts throughout purification, the subunit composition appears to be A, B, C (or C and C') and D . The 5' nontranscribed region of DPB3 contained the MulI-related sequence ACGCGA, while the 0.9-kb DPB3 transcript accumulated periodically during the cell cycle and peaked at the G1/S boundary . The level of DPB3 transcript thus appears to be under the same cell cycle control as those of POL2, DPB2 and other DNA replication genes . DPB3 was mapped to chromosome II, 30 cM distal to his7.

Nucleic Acids Res, 1991 Sep 25, 19(18), 4949 - 53
Direct introduction and transient expression of capped and non-capped RNA in Saccharomyces cerevisiae; Russell PJ et al.; We report the introduction of functional RNA molecules into yeast spheroplasts . Plasmids containing the firefly luciferase coding region were transcribed to yield RNAs suitable for introduction into yeast cells and direct assay of their translation products . The 5' noncoding regions of the RNAs were derived either from the 5' noncoding regions of firefly luciferase, poliovirus, or yeast virus-like-particle (VLP) L-A or M1 RNAs . Capped and non-capped mRNAs were made by T7 RNA polymerase-directed transcription and introduced into yeast spheroplasts . The peak time of luciferase transient expression from introduced RNAs was 2-4 h after their introduction . In contrast, transient expression of luciferase from a non-replicative, luciferase-encoding plasmid introduced into the cells was maximal at 16 h . For capped mRNAs, luciferase activity increased linearly with transcript amount for both yeast and human (HeLa) cells . Although non-capped luciferase mRNAs were expressed more efficiently following introduction into yeast than into HeLa cells, the 5' noncoding sequences from yeast double-stranded (ds)RNA VLP RNAs conferred no greater apparent cap-independence than non-VLP RNA sequences in this transient expression assay . The RNA transient expression system will allow the study of translation of capped and non-capped RNAs in yeast cells and of the replicative cycle of yeast virus-like RNA genomes.

J Biol Chem, 1991 Sep 25, 266(27), 18287 - 93
Molecular characterization of Saccharomyces cerevisiae URA6 gene . DNA sequence, mutagenesis analysis, and cell cycle regulation relevant to its suppression mechanism to cdc8 mutation; Jiang ZR et al.; Yeast SOC8 DNA fragment was isolated as a wild type dominant suppressor of cdc8 mutation . We have used Bal31 deletion analysis to define the minimal 1 kilobase HpaI-NcoI DNA element required for complementing the cdc8 mutation . The complementing sequence harbored a multicopy plasmid also enhanced by uridine monophosphate kinase in crude extracts . DNA sequence analysis revealed an open reading frame encoding a protein with a molecular weight of 24,949 . Since our SOC8 sequence was identical to that of URA6 gene, which encodes uridine monophosphate kinase, we conclude that SOC8 is allelic with URA6, and we use the term URA6 hereafter . Northern blotting experiments showed that the size of mRNA is about 0.9 kibobases . Primer extension experiments showed multiple transcriptional starting sites primarily located at -160 The size and the deduced amino acid composition are consistent with information obtained from purified uridine monophosphate kinase . Thus, both molecular genetic and biochemical evidence supports a notion that the URA6 is SOC8 encoding a yeast uridine monophosphate kinase . Mutagenesis analysis of its putative nucleotide-binding site, altering essential lysine to glutamic acid, resulted in loss of its uridine monophosphate kinase activity . Complementation analysis studies indicated that the mutated ura6 gene abolished its ability to complement ura6 mutant cells; nor could it suppress cdc8 mutation . Unlike CDC8, the mRNA level of the URA6 gene did not fluctuate throughout the cell cycle; presumably, the temporal order of these two enzymatic activities might be different during cell cycle progression . These data may explain an incomplete suppression of cdc8 by URA6, as previously observed . Taken together, the results support our previous speculation that the suppression of the cdc8 mutation mechanisms by URA6 is due to the provision of the trans-acting dTMP kinase activity to complement the cdc8 defect.

FEBS Lett, 1991 Sep 23, 290(1-2), 43 - 8
In vitro activation of the Saccharomyces cerevisiae Ras/adenylate cyclase system by glucose and some of its analogues; Pardo LA et al.; Using crude membrane preparations of Saccharomyces cerevisiae, we have demonstrated that glucose and glucose analogues which are not efficiently phosphorylated activate the guanine nucleotide-dependent adenylate cyclase in vitro . The activation appears to be mediated by the Ras proteins . Moreover, data are presented indicating that glucose and its analogues activate adenylate cyclase by stimulating the exchange of guanine nucleotides at its regulatory component . Thus, it has been possible to show the action of a physiological effector on the nucleotide exchange reaction in a member of the ras superfamily.

FEBS Lett, 1991 Sep 23, 290(1-2), 38 - 42
Glycogen hyperaccumulation in Saccharomyces cerevisiae ras2 mutant . A biochemical study; Fernandez-Banares I et al.; The mechanism by which yeast ras2 mutant hyperaccumulates glycogen has been investigated . Total glycogen synthase activity was between 2.5 and 1.3 times higher in the ras2 mutant than in an isogenic strain . In addition, while in the normal strain the glycogen synthase activation state decreased along the exponential phase, in the mutant strain the opposite behaviour was observed: glycogen synthase activation state rose continuously reaching full activation at the beginning of the stationary phase . Glycogen phosphorylase a activity was up to 40 times higher in the mutant than in the normal strain . Glucose 6-phosphate and fructose 2,6-bisphosphate levels were slightly more elevated in the mutants . The increase in total glycogen synthase and, particularly, the full activation of this enzyme may explain glycogen hyperaccumulation in the ras2 mutant even in the presence of elevated levels of glycogen phosphorylase a.

