Mol Gen Genet, 1988 Apr, 212(1), 124 - 8 The Escherichia coli proB gene corrects the proline auxotrophy of Saccharomyces cerevisiae pro1 mutants; Orser CS et al.; We constructed plasmids carrying the Escherichia coli proB gene that encodes gamma-glutamyl kinase, under the control of the yeast GAL1 promoter . This construction was carried out with both the wild-type proB+ gene and a mutant allele, proB74, that specifies an enzyme resistant to feedback inhibition by proline . Yeast pro1 mutants harboring these plasmids are proline prototrophs . We conclude that the pro1 mutation results in a deficiency in the gamma-glutamyl kinase activity in Saccharomyces cerevisiae . Expression of the proB74 allele in yeast resulted in enhanced resistance to the proline analogue L-azetidine-2-carboxylate and in a 2.4-fold elevation of the intracellular free proline levels . This result suggests that gamma-glutamyl kinase is the rate limiting step in proline biosynthesis in yeast. J Bacteriol, 1988 Apr, 170(4), 1878 - 86 Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis; Klig LS et al.; A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis . Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype . CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level . Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme . This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis . Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors . The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase . Phosphatidylinositol synthase was not affected in the cdg1 mutant. Mol Cell Biol, 1988 Apr, 8(4), 1421 - 31 Functional organization of the retrotransposon Ty from Saccharomyces cerevisiae: Ty protease is required for transposition; Youngren SD et al.; We used several mutations generated in vitro to further characterize the functions of the products encoded by the TyB gene of the transpositionally active retrotransposon TyH3 from Saccharomyces cerevisiae . Mutations close to a core protein domain of TyB, which is homologous to retroviral proteases, have striking effects on Ty protein processing, the physiology of Ty viruslike particles, and transposition . The Ty protease is required for processing of both TyA and TyB proteins . Mutations in the protease resulted in the synthesis of morphologically and functionally aberrant Ty viruslike particles . The mutant particles displayed reverse transcriptase activity, but did not synthesize Ty DNA in vitro . Ty RNA was present in the mutant particles, but at very low levels . Transposition of a genetically tagged element ceased when the protease domain was mutated, demonstrating that Ty protease is essential for transposition . One of these mutations also defined a segment of TyB encoding an active reverse transcriptase . These results indicate that the Ty protease, like its retroviral counterpart, plays an important role in particle assembly, replication, and transposition of these elements. Gene, 1988 Mar 31, 63(2), 175 - 85 Mapping and sequencing of the dihydrofolate reductase gene (DFR1) of Saccharomyces cerevisiae; Barclay BJ et al.; The dihydrofolate reductase gene (DFR1) from Saccharomyces cerevisiae has been mapped and sequenced . The gene was isolated on an 8.8-kb BamHI fragment from a yeast genomic library by screening of Escherichia coli transformants for resistance to trimethoprim . A 1.8-kb SalI-BamHI fragment which was able to confer methotrexate resistance in yeast also complemented an E . coli DHFR-deficient (folA) mutant . Nucleotide sequence analysis revealed that the yeast DFR1 gene encoded a polypeptide with a predicted Mr of 24230 . The deduced sequence of 211 amino acid residues showed considerable homology with DHFRs from both bacterial and animal sources . The codon bias index of the DFR1 coding region is 0.0083, which indicates a random pattern of codon usage . The upstream region contains two consensus sequences required for binding of the yeast's positive regulatory factor, GCN4, suggesting that the DFR1 gene might be subject to the amino acid general control . Several potential 'TATA' boxes are located in the sequence 5' to the gene . Located in the 3' flanking region are homologies with several canonical sequences thought to be required for efficient transcription termination in yeast . We also mapped the DFR1 gene to a position 1.4 cM proximal to the MET7 locus on chromosome XV. Biochem Biophys Res Commun, 1988 Mar 30, 151(3), 1346 - 51 Accumulation of processing intermediates of the RAS2 protein in strain 112 of Saccharomyces cerevisiae; Breviario D et al.; Strain 112 (RAS1 RAS2) contains