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