J Biol Chem, 1991 Sep 15, 266(26), 17537 - 44
TIP 1, a cold shock-inducible gene of Saccharomyces cerevisiae; Kondo K et al.; Using differential hybridization, genes whose expression is induced at low temperatures were identified in yeast Saccharomyces cerevisiae . One of these genes that corresponds to an mRNA that is induced 6-8-fold within 2 h after shifting the culture temperature from 30 to 10 degrees C was further characterized . Surprisingly, its expression was also induced by heat shock, and thus the gene was designated TIP 1 (temperature shock-inducible protein gene) . Southern hybridization analysis demonstrated that there are several genes homologous to the TIP 1 gene on the yeast genome . A TIP 1 disruption mutation exerted an observable effect neither on growth nor on viability after being exposed to freezing temperatures . The TIP 1 gene encodes a protein of 210 amino acid residues with a molecular weight of 20,727, containing 20.0% alanine and 23.3% serine . The TIP 1 protein has a typical signal peptide at the amino-terminal end and an extremely hydrophobic sequence at the carboxyl-terminal end . The TIP 1 protein is thus likely to be secreted across the membrane and anchored on the outside surface of the plasma membrane . These results indicate that the TIP 1 protein is a new type of stress inducible protein in yeast.

Nucleic Acids Res, 1991 Sep 11, 19(17), 4761 - 6
Base mismatch-specific endonuclease activity in extracts from Saccharomyces cerevisiae; Chang DY et al.; An endonuclease activity (called MS-nicking) for all possible base mismatches has been detected in the extracts of yeast, Saccharomyces cerevisiae . DNAs with twelve possible base mismatches at one defined position are cleaved at different efficiencies . DNA fragments with A/G, G/A, T/G, G/T, G/G, or A/A mismatches are nicked with greater efficiencies than C/T, T/C, C/A, and C/C . DNA with an A/C or T/T mismatch is nicked with an intermediate efficiency . The MS-nicking is only on one particular DNA strand, and this strand disparity is not controlled by methylation, strand break, or nature of the mismatch . The nicks have been mapped at 2-3 places at second, third, and fourth phosphodiester bonds 5' to the mispaired base; from the time course study, the fourth phosphodiester bond probably is the primary incision site . This activity may be involved in mismatch repair during genetic recombination.

J Mol Biol, 1991 Sep 5, 221(1), 225 - 37
Identification and functional analysis of the nuclear localization signals of ribosomal protein L25 from Saccharomyces cerevisiae; Schaap PJ et al.; The regions of the large subunit ribosomal protein L25 from Saccharomyces cerevisiae responsible for nuclear localization of the protein were identified by constructing fusion genes encoding various segments of L25 linked to the amino terminus of beta-galactosidase . Indirect immunofluorescence of yeast cells expressing the fusions demonstrated that amino acid residues 1 to 17 as well as 18 to 41 of L25 promote import of the reporter protein into the nucleus . Both nuclear localization signal (NLS) sequences appear to consist of two distinct functional parts: one showed relatively weak nuclear targeting activity, whereas the other considerably enhances this activity but does not promote nuclear import by itself . Microinjection of in vitro prepared intact and N-terminally truncated L25 into Xenopus laevis oocytes demonstrated that the region containing the two NLS sequences is indeed required for efficient nuclear localization of the ribosomal protein . This conclusion was confirmed by complementation experiments using a yeast strain that conditionally expresses wild-type L25 . The latter experiments also indicated that amino acid residues 1 to 41 of L25 are required for full functional activity of yeast 60 S ribosomal subunits . Yeast cells expressing forms of L25 that lack this region are viable, but show impaired growth and a highly abnormal cell morphology.

J Biol Chem, 1991 Sep 5, 266(25), 16808 - 12
The periodically expressed TMP1 gene of Saccharomyces cerevisiae is subject to START-dependent and START-independent regulation; Poon PP et al.; The rate of transcription of the Saccharomyces cerevisiae gene encoding thymidylate synthase (TMP1) fluctuates periodically during the cell cycle . The simplest explanation for this pattern of expression is that transcription occurs during the late G1 and early S phases and does not occur during other stages of the cell cycle . In this report, however, we show that TMP1 is subject to regulation that results in at least three different levels of expression: essentially nondetectable expression during stationary phase (G0), moderate level expression in START-arrested growing cells (START-independent), and a high level of expression in proliferating cells (START-dependent) . Our analysis also shows that upstream elements important for START-independent expression and required for START-dependent expression are located within a 37-base region.

FEBS Lett, 1991 Sep 2, 289(1), 54 - 8
Purification and partial characterization of ubiquitin-activating enzyme from Saccharomyces cerevisiae; Hoefer M et al.; Ubiquitin-activating enzyme was purified from the yeast Saccharomyces cerevisiae by covalent affinity chromatography on ubiquitin-Sepharose followed by HPLC anion-exchange chromatography . Enzyme activity was monitored by the ubiquitin-dependent ATP: 32PPi exchange assay . The purified enzyme has a specific activity of 1.5 mumol 32PPi incorporated into ATP.min-1.mg-1 at 37 degrees C and pH 7.0 under standard conditions for substrate concentrations as described by Ciechanover et al . (1982) J . Biol . Chem . 257, 2537-2542 . The catalytic activity showed a maximum at pH 7.0 . Its molecular weight both in non-denaturing and in SDS-gel electrophoresis was estimated to be 115 kDa, suggesting a monomeric form . The isoelectric point determined by gel electrofocusing was approximately 4.7 . Two protein bands differing slightly in electrophoretic mobility could be distinguished when SDS gels were loaded with very small amounts of purified E1 and immunoblotted, the one with higher molecular weight being clearly predominant . The same two bands were also found in anti-E1 immunoblots of crude yeast lysates prepared under broad protease inhibition.

Mutat Res, 1991 Sep-Oct, 250(1-2), 239 - 49
The influence of solvent stress on MMS-induced genetic change in Saccharomyces cerevisiae; Zimmermann FK et al.; MMS induced mitotic recombination but not mitotic chromosome loss when tested in pure form in strain D61.M of Saccharomyces cerevisiae, confirming previous results of Albertini (1991), whereas in Aspergillus nidulans it also induced chromosomal malsegregation in addition to mitotic recombination (Kafer, 1988) . However, induction of mitotic chromosome loss was observed in combination with strong inducers of chromosome loss such as the aprotic polar solvents ethyl acetate and to a lesser extent methyl ethyl ketone but not with gamma-valerolactone and propionitrile . In addition to this, 4 solvents, dimethyl formamide, dimethyl sulfoxide, dioxane and pyridine, enhanced the MMS-induced mitotic recombination in strain D61.M . An enhancement of MMS-induced mitotic recombination and reverse mutation could be demonstrated for ethyl acetate and gamma-valerolactone in yeast strain D7.