a naturally occurring mutation which significantly retards processing of the RAS2 gene product . This mutation, resulting in the accumulation of precursor forms of RAS2 protein, has been assigned by genetic analysis to a single chromosomal locus distinct from the RAS2 locus . In addition to the known precursor molecule of 41000 daltons (p41), 112 cells accumulate within the soluble fraction an intermediate form of RAS2 (p40-1), which migrates, in SDS-polyacrylamide gel, between p41 and the fully processed, membrane-bound 40,000 daltons (p40) product . We propose for RAS2 protein processing the following sequence of events: p41 greater than p40-1 greater than p40 where p40-1 represents a RAS2 intermediate required for the targeting of the protein to the plasma membrane. Biochim Biophys Acta, 1988 Mar 30, 933(1), 212 - 22 The definition of mitochondrial H+ ATPase assembly defects in mit- mutants of Saccharomyces cerevisiae with a monoclonal antibody to the enzyme complex as an assembly probe; Hadikusumo RG et al.; mit- mutants with genetically defined mutations in the mitochondrial structural genes of the H+-ATPase membrane subunits 6, 8 and 9 were analysed to determine the H+-ATPase assembly defects that resulted as a consequence of the mutations . These include mutants which do not synthesize one of the membrane subunits and mutants which can synthesize these subunits, but in an altered form . Protein subunits which can still be assembled to the defective H+-ATPase in these mutants were determined by immunoprecipitation using a monoclonal antibody to the beta-subunit of the enzyme complex . The results suggest that the assembly pathway of the mitochondrially synthesized H+-ATPase subunits involves the sequential addition of subunits 9, 8 and 6 to a membrane-bound F1-sector . In addition to subunits of the F0- and F1-sectors, two other polypeptides (Mr = 18,000 and Mr = 25,000) are associated with the yeast H+-ATPase . These polypeptides were not observed in the immunoprecipitates obtained from mutants in which the F0-sector is not properly assembled. FEBS Lett, 1988 Mar 14, 229(2), 383 - 7 Sequence of the gene encoding phosphoglycerate mutase from Saccharomyces cerevisiae; White MF et al.; The gene encoding yeast phosphoglycerate mutase was isolated, and its sequence was determined . The gene specifies a protein of 246 amino acids, and contains no introns . The sequence shows a strong codon bias . The upstream untranslated portion of the gene contains a CT-rich block such as is found in many highly expressed yeast genes, but does not have the associated CAAG sequence. Biochim Biophys Acta, 1988 Mar 11, 968(3), 408 - 17 Guanine nucleotide regulation of adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae; Becker JM et al.; Adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae was examined . Among various permeabilization procedures, including organic solvents, detergents and other reagents, dimethylsulfoxide (DMSO) and digitonin treatments resulted in the highest recovery of adenylate cyclase activity . Incubation of cells at 30 degrees C with digitonin at 0.01% to 0.1%, or DMSO at 20% to 40% for 15 to 30 min gave optimal adenylate cyclase activity . The enzyme activity in digitonin-permeabilized cells could be supported only by Mn2+, whereas Mg2+ with or without guanine nucleotides did not support cyclase activity . DMSO-permeabilized cells exhibit efficient Mn2+- and Mg2+/Gpp{NH}p-dependent stimulation . Furthermore, digitonin added to yeast membranes at a 1:50 detergent to protein ratio (w/w) abolishes guanyl nucleotide regulation without significantly affecting the Mn2+-supported cyclase activity . The superiority of DMSO is further supported by the fact that recovery of adenylate cyclase activity is better in the DMSO-treated cells than in the digitonin-treated cells . DMSO most probably causes less disturbance of the fabric of the native cell . We conclude that digitonin, but not DMSO, uncouples the catalytic unit of adenylate cyclase from the regulatory GTP binding (ras) proteins. Biochim Biophys Acta, 1988 Mar 4, 959(1), 9 - 19 Inhibition of sterol biosynthesis in Saccharomyces cerevisiae by N,N-diethylazasqualene and derivatives; Balliano G et al.; The ability of some azasqualene derivatives to inhibit yeast cell growth was compared with their inhibition activity on squalene-2,3-oxide cyclase (EC 5.4.99.7) both in living cells and in microsome preparations . Among the compounds tested, N,N-diethylazasqualene showed the best correlation between the activity on squalene-2,3-oxide cyclase and its inhibition of yeast growth . The N-oxide derivative, N,N-diethylazasqualene N-oxide, which was as active as the amine in microsomes, was much less active in living cells, probably because it could not easily penetrate the cell wall . Kinetic analysis of the inhibitory activity of compounds on squalene-2,3-oxide cyclase revealed a sharp difference between N,N-diethylazasqualene and its N-oxide; the former showed a non-competitive-type inhibition, whereas the latter behaved as a competitive inhibitor. Curr Genet, 1988 Mar, 13(3), 207 - 17 Regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae; Holmberg S et al.; The threonine deaminase gene (ILV1) of Saccharomyces cerevisiae has been designated "multifunctional" since Bollon (1974) indicated its involvement both in the catalysis of the first step in isoleucine biosynthesis and in the regulation of the isoleucine-valine pathway . Its role in regulation is characterized by a decrease in the activity of the five isoleucine-valine enzymes when cells are grown in the presence of the three branched-chain amino acids, isoleucine, valine and leucine (multivalent repression) . We have demonstrated that the regulation of AHA reductoisomerase (encoded by ILV5) and branched-chain amino acid transaminase is unaffected by the deletion of ILV1, subsequently revealing that the two enzymes can be regulated in the absence of threonine deaminase . Both threonine deaminase activity and ILV1 mRNA levels increase in mutants (gcd2 and gcd3) having constitutively depressed levels of enzymes under the general control of amino acid biosynthesis, as well as in response to starvation for tryptophan and branched-chain amino acid imbalance . Thus, the ILV1 gene is under general amino acid control, as is the case for both the ILV5 and the transaminase gene . Multivalent repression of reductoisomerase and transaminase can be observed in mutants defective in general control (gcn and gcd), whereas this is not the case for threonine deaminase . Our analysis suggests that repression effected by general control is not complete in minimal medium . Amino acid dependent regulation of threonine deaminase is only through general control, while the branched-chain amino acid repression of AHA reducto isomerase and the transaminase is caused both by general control and an amino acid-specific regulation. Genetika, 1988 Mar, 24(3), 568 - 71 {Ploidy of a mutant Saccharomyces cerevisiae defective in homologous recombination}; Semenova VD et al.; The ploidy of a mutant of Saccharomyces cerevisiae defective in recombination (Rec-) has been determined using tetrad analysis and flowing fluorometry . Evidence is obtained that the effect of the Rec- phenotype, i.e . the increase of the stability of plasmids with 2 mkm DNA ori replication in the yeast cirO cells is not the result of the diploidy in cells of the Rec- mutant developed in the process of transformation. Mol Cell Biol, 1988 Mar, 8(3), 1309 - 18 The a-factor pheromone of Saccharomyces cerevisiae is essential for mating; Michaelis S et al.; The Saccharomyces cerevisiae pheromone a-factor is produced by a cells and interacts with alpha cells to cause cell cycle arrest and other physiological responses associated with mating . Two a-factor structural genes, MFA1 and MFA2, have been previously cloned with synthetic probes based on the a-factor amino acid sequence (A . Brake, C . Brenner, R . Najarian, P . Laybourn, and J . Merryweather, cited in M.-J . Gething {ed.}, Protein transport and secretion, 1985) . We have examined the function of these genes in a-factor production and mating by construction and analysis of chromosomal null mutations . mfa1 and mfa2 single mutants each exhibited approximately half the wild-type level of a-factor activity and were proficient in mating, whereas the mfa1 mfa2 double mutant produced no a-factor and was unable to mate . These results demonstrate that both genes are functional, that each gene makes an equivalent contribution to the a-factor activity and mating capacity of a cells, and that a-factor plays an essential role in mating . Strikingly, exogenous a-factor did not alleviate the mating defect of the double mutant, suggesting that an a cell must be producing a-factor to be an effective mating partner. Mol Cell Biol, 1988 Mar, 8(3), 1253 - 8 A DNA sequence conferring high postmeiotic segregation frequency to heterozygous deletions in Saccharomyces cerevisiae is related to sequences associated with eucaryotic recombination hotspots; White JH et al.; The meiotic behavior of two graded series of deletion mutations in the ADE8 gene in Saccharomyces cerevisiae was analyzed to investigate the molecular basis of meiotic recombination . Postmeiotic segregation (PMS) was observed for a subset of the deletion heterozygosities, including deletions of 38 to 93 base pairs . There was no clear relationship between deletion length and PMS frequency . A common sequence characterized the novel joint region in the alleles which displayed PMS . This sequence is related to repeated sequences recently identified in association with recombination hotspots in the human and mouse genomes . We propose that these particular deletion heterozygosities escape heteroduplex DNA repair because of fortuitous homology to a binding site for a protein. Mol Cell Biol, 1988 Mar, 8(3), 1179 - 85 Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae; Morrison A et al.; The RAD6 gene of Saccharomyces cerevisiae, which is required for normal tolerance of DNA damage and for sporulation, encodes a 172-residue protein whose 23 carboxyl-terminal residues are almost all acidic . We show that this polyacidic sequence appends to RAD6 protein as a polyanionic tail and that its function in vivo does not require stoichiometry of length . RAD6 protein was purified to near homogeneity from a yeast strain carrying a RAD6 overproducing plasmid . Approximately the first 150 residues of RAD6 protein composed a structural domain that was resistant to proteinase K and had a Stokes radius typical of a globular protein of its calculated mass . The carboxyl-terminal polyacidic sequence was sensitive to proteinase K, and it endowed RAD6 protein with an aberrantly large Stokes radius that indicates an asymmetric shape . We deduce that RAD6 protein is monomeric and comprises a globular domain with a freely extending polyacidic tail . We tested the phenotypic effects of partial or complete deletion of the polyacidic sequence, demonstrating the presence of the shortened proteins in the cell by using antibody to RAD6 protein . Removal of the entire polyacidic sequence severely reduced sporulation but only slightly affected survival after UV irradiation or UV-induced mutagenesis . Strains with deletions of all but the first 4 or 15 residues of the polyacidic sequence were phenotypically almost wild type or wild type, respectively . We conclude that the intrinsic activity of RAD6 protein resides in the globular domain, that the polyacidic sequence has a stimulatory or modifying role evident primarily in sporulation, and that only a short section apparently functions as effectively as the entire polyacidic sequence. Mol Gen Genet, 1988 Mar, 211(3), 455 - 8 Mitotic gene conversion of large DNA heterologies in Saccharomyces cerevisiae; Aguilera A; Gene conversion of large DNA heterologous fragments has been shown to take place efficiently in Saccharomyces cerevisiae . It has been found that a 2.6 kb LEU2 DNA fragment in a multicopy plasmid was replaced by a 3.1 kb PGI1 chromosomal DNA fragment, when both fragments were flanked by homologous DNA regions . Gene conversion was asymmetric in a total of 481 recombinants analyzed . In contrast, truncated PGI1 or LEU2 genes in multicopy plasmids, gave no recombinants that restored a complete plasmid copy of these genes in a total of 242 recombinants studied, confirming that a conversion tract is disrupted by a heterologous region . The asymmetry of the events detected suggest that gene conversion of large DNA heterologies involves a process whereby a gap first covers one heterologous fragment and then this is followed by new DNA synthesis using the other heterologous fragment as a template . Therefore, it is likely that large DNA heterologies are converted by a double-strand gap repair mechanism. Mol Gen Genet, 1988 Mar, 211(3), 430 - 4 The sporulation capable (sca) mutation of Saccharomyces cerevisiae is an allele of the SIR2 gene; Margolskee JP; We have used the special properties of the spo13-1 mutation in order to study the regulation of yeast meiosis by the mating type loci . We have found that both the rme1-1 mutation and the sca mutation allow haploid meiosis in spo13-1 strains . Therefore, haploid meiosis is regulated in the same manner as diploid meiosis . Unlike rme1-1, the sca mutation allows meiosis through derepression of the silent mating type cassettes; sca strains can sporulate only because they express both MATa and MAT alpha information . We have found further that sca is an allele of SIR2, one of the genes involved in repression of the silent cassettes . Therefore, the RME1 gene is the only known candidate for a master negative regulator through which the MAT locus controls meiosis. Genetics, 1988 Mar, 118(3), 