J Steroid Biochem Mol Biol, 1991 Sep, 39(3), 291 - 7
High level expression of biologically active estrogen receptor in Saccharomyces cerevisiae; McDonnell DP et al.; Biochemical over-expression of the human estrogen receptor was achieved using a Saccharomyces cerevisiae expression system . The receptor was produced as a novel ubiquitin fusion protein . This fusion protein is short lived in the cell and is processed to produce unfused receptor shortly after folding . Conventional high copy expression plasmids produced receptor to about 0.04% of the total soluble protein . By incorporating a defective leu2 allele into these vectors, an additional 5-fold increase in receptor production was obtained . The recombinant receptor was undergraded, soluble and biologically active . Conventional methods of disrupting cells using glass beads had a detrimental effect on the ability of the receptor to bind hormone . Enzymatic digestion of the cell wall followed by hypotonic shock liberates the receptor that quantitatively binds estrogen.

Mol Cell Biol, 1991 Sep, 11(9), 4726 - 31
A codon change in beta-tubulin which drastically affects microtubule structure in Drosophila melanogaster fails to produce a significant phenotype in Saccharomyces cerevisiae; Praitis V et al.; The relative uniformity of microtubule ultrastructure in almost all eukaryotic cells is thought to be a consequence of the conserved elements of tubulin sequence . In support of this idea, a mutation in a beta-tubulin gene of Drosophila melanogaster, occurring at a highly conserved position, produces U-shaped microtubules, suggesting a defect in either nucleation or packing during assembly (M . T . Fuller, J . H . Caulton, J . A . Hutchens, T . C . Kaufman, and E . C . Raff, J . Cell Biol . 104:385-394, 1987, and J . E . Rudolph, M . Kimble, H . D . Hoyle, M . A . Subler, and E . C . Raff, Mol . Cell . Biol . 7:2231-2242, 1987) . Surprisingly, we find that introducing the same mutation into the sole beta-tubulin gene of Saccharomyces cerevisiae has virtually no consequences for microtubule structure or function in that organism.

Biochem J, 1991 Sep 1, 278 ( Pt 2), 393 - 7
Characteristics of Nicotiana tabacum nitrate reductase protein produced in Saccharomyces cerevisiae; Truong HN et al.; Tobacco nitrate reductase (NR) produced in yeast retains cytochrome c reductase activity, but not NR activity . Biochemical data suggest that the haem and FAD domains are functional, and that the molybdenum cofactor (MoCo) domain is inactive owing to the absence of MoCo in yeast . The native form of the produced NR is dimeric . Thus MoCo is not involved in NR dimerization in higher plants, contrary to current assumptions.

J Cell Biol, 1991 Sep, 114(6), 1167 - 78
PAS3, a Saccharomyces cerevisiae gene encoding a peroxisomal integral membrane protein essential for peroxisome biogenesis; Hohfeld J et al.; Saccharomyces cerevisiae pas3-mutants are described which conform the pas-phenotype recently reported for the peroxisomal assembly mutants pas1-1 and pas2 (Erdmann, R., M . Veenhuis, D . Mertens, and W.-H Kunau, 1989, Proc . Natl . Acad . Sci . USA . 86:5419-5423) . The isolation of pas3-mutants enabled us to clone the PAS3 gene by functional complementation . DNA sequence analysis revealed a 50.6-kD protein with at least one domain of sufficient length and hydrophobicity to span a lipid bilayer . To verify these predictions antibodies were raised against a truncated portion of the PAS3 coding region overexpressed in E . coli . Pas3p was identified as a 48 kD peroxisomal integral membrane protein . It is shown that a lack of this protein causes the peroxisome-deficient phenotype and the cytosolic mislocalization of peroxisomal matrix enzymes . Based on protease digestion experiments Pas3p is discussed to be anchored in the peroxisomal membrane by its amino-terminus while the bulk of the molecule is exposed to the cytosol . These findings are consistent with the possibility that Pas3p is one component of the peroxisomal import machinery.

Eur J Biochem, 1991 Sep 1, 200(2), 337 - 43
Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae . Study of its functional organisation by deletion analysis; Eriani G et al.; Aspartyl-tRNA synthetase (AspRS) from yeast, a homodimer of 125 kDa, was shortened by several residues from the C- and N-termini, via site-directed mutagenesis, to examine the contribution of the removed peptides to the enzyme properties . This study showed that the N-terminal sequence up to amino acid 70 (which confers peculiar ionic properties to the protein) is dispensable for activity . Domains located beyond amino acid 70 appeared to have increasing catalytic importance; the removal of 80 or 90 residues affected the Km values for ATP and deletions of 101 or 140 amino acids profoundly modified the physiochemical properties of AspRS, and by consequence, its structural organisation (extraction of the mutated proteins out of the cells required the presence of SDS) . On the C-terminal side, very limited modifications readily affected the enzyme properties . Deletion of as few as three residues increased the Km for ATP and reduced the aminoacylation kcat as well as the thermostability of the adenylate synthesis activity; the kcat of this step was impaired after deletion of two further residues . Finally, shortening the C-terminal decapeptide completely inactivated AspRS, whilst affecting neither its affinity for tRNAAsp nor its dimerisation capacity . These data reveal the role of the C-terminal decapeptide as a determinant in both reactions catalysed by AspRS . This peptide is involved in ATP binding, stabilising the functional conformation of the amino-acid-activating domain and probably maintaining the tRNA-acceptor end in a reactive position with regard to the activated amino acid.

J Bacteriol, 1991 Sep, 173(18), 5918 - 20
Null mutants of Saccharomyces cerevisiae Cu,Zn superoxide dismutase: characterization and spontaneous mutation rates; Gralla EB et al.; Deletion-replacement mutations of the Saccharomyces cerevisiae Cu,Zn superoxide dismutase gene were constructed . They were exquisitely sensitive to redox cycling drugs and showed slight sensitivity to other agents . The aerobic spontaneous mutation rate was three- to fourfold higher in sod1 delta 1 mutants, while the anaerobic rate was similar to that of the wild type.

Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7585 - 9
Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae; Schiestl RH et al.; DNA fragments (generated by BamHI treatment) with no homology to the yeast genome were transformed into Saccharomyces cerevisiae . When the fragments were transformed in the presence of the BamHI enzyme, they integrated into genomic BamHI sites . When the fragments were transformed in the absence of the enzyme, they integrated into genomic G-A-T-C sites . Since the G-A-T-C sequence is present at the ends of BamHI fragments, this results indicates that four base pairs of homology are sufficient for some types of mitotic recombination.

Mol Cell Biol, 1991 Sep, 11(9), 4555 - 60
Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae; Woontner M et al.; We report an improved in vitro transcription system for Saccharomyces cerevisiae . Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M . Woontner and J . A . Jaehning, J . Biol . Chem . 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts . We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation . Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16) . Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription . Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.

Glycobiology, 1991 Sep, 1(4), 387 - 91
Specificity of the mannosyltransferase which initiates outer chain formation in Saccharomyces cerevisiae; Reason AJ et al.; The in vitro specificity of the alpha 1-6 mannosyltransferase that initiates outer chain formation in Saccharomyces cerevisiae (Romero and Herscovics, J . Biol . Chem., 264, 1946-1950, 1989) was reassessed by fast atom bombardment mass spectrometry (FAB-MS) . A particulate fraction from the mnn1 mutant was incubated with GDP-mannose and either Man9GlcNAc (M9T) isolated from thyroglobulin or Man8GlcNAc (M8Y) obtained by treatment of the M9T with the yeast specific mannosidase . The Man10GlcNAc (M10Y) and Man9GlcNAc (M9Y) oligosaccharides thus obtained, and the substrate oligosaccharides, were peracetylated or perdeuteroacetylated and submitted to FAB-MS using meta-nitrobenzylalcohol as the matrix . The latter was chosen as the matrix because it enhances the abundance of high-mass-fragment ions of peracetylated oligosaccharides and thereby facilitates the assignment of branching patterns . The results indicate that the alpha 1-6 mannosyltransferase catalyses the addition of mannose to the alpha 1-3 mannose residue, and thus provide additional new evidence to support the revised structure of yeast mannoproteins proposed by Hernandez et al . (J . Biol . Chem., 264, 11849-11856, 1989) . {formula: see text} where Gn is N-acetylglucosamine, M is mannose and M is mannose added by the enzyme.

Glycobiology, 1991 Sep, 1(4), 367 - 73
Partial purification of a mannosyltransferase involved in the O-mannosylation of glycoproteins from Saccharomyces cerevisiae; Sharma CB et al.; The mannosyltransferase that catalyses the transfer of mannose from dolichyl-phosphate-mannose (Dol-P-Man) to the hydroxyl group of serine/threonine residues in the acceptor peptide (Tyr-Asn-Pro-Thr-Ser-Val) was partially purified approximately 150-fold from the microsomal membrane fraction of Saccharomyces cerevisiae . The membrane-bound enzyme was solubilized with 0.5% Triton X-100 at a protein:detergent ratio of 2:1, and was then purified by ion-exchange chromatography on DEAE-cellulose, followed by hydroxyapatite column chromatography . The partially purified enzyme had a pH optimum of 7.2 and required Mg2+ at an optimum concentration of 10 mM for activity . The apparent mol . wt of the enzyme, as estimated by gel filtration on Sephacryl S-300, was approximately 125 kDa . The activity of the partially purified enzyme was greatly stimulated by phosphatidylcholine (PC), while other naturally occurring phosphoglycerides had no significant effect . The extent of activation of mannosyltransferase activity was greatly affected by the number of carbons and the degree of saturation/unsaturation of the fatty acid substituents, as well as by their position on the glycerol moiety of the PC molecule . Maximum stimulation of the mannosyltransferase activity was induced by a PC derivative in which both sn-1 and sn-2 positions on the glycerol moiety were occupied by C12:0 fatty acids . In general, mannosyltransferase was found to exhibit greater specificity for the L-alpha-PC derivatives in which the sn-2 position of the glycerol contained a saturated fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Biol Int Rep, 1991 Sep, 15(9), 863 - 73
Biosynthesis of glycophosphoinositol anchors in Saccharomyces cerevisiae; Conzelmann A et al.; Numerous membrane glycoproteins of Saccharomyces cerevisiae are posttranslationally modified by the addition of a glycophosphatidylinositol (GPI) . These proteins can be detected most easily by metabolic labelling of yeast cells with 3H-myoinositol or 3H-palmitate . This report summarizes what little is known about the identity, biosynthesis and cellular localization of GPI-modified glycoproteins in Saccharomyces cerevisiae as well as what could be learned from the system with respect to the biosynthesis of GPI's in general.

Mol Endocrinol, 1991 Sep, 5(9), 1239 - 45
The tissue-specific mammalian transcription factor, Pit-1, activates transcription in Saccharomyces cerevisiae; Ding Y et al.; Pit-1 is a tissue-specific transcription factor which binds to specific DNA sequences within 5' flanking regions of the PRL and GH genes and activates the transcription of these genes . Previous studies have shown that expression of Pit-1 is necessary to activate transcription from the PRL or GH promoters in heterologous mammalian cells . In the present study the ability of Pit-1 expression vectors to activate expression of reporter genes in Saccharomyces cerevisiae was examined . The test system used Pit-1 expression vectors and an indicator plasmid containing multiple copies of a Pit1-binding site as a replacement for the upstream activator sequence of the CYC1 promoter . Significant activation of indicator plasmid expression was detected only in the presence of functional Pit-1 expression vectors . In both mammalian and yeast cells, amino-terminal deletions of the Pit-1 coding sequence produced similar and gradual loss of transcriptional activation . This finding indicates that similar or identical regions of Pit-1 are required for transcriptional activation in mammalian and yeast cells . Although synthetic DNA elements containing multiple copies of a single Pit-1-binding site were sufficient to permit Pit-1-mediated transcriptional activation in both yeast and mammalian cells, DNA fragments representing the proximal region or distal enhancer region of the PRL gene were transcriptionally active only in mammalian cells . These studies establish the ability of Pit-1 to stimulate transcription in the absence of other tissue-specific factors and provide a system for further genetic studies of Pit-1 structure/function relationships as well as evaluation of target sequences necessary for Pit-1 action.