411 - 5 Maintenance of the 2 micron circle plasmid of Saccharomyces cerevisiae by sexual transmission: an example of a selfish DNA; Futcher B et al.; Many eukaryotic mobile elements have been identified, but few have any obvious function . This has led to the proposal that many such elements may be parasitic DNA . We have used the 2 micron circle plasmid of Saccharomyces cerevisiae as a model system to investigate the maintenance of a cryptic genetic element . We find that under certain conditions this plasmid can spread through experimental populations despite demonstrable selection against it . This spread is dependent upon outbreeding, suggesting that cell to cell transmission of the plasmid during the yeast sexual cycle can counterbalance selection, and maintain the plasmid in populations . This result provides experimental support for the idea that some mobile elements may be parasitic DNA. DNA, 1988 Mar, 7(2), 117 - 26 Regulated secretion of MuGM-CSF in Saccharomyces cerevisiae via GAL1:MF alpha 1 prepro sequences; Shaw KJ et al.; Murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was expressed in Saccharomyces cerevisiae using a novel regulated secretion system . This system involves the fusion of the GAL1 upstream regulatory region to the signal sequence of the alpha mating pheromone, and the integration of this GAL1:MF alpha 1 prepro:MuGM-CSF construct into the yeast chromosome . These constructs were very stable under both selective and nonselective conditions: after 30 generations of growth no plasmid loss was observed . The expression and secretion of MuGM-CSF were analyzed by biological assays and Western blots of yeast culture medium and yeast cell extracts . Expression of MuGM-CSF was regulated by galactose induction . In addition, expression levels were proportional to the number of tandem copies of the gene inserted into the yeast chromosome. J Cell Biol, 1988 Mar, 106(3), 557 - 66 Histone H3 and H4 gene deletions in Saccharomyces cerevisiae; Smith MM et al.; The genome of haploid Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 genes . Strains with deletions of each of these loci were constructed by gene replacement techniques . Mutants containing deletions of either gene set were viable, however meiotic segregants lacking both histone H3 and H4 gene loci were inviable . In haploid cells no phenotypic expression of the histone gene deletions was observed; deletion mutants had wild-type growth rates, were not temperature sensitive for growth, and mated normally . However, diploids homozygous for the H3-H4 gene deletions were slightly defective in their growth and cell cycle progression . The generation times of the diploid mutants were longer than wild-type cells, the size distributions of cells from exponentially growing cultures were skewed towards larger cell volumes, and the G1 period of the mutant cells was longer than that of the wild-type diploid . The homozygous deletion of the copy-II set of H3-H4 genes in diploids also increased the frequency of mitotic chromosome loss as measured using a circular plasmid minichromosome assay. Yeast, 1988 Mar, 4(1), 41 - 6 Purification of isocitrate lyase from Saccharomyces cerevisiae; Lopez-Boado YS et al.; Isocitrate lyase purified to homogeneity from Saccharomyces cerevisiae was composed of four identical subunits with a molecular mass of 75 kDa . The enzyme was most active at pH 7.0 in the presence of 5 mM-Mg2+ . The Km value for threo-Ds-isocitrate was 1.4 mM . Isocitrate lyase was shown to be thermostable at 50 degrees C for 60 min at a high salt concentration, but rapidly lost activity at -20 degrees C or by dialysis. J Gen Microbiol, 1988 Mar, 134 ( Pt 3), 785 - 90 Regulation of trehalase activity during the cell cycle of Saccharomyces cerevisiae; Van Doorn J et al.; Synchronous cultures of Saccharomyces cerevisiae prepared by selection of small unbudded cells from an elutriating rotor were used to measure trehalase activity during the cell cycle . After the small cells had been removed from the rotor, the remainder was used to prepare asynchronous control cultures . Both synchronous and control cultures were studied for two cell cycles . In asynchronous cultures the trehalase activity of crude cell lysates rose continuously . In synchronized populations trehalase activity increased from the beginning of budding onwards . However, around the period of cell division the enzyme activity dropped rapidly but transiently by more than 5-fold . The same changes were found during the second budding cycle . Measurements of invertase and glucose-6-phosphate dehydrogenase