J Membr Biol, 1991 Sep, 123(3), 261 - 8
Characterization of constitutive exocytosis in the yeast Saccharomyces cerevisiae; Lew DJ et al.; Constitutive exocytosis was investigated in the yeast Saccharomyces cerevisiae using temperature-sensitive mutant (sec) strains which do not allow vesicle fusion to the plasma membrane at the restrictive temperature . Secretory vesicles were accumulated in the cell at the restrictive temperature and then protein synthesis was blocked with cycloheximide . Upon returning the cells to the permissive temperature the contents of the accumulated vesicles were secreted . This allowed the study of constitutive exocytosis independent of the processes responsible for vesicular biosynthesis . Neither the kinetics nor magnitude of exocytosis were affected by removal of external Ca2+ or perturbations of cytosolic Ca2+ . This suggests that in those systems where calcium is required for exocytosis it is a regulatory molecule and not part of the mechanism of membrane fusion . Release occurred over a very broad range of pH and in media with different ionic compositions, suggesting that ionic and potential gradients across the plasma membrane play no role in exocytosis in yeast . High osmolarity inhibited the rate, but not the extent, of release . A novel inhibitory effect of azide was detected which occurred only at low pH . Vanadate also inhibited release in a pH-independent manner . Secretion occurred at the same rate in cells with or without accumulated vesicles . This infers a rate-limiting step following vesicle accumulation, perhaps a limiting number of release sites on the plasma membrane.

Biochem Int, 1991 Sep, 25(2), 339 - 48
Characterization of mRNAs and coding potential of the PET54 gene from Saccharomyces cerevisiae; Burke KA et al.; The nuclear PET54 gene in yeast controls expression of two mitochondrial genes: COX1 at the level of pre-mRNA splicing and COX3 at the level of mRNA translation . Two size classes (1.6 and 1.1 kb) of transcripts that contain the PET54 coding region are produced in vivo . Relative to the majority of yeast mRNAs analyzed so far, the 5' untranslated leader region of the 1.6 kb transcript is unusually long (254 bases), while that for the major 1.1 kb transcript is unusually short (1 base) . The majority of each class of PET54 mRNA was associated with polysomes in vivo . The possibility that two polypeptides are produced in vivo from the 1.1 kb PET54 mRNA was raised by the work of Sedman et al . {J . Virol . 64: 453-457, 1990}, which showed that translation initiation at a downstream AUG occurs with increased efficiency when the upstream AUG is located very close to the 5' end of the mRNA . However, two sensitive assays for production of a second polypeptide, which is predicted to be 22 kD, were employed and no second polypeptide was detected . Furthermore, a nonsense mutation introduced near the beginning of the PET54 open reading frame abolished both COX1 and COX3 gene expression . These results indicate that the PET54 gene encodes predominantly a single functional polypeptide that is employed for expression of both the COX1 and COX3 genes of mitochondrial DNA.

Eur J Biochem, 1991 Sep 1, 200(2), 359 - 67
The role of the internal hydrogen bond network in first-order protein electron transfer between Saccharomyces cerevisiae iso-1-cytochrome c and bovine microsomal cytochrome b5; Whitford D et al.; An internal water molecule (designated WAT166) is found in iso-1-cytochrome c which is part of a redox-state-dependent hydrogen bond network . The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site-directed mutagenesis leading to structural perturbations and associated changes in redox potential . Using saturation transfer 1H-NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso-1-cytochromes c in which the position of this water molecule is altered . In particular, the reverse electron transfer rate is measured within a complex formed between either wild-type or variant yeast iso-1-cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5 . For three variants of yeast iso-1-cytochrome c the rate constants measured by saturation transfer are wild-type (Asn52, E0 = 270 mV, kex = 0.3 s-1), Asn52----Ala (E0 = 240 mV, kex = 0.6 s-1), Asn52----Ile (E0 = 220 mV, kex = 1.0 s-1) . The first-order rates are compared with that of a fourth variant Phe82----Gly which has been measured previously (E0 = 220 mV, kex = 0.7 s-1) . An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis . So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein . Studies of the activation parameters suggests that a variation of temperature changes both delta G* and also the prefactor . These data are discussed in terms of models involving dynamic molecular recognition between proteins.

J Bacteriol, 1991 Sep, 173(17), 5275 - 9
Isolation and characterization of a dinucleoside triphosphatase from Saccharomyces cerevisiae; Brevet A et al.; An enzyme able to cleave dinucleoside triphosphates has been purified 3,750-fold from Saccharomyces cerevisiae . Contrary to the enzymes previously shown to catabolize Ap4A in yeast, this enzyme is a hydrolase rather than a phosphorylase . The dinucleoside triphosphatase molecular ratio estimated by gel filtration is 55,000 . Dinucleoside triphosphatase activity is strongly stimulated by the presence of divalent cations . Mn2+ displays the strongest stimulating effect, followed by Mg2+, Co2+, Cd2+, and Ca2+ . The Km value for Ap3A is 5.4 microM (50 mM Tris-HCl {pH 7.8}, 5 mM MgCl2, and 0.1 mM EDTA; 37 degrees C) . Dinucleoside polyphosphates are substrates of this enzyme, provided that they contain more than two phosphates and that at least one of the two bases is a purine (Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, m7Gp3A, m7Gp3G, Ap4A, Ap4G, Ap4C, Ap4U, Gp4G, and Ap5A are substrates; AMP, ADP, ATP, Ap2A, and Cp4U are not) . Among the products, a nucleoside monophosphate is always formed . The specificity of cleavage of methylated dinucleoside triphosphates and the molecular weight of dinucleoside triphosphatase indicate that this enzyme is different from the mRNA decapping enzyme previously characterized (A . Stevens, Mol . Cell . Biol . 8:2005-2010, 1988).