activities in the same synchronous and asynchronous cultures revealed a continuous increase for both enzymes . Incubation of cell lysates with cAMP-dependent protein kinase before assaying for trehalase resulted in a 2-fold enhancement of enzyme activity in asynchronous control cultures . In synchronized cells this treatment also led to a significant stimulation of trehalase activity, and largely abolished the cell-cycle-dependent oscillatory pattern of enzyme activity . These results suggest that the activity of trehalase during the cell cycle is regulated, presumably at the post-translational level, by a phosphorylation-dephosphorylation mechanism. Yeast, 1988 Mar, 4(1), 47 - 59 Expression of env sequences of the bovine leukemia virus (BLV) in the yeast Saccharomyces cerevisiae; Brantl S et al.; DNA sequences of the envelope (env) gene of the bovine leukemia virus (BLV) were expressed in the yeast Saccharomyces cerevisiae . Two yeast promoters, the repressible PHO5 promoter and the constitutive PGK promoter, were used to construct four expression plasmids comprising either a sequence of the surface antigen gp51 or a (gp51 + gp30) sequence . The expressed heterologous gene products were characterized by Western blot analysis and competitive radioimmunoassay . By means of Northern blot analysis the steady-state level of env-specific mRNA was analysed . The highest expression rate was obtained from recombinant plasmid YEpSG 94 comprising a gp51 sequence--a 630 base pair fragment containing 70% of the gp51 but lacking the N terminus--as well as the PHO5 promoter including PHO5 signal sequence and the PHO5 terminator . The recombinant gp51 was partially glycosylated but the PHO5 signal peptide did not seem to be cleaved off . No immunoreactive material could be found in the periplasm or in the culture medium . By means of monoclonal antibodies directed against eight different epitopes of viral gp51, all four sequential antigenic determinants were detected in the AH 216 (YEpSG 94) expression product. Genetics, 1988 Mar, 118(3), 401 - 10 Physical lengths of meiotic and mitotic gene conversion tracts in Saccharomyces cerevisiae; Judd SR et al.; Physical lengths of gene conversion tracts for meiotic and mitotic conversions were examined, using the same diploid yeast strain in all experiments . This strain is heterozygous for a mutation in the URA3 gene as well as closely linked restriction site markers . In cells that had a gene conversion event at the URA3 locus, it was determined by Southern analysis which of the flanking heterozygous restriction sites had co-converted . It was found that mitotic conversion tracts were longer on the average than meiotic tracts . About half of the tracts generated by spontaneous mitotic gene conversion included heterozygous markers 4.2 kb apart; none of the meiotic conversions included these markers . Stimulation of mitotic gene conversion by ultraviolet light or methylmethanesulfonate had no obvious effect on the size or distribution of the tracts . Almost all conversion tracts were continuous. J Bacteriol, 1988 Mar, 170(3), 1399 - 402 Identification of a third nuclear protein-coding gene required specifically for posttranscriptional expression of the mitochondrial COX3 gene is Saccharomyces cerevisiae; Kloeckener-Gruissem B et al.; A third nuclear protein-coding gene termed PET122 has been shown to be required for a post-transcriptional step in expression of the mitochondrial COX3 gene is Saccharomyces cerevisiae . pet122 mutants fail to produce cytochrome c oxidase subunit III, which is the polypeptide product of the COX3 gene, but produce normal amounts of mature COX3 mRNA . A strain bearing the pet122-1 allele is amber suppressible and correctly processes the 5' end of COX3 mRNA . Therefore, the PET122 gene product is a protein required for the expression of COX3 at some step after transcription and 5'-end processing of its transcript. J Theor Biol, 1988 Feb 21, 130(4), 481 - 92 A mathematical model of recombinational amplification of the 2 mu plasmid in the yeast Saccharomyces cerevisiae; Wittrup KD et al.; A mathematical model of 2 mu plasmid recombinational amplification in Saccharomyces cerevisiae has been developed, based on mechanisms of 2 mu recombination and replication presented in the literature . A probabilistic description reveals the limits inherent in the recombinational mode of plasmid amplification . These limits correspond well with values calculated from reported results . In the model, copy number control is effected by the constitutive expression of a repressor of recombinase expression . Estimation of the model parameters is accomplished via a set of heuristic rules which restrict the feasible parameter space considerably . It is demonstrated that many parameter sets arbitrarily chosen from the feasible parameter space reproduce the observed characteristics of 2 mu plasmid amplification: rapid correction of downward copy number deviations, with a lack of strict control of steady-state copy number. Eur J Biochem, 1988 Feb 15, 172(1), 205 - 11 Modifications of oxidative phosphorylations in mitochondria isolated from a mutant of Saccharomyces cerevisiae . Possible alterations of the phosphate transport; Manon S et al.; Mutants of Saccharomyces cerevisiae were isolated which supported two unlinked nuclear mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial one conferring paromomycin sensitivity . Mitochondria isolated from such a mutant exhibited modifications of several phosphate-requiring functions: (a) kinetic parameters of the phosphate dependence of ATP synthesis were modified; (b) in the absence of phosphate the inner mitochondrial membrane exhibited a high proton leakage; (c) mutant mitochondria always exhibited a poor respiratory control and required tenfold more phosphate to reach a maximal state 3 of respiration; (d) phosphate transport, as measured by swelling experiments, was mersalyl-insensitive and, consequently, state 3 of the respiration and ATP synthesis remained less mersalyl-sensitive than in wild-type mitochondria . Analysis of the mitochondrial metabolism of diploid and segregant strains indicates that these modifications are related to the cryosensitive phenotype; however, at present, a cooperative effect of the mitochondrial mutation cannot be eliminated . It is proposed that the phosphate carrier itself or a regulatory element was modified. Eur J Biochem, 1988 Feb 15, 172(1), 179 - 84 Inactivation of the gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase in Saccharomyces cerevisiae; Maarse AC et al.; The single nuclear gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase (complex III) in Saccharomyces cerevisiae has been inactivated by a one-step gene disruption procedure . Inactivation results in a loss of ubiquinol-cytochrome-c oxidoreductase activity (less than 1% wild type) and respiratory deficiency . Cells lacking the 11-kDa protein also display lowered steady-state levels of other complex-III subunits encoded by nuclear genes including the 14-kDa subunit VII and the Rieske Fe-S protein and of the mitochondrially encoded cytochrome b . The steady-state levels of the transcripts from the genes encoding these proteins are however not reduced . The results strongly imply that the 11-kDa protein plays an important role in regulating the synthesis of complex III at the post-transcriptional level, most likely assembly . Separation of chromosomes by pulsed-field gel electrophoresis of DNA of wild-type and of the mutant lacking the 11-kDa-protein gene followed by Southern blot analysis reveals that the latter gene is located on chromosome X rather than on XII as reported by Van Loon et al . {Mol . Gen . Genet . 197 (1984) 219-224}. Biochem Biophys Res Commun, 1988 Feb 15, 150(3), 1144 - 8 Cyclic AMP control of GTP pools in Saccharomyces cerevisiae; Pall ML; Previous studies have shown that GTP and cyclic AMP have similar effects on the regulation of sporulation in the yeast Saccharomyces cerevisiae . Declines in either nucleotide can trigger sporulation . These results raise the question whether either nucleotide influences the pool of the other . The current study shows that a cyclic AMP deficiency produces a decline in GTP pools and cyclic AMP readdition quickly increases GTP pools . UTP but not CTP shows a similar pattern of control to that shown by GTP . These results suggest that cyclic AMP effects on sporulation and possibly other cell properties may be mediated in part or in whole by GTP . They provide support for the hypothesis that GTP has a general role in stimulating cellular growth and proliferation. J Biochem (Tokyo), 1988 Feb, 103(2), 321 - 6 Secretion of an active nonglycosylated form of the repressible acid phosphatase of Saccharomyces cerevisiae in the presence of tunicamycin at low temperatures; Mizunaga T et al.; The role of mannan chains in the formation and secretion of active acid phosphatase of yeast (Saccharomyces cerevisiae), a repressible cell surface mannoprotein, was studied in yeast protoplast systems by using tunicamycin at various temperatures . At 30 degrees C, tunicamycin-treated protoplasts did not produce active acid phosphatase; however, at 25 or 