Mol Cell Biol, 1991 Sep, 11(9), 4455 - 65
Physiological and genetic analysis of the carbon regulation of the NAD-dependent glutamate dehydrogenase of Saccharomyces cerevisiae; Coschigano PW et al.; We found that cells of Saccharomyces cerevisiae have an elevated level of the NAD-dependent glutamate dehydrogenase (NAD-GDH; encoded by the GDH2 gene) when grown with a nonfermentable carbon source or with limiting amounts of glucose, even in the presence of the repressing nitrogen source glutamine . This regulation was found to be transcriptional, and an upstream activation site (GDH2 UASc) sufficient for activation of transcription during respiratory growth conditions was identified . This UAS was found to be separable from a neighboring element which is necessary for the nitrogen source regulation of the gene, and strains deficient for the GLN3 gene product, required for expression of NAD-GDH during growth with the activating nitrogen source glutamate, were unaffected for the expression of NAD-GDH during growth with activating carbon sources . Two classes of mutations which prevented the normal activation of NAD-GDH in response to growth with nonfermentable carbon sources, but which did not affect the nitrogen-regulated expression of NAD-GDH, were found and characterized . Carbon regulation of GDH2 was found to be normal in hxk2, hap3, and hap4 strains and to be only slightly altered in a ssn6 strain; thus, in comparison with the regulation of previously identified glucose-repressed genes, a new pathway appears to be involved in the regulation of GDH2.

Gene, 1991 Aug 30, 105(1), 83 - 90
Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae; Lopes TS et al.; Targeted integration of the yeast plasmid pMIRY2 into the ribosomal DNA (rDNA) of Saccharomyces cerevisiae by homologous recombination results in transformants carrying 100-200 copies of the plasmid per cell which are stably maintained over a large number of generations {Lopes et al., Gene 79 (1989) 199-206} . These properties make pMIRY2 an attractive vector for high-level production of (heterologous) proteins by yeast cells . We have investigated the mechanism underlying high-copy-number (hcn) integration of pMIRY-type plasmids and show that either targeting to a location outside the rDNA locus or use of the wild-type LEU2, instead of the deficient LEU2d gene, as selection marker reduces the copy number to the low value characteristic of standard integrating (YIp-type) yeast plasmids . Further experiments demonstrate that the hcn of pMIRY-type plasmids is achieved by amplification of a small number of copies initially integrated into the rDNA locus . Amplification depends upon the strong selection pressure created by the extremely low expression of the deficient LEU2d gene, but not on the presence of this gene per se . The hcn integration also occurs when either the TRP1 or URA3 gene is used as the selection marker, provided expression of the marker gene is severely curtailed, e.g., by removal of most of its 5'-flanking region.

Gene, 1991 Aug 30, 105(1), 139 - 40
Nucleotide sequence of the RAD57 gene of Saccharomyces cerevisiae; Kans JA et al.; We have determined the nucleotide (nt) sequence of the RAD57 gene of Saccharomyces cerevisiae . RAD57 contains an open reading frame of 1380 bp . The deduced amino acid sequence of 460 residues contains a potential nt-binding sequence and shows significant similarity to the preliminary sequence of RAD51.

Gene, 1991 Aug 30, 105(1), 73 - 81
Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae; Hickey DR et al.; Genes encoding tuna, pigeon, and horse cytochromes c were constructed with synthetic oligodeoxyribonucleotides having preferred codons and portions of the iso-1-cytochrome c-encoding gene from the yeast Saccharomyces cerevisiae . The genes were ligated into an expression vector, which contains the normal 5'- and 3'-untranslated regions of the yeast iso-1-cytochrome c gene, and were integrated in single copy into the chromosome . Yeast strains were also constructed with multiple integrated copies of the pigeon gene . The heterologous and normal mRNA levels of the single-copy strains were equivalent . Although the N-terminal methionines were completely cleaved in the heterospecific proteins, the levels of trimethylation of Lys72 and acetylation of N-terminal glycines ranged from 39-78% and 10-70%, respectively . Horse cytochrome c was produced at a nearly normal level, whereas the pigeon and tuna cytochromes c were produced at approx . 40% of the normal levels . The levels of the cytochromes c and growth of the mutant yeast strains indicated that the heterospecific cytochromes c had approx . 50% specific activity in vivo.

FEBS Lett, 1991 Aug 19, 288(1-2), 86 - 90
Acquisition of thermotolerance in Saccharomyces cerevisiae without heat shock protein hsp 104 and in the absence of protein synthesis; De Virgilio C et al.; Acquisition of thermotolerance in response to a preconditioning heat treatment at 40 degrees C was studied in mutants of the yeast Saccharomyces cerevisiae lacking a specific heat shock protein or the ability to synthesize proteins at 40 degrees C . A mutant carrying a deletion of heat shock protein hsp 104 and the corresponding wildtype strain were both highly sensitive to heat stress at 50.4 degrees C without preconditioning but both acquired almost the same level of thermotolerance after 60 min of preconditioning . Both strains showed equal induction of trehalose-6-phosphate synthase and accumulated equal levels of trehalose during the treatment . The conditional mutant ts--187 synthesized no proteins during the preconditioning heat treatment but nevertheless acquired thermotolerance, albeit to a lesser degree than the corresponding wildtype strain . Induction of trehalose-6-phosphate synthase and accumulation of trehalose were reduced to a similar extent . These results show that acquisition of thermotolerance and accumulation of trehalose are closely correlated during heat preconditioning and are modulated by protein synthesis but do not require it.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7308 - 12
Two essential components of the Saccharomyces cerevisiae transcription factor TFIIIB: transcription and DNA-binding properties; Kassavetis GA et al.; RNA polymerase III transcription factor TFIIIB from Saccharomyces cerevisiae contains at least two polypeptides, with apparent masses of 90 and 70 kDa, that were previously identified by photocrosslinking to DNA . It is shown here that TFIIIB can be chromatographically separated into two components, each of which is required for efficient tRNA gene transcription . DNA-protein photocrosslinking experiments show these two components separately contain the 90- and 70-kDa TFIIIB-specific polypeptides . The 70-kDa component forms a heparin-sensitive complex with transcription factor TFIIIC and DNA, stabilizes TFIIIC interaction with the tRNA gene promoter elements, and protects against DNase I digestion in the 3' portion of the upstream DNA sequence that is occupied by TFIIIB . The 90-kDa component of TFIIIB, which only detectably interacts with the TFIIIC-DNA complex when the 70-kDa component is also present, generates the complete DNase I protection pattern of TFIIIB and bestows heparin-insensitivity on the TFIIIB-DNA complex . The resolution of TFIIIB into two functional components further defines the probable steps and interactions involved in the formation of stable transcription complexes.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7026 - 30
An approach for isolation of mutants defective in 35S ribosomal RNA synthesis in Saccharomyces cerevisiae; Nogi Y et al.; We have developed a method to isolate mutants of Saccharomyces cerevisiae that are primarily defective in the transcription of 35S ribosomal RNA (rRNA) genes by RNA polymerase I . The method uses a system in which the 35S rRNA gene is fused to the GAL7 promoter and is transcribed by RNA polymerase II under control of the GAL regulatory system . Chromosomal mutations affecting components specifically involved in synthesis of 35S rRNA by RNA polymerase I can be suppressed by this hybrid gene in the presence of inducer (galactose) but not in its absence . We looked for mutants the growth of which depended on the presence of plasmid expressing the hybrid gene . For this purpose, we used a red/white-colony color assay as the initial screen followed by a test for galactose-dependent growth . We have thus isolated many mutants and identified at least nine genes (RRN1-RRN9) involved in 35S rRNA synthesis, two of which correspond to known RNA polymerase I subunit genes RPA190 and RPA135.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 6986 - 90
Expression of RNase P RNA in Saccharomyces cerevisiae is controlled by an unusual RNA polymerase III promoter; Lee JY et al.; The RNA subunit of Saccharomyces cerevisiae nuclear RNase P is encoded by a single-copy, essential gene, RPR1 . The 369-nucleotide mature form of the RNA has an apparent precursor with an 84-nucleotide 5' leader and approximately 33 nucleotides of additional 3' sequence . Analysis of RPR1 transcription in a strain with a temperature-sensitive lesion in RNA polymerase III shows that the gene is transcribed in vivo by RNA polymerase III . Examination of potential promoter regions using both progressive upstream deletions and point mutations indicates that at least two sequences contained within the 5' leader region are essential for expression in vivo, while sequences farther upstream influence efficiency . The required leader elements resemble tRNA gene-like A-box and B-box internal promoters in sequence and spacing . As in the tRNA genes, transcription factor TFIIIC binds to this region in vitro and binding is severely reduced by either A-box or B-box point mutations that impair expression in vivo . It thus appears that the yeast RNase P RNA gene has adopted a promoter strategy that places an RNA polymerase III "internal" promoter upstream of the mature structural domain to help drive transcription.

J Biol Chem, 1991 Aug 15, 266(23), 15549 - 54
Cloning and characterization of a Saccharomyces cerevisiae gene encoding a new member of the ubiquitin-conjugating protein family; Qin S et al.; Ubiquitin-conjugating enzymes (E2s), which participate in the post-translational conjugation of ubiquitin to proteins, are encoded by a multigene family in the yeast Saccharomyces cerevisiae . E2s function in a variety of cellular activities including intracellular proteolysis, DNA repair, sporulation, and cell cycle traverse . Here, we report the cloning and characterization of a new member of the yeast UBC gene family, UBC8 . UBC8 encodes a 206-amino acid protein containing a highly acidic carboxyl terminus . The primary structure of the protein is similar to that of all other known E2s, with the highest homology being to the E2 (23 kDa) of wheat germ . Haploid strains in which the UBC8 gene is disrupted are viable, and the disruption does not produce any obvious phenotype . The UBC8 protein, produced in Escherichia coli, forms thiol ester adducts with ubiquitin and, apparently, diubiquitin, but does not transfer ubiquitin to histones.

Biochem Biophys Res Commun, 1991 Aug 15, 178(3), 1167 - 75
Rat liver c-erb A beta 1 thyroid hormone receptor is a constitutive activator in yeast (Saccharomyces cerevisiae): essential role of domains D,E and F in hormone-independent transcription; Ohashi H et al.; To assess thyroid hormone receptor (TR)-mediated activation of transcription in yeast in the presence or absence of thyroid hormone (T3), we developed a co-expression system using a TR-beta 1 expression vector and a reporter plasmid containing a 16 base pair palindromic thyroid hormone response element (TRE) upstream from a proximal CYC1 promoter that was fused to the beta-galactosidase lac Z gene of Escherichia coli . Although TR-beta 1 functions as a repressor in most mammalian systems, using our system we observed a unique thyroid hormone-independent transcriptional response indicating that wild TR-beta 1 acted as a constitutive activator in yeast; the addition of 1 microM T3 induced a moderate but significant (p less than 0.01) 25-40% further increase in transcriptional activity . Using a series of rat TR-beta 1 mutant constructs, we found that deletion of domain D and portions of E completely eliminated transcriptional activity, whereas truncations of domain F and E permitted a partial (20-40%) response compared to wild TR-beta 1 in the presence or absence of T3 . These observations indicate that TR-beta 1 functions as an activator in yeast and that domains D,E and F play important interactive roles in its hormone-independent gene activation with the D domain likely being the most essential . Furthermore, our results suggest that the different transcriptional property of TR-beta 1 in yeast compared to mammalian cells i.e . activator vs repressor function, is likely determined by transcriptional factor differences which are dependent upon cellular origin.

Biochemistry, 1991 Aug 13, 30(32), 8092 - 6
Isolation of temperature-sensitive DNA polymerase III from Saccharomyces cerevisiae cdc2-2; Blank A et al.; DNA polymerase III of the yeast Saccharomyces cerevisiae has been reported to be encoded at the CDC2 locus based on two observations . First, the CDC2 gene has homology to known DNA polymerase genes {Boulet et al . (1989) EMBO J . 8, 1849-1854}, and second, the mutants cdc2-1 and cdc2-2 yield little or no DNA polymerase III activity in vitro {Boulet et al . (1989); Sitney et al . (1989) Cell 56, 599-605} . We describe here the isolation of temperature-sensitive DNA polymerase III from cdc2-2 strains . Our results provide direct experimental confirmation of the previously inferred gene/enzyme relationship and verify the conclusion that DNA polymerase III is required to replicate the genome . We isolated DNA polymerase III from two cdc2-2 strains, one containing the wild-type allele for DNA polymerase I (CDC17) and the other a mutant DNA polymerase I allele (cdc17-1) . Yields from cdc2-2 cells of both DNA polymerase III activity and an associated 3'-5'-exonuclease activity {exonuclease III; Bauer et al . (1988) J . Biol . Chem . 263, 917-924} were decreased relative to yields from CDC2 cells . DNA polymerase III activity from cdc2-2 strains is thermolabile, displaying at least a 4-fold reduction in half-life at 44 degrees C . The activity is also labile at 37 degrees C, a temperature which is restrictive for growth of cdc2-2 but not CDC2 strains . At 23 degrees C, a temperature which is permissive for growth of both cdc2-2 and CDC2 strains, the mutant and wild-type DNA polymerase III activities display equal stability . These observations provide a demonstrable biochemical basis for the thermosensitive phenotype of cdc2-2 cells.

J Biol Chem, 1991 Jul 25, 266(21), 13859 - 65
Phosphatidylinositol 4-kinase from Saccharomyces cerevisiae . Kinetic analysis using Triton X-100/phosphatidylinositol-mixed micelles; Buxeda RJ et al.; Phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified from Saccharomyces cerevisiae by an improved procedure over that previously reported (Belunis, C.J., Bae-Lee, M., Kelley, M.J., and Carman, G.M . (1988) J . Biol . Chem . 263, 18897-18903) for the enzyme . The molecular mass of the enzyme was 45 kDa . The 35-kDa protein previously identified as PI 4-kinase was a proteolysis product of the 45-kDa protein . A detailed kinetic analysis of the purified enzyme was performed with Triton X-100/phosphatidylinositol-mixed micelles according to the "surface dilution" (Deems, R.A., Eaton, B.R., and Dennis, E.A . (1975) J . Biol . Chem . 250, 9013-9020) and "dual phospholipid" (Hendrickson, H.S., and Dennis, E.A . (1984) J . Biol . Chem . 259, 5734-5739) kinetic models . Phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol at concentrations of phosphatidylinositol below 0.1 mM . Above 0.1 mM activity was only dependent on the surface concentration of phosphatidylinositol . The enzyme more closely followed the dual phospholipid model where the enzyme associated with Triton X-100 micelles when phosphatidylinositol was present . The interfacial Michaelis constant (KmB) for phosphatidylinositol was 0.0036 mol fraction and the dissociation constant (KsA) for phosphatidylinositol in the micelle surface was 0.26 mM . The results of glycerol gradient centrifugation studies showed that the enzyme was physically associated with Triton X-100/phosphatidylinositol micelles.

Mol Cell Biol, 1991 Aug, 11(8), 4266 - 73
TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae; Ko CH et al.; We describe the cloning and molecular analysis of TRK2, the gene likely to encode the low-affinity K+ transporter in Saccharomyces cerevisiae . TRK2 encodes a protein of 889 amino acids containing 12 putative membrane-spanning domains (M1 through M12), with a large hydrophilic region between M3 and M4 . These structural features closely resemble those contained in TRK1, the high-affinity K+ transporter . TRK2 shares 55% amino acid sequence identity with TRK1 . The putative membrane-spanning domains of TRK1 and TRK2 share the highest sequence conservation, while the large hydrophilic regions between M3 and M4 exhibit the greatest divergence . The different affinities of TRK1 trk2 delta cells and trk1 delta TRK2 cells for K+ underscore the functional independence of the high- and low-affinity transporters . TRK2 is nonessential in TRK1 or trk1 delta haploid cells . The viability of cells containing null mutations in both TRK1 and TRK2 reveals the existence of an additional, functionally independent potassium transporter(s) . Cells deleted for both TRK1 and TRK2 are hypersensitive to low pH; they are severely limited in their ability to take up K+, particularly when faced with a large inward-facing H+ gradient, indicating that the K+ transporter(s) that remains in trk1 delta trk2 delta cells functions differently than those of the TRK class.

Mol Cell Biol, 1991 Aug, 11(8), 4235 - 43
Cloning and characterization of a gene which determines osmotic stability in Saccharomyces cerevisiae; Stateva LI et al.; The srb1-1 mutation of Saccharomyces cerevisiae is an ochre allele which renders the yeast dependent on an osmotic stabilizer for growth and gives the cells the ability to lyse on transfer to hypotonic conditions . A DNA fragment which complements both of these phenotypic effects has been cloned . This clone contains a functional gene which is transcribed into a 2.3-kb polyadenylated mRNA molecule . Transformation of yeast strains carrying defined suppressible alleles demonstrated that the cloned fragment does not contain a nonsense suppressor . Integrative transformation and gene disruption experiments, when combined with classical genetic analysis, confirmed that the cloned fragment contained the wild-type SRB1 gene . The integrated marker was used to map SRB1 to chromosome XV by Southern hybridization and pulsed-field gel electrophoresis . A disruption mutant created by the insertion of a TRP1 marker into SRB1 displayed only the lysis ability phenotype and was not dependent on an osmotic stabilizer for growth . Lysis ability was acquired by growth in (or transfer to) an osmotically stabilized environment, but only under conditions which permitted budding . It is inferred that budding cells lyse with a higher probability and that weak points in the wall at the site of budding are involved in the process . The biotechnological potential of the cloned gene and the disruption mutant is discussed.

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