20 degrees C they formed and secreted active enzyme . This form of acid phosphatase gave 59-, 57-, and 55-kDa bands on SDS-PAGE which neither bound to concanavalin A Sepharose, nor changed in molecular weight upon treatment with endoglycosidase H, indicating that the peptides are nonglycosylated . The nonglycosylated form, like its glycosylated counterpart, is a dimer on the basis of gel permeation chromatography . The Km for para-nitrophenyl-phosphate and Ki for inorganic phosphate of both glycosylated and nonglycosylated acid phosphatases were almost the same . These results suggested that 1) the conformation of the nonglycosylated acid phosphatase secreted at low temperatures is probably identical with that of the glycosylated one, and 2) the conformation of acid phosphatase is very important for its secretion . The rate of intracellular transport of nonglycosylated acid phosphatase is about one-fourth that of the glycosylated enzyme, indicating that glycosylation facilitates the transport of acid phosphatase proteins. Genes Dev, 1988 Feb, 2(2), 160 - 72 Depletion of Saccharomyces cerevisiae ribosomal protein L16 causes a decrease in 60S ribosomal subunits and formation of half-mer polyribosomes; Rotenberg MO et al.; We constructed yeast strains containing deletion-insertion null alleles of the RPL16A or RPL16B genes encoding the 60S ribosomal subunit protein L16 to determine the role of L16 in the synthesis and function of ribosomes . Strains lacking a functional RPL16A gene grow as rapidly as wild type, whereas those containing a null allele of RPL16B grow more slowly than wild type . RNA analysis using RPL16 probes revealed that both RPL16 genes are transcribed and that RPL16B transcripts accumulate to twice the level of RPL16A transcripts . No evidence was obtained for the occurrence of dosage compensation at the level of RPL16 mRNA accumulation in either mutant . Strains lacking both RPL16 genes are apparently inviable, demonstrating that L16 is an essential yeast ribosomal protein . Introduction of an extra copy of either RPL16 gene into rpl16b mutants restored wild-type growth rates, indicating that the two forms of the L16 protein are interchangeable . rpl16 mutants are deficient in 60S ribosomal subunits relative to 40S subunits . 43S preinitiation complexes accumulate in half-mer polyribosomes in the absence of sufficient 60S subunits . We postulate that the slow-growth phenotype of rpl16 mutants results from the perturbation of initiation of protein synthesis. Mol Cell Biol, 1988 Feb, 8(2), 945 - 54 Comparison of the structure and cell cycle expression of mRNAs encoded by two histone H3-H4 loci in Saccharomyces cerevisiae; Cross SL et al.; The haploid genome of Saccharomyces cerevisiae contains two nonallelic sets of histone H3 and H4 gene pairs, termed the copy I and copy II loci . The structures of the mRNA transcripts from each of these four genes were examined by nuclease protection and primer extension mapping . For each gene, several species of mRNAs were identified that differed in the lengths of their 5' and 3' untranslated regions . The cell cycle accumulation pattern of the H3 and H4 mRNAs was determined in cells from early-exponential-growth cultures fractionated by centrifugal elutriation . The RNA transcripts from all four genes were regulated with the cell division cycle, and transcripts from the nonallelic gene copies showed tight temporal coordination . Cell cycle regulation did not depend on selection of a particular histone mRNA transcript since the ratio of the multiple species from each gene remained the same across the division cycle . Quantitative measurements showed significant differences in the amounts of mRNA expressed from the two nonallelic gene sets . The mRNAs from the copy II H3 and H4 genes were five to seven times more abundant than the mRNAs from the copy I genes . There was no dosage compensation in the steady-state levels of mRNA when either set of genes was deleted . In particular, there was no increase in the amount of copy I H3 or H4 transcripts in cells in which the high-abundance copy II genes were deleted. Mol Cell Biol, 1988 Feb, 8(2), 938 - 44 Suppression of chromosomal mutations affecting M1 virus replication in Saccharomyces cerevisiae by a variant of a viral RNA segment (L-A) that encodes coat protein; Uemura H et al.; For the maintenance of "killer" M1 double-stranded RNA in Saccharomyces cerevisiae, more than 30 chromosomal genes are required . The requirement for some of these genes can be completely suppressed by a cytoplasmic element, {B} (for bypass) . We have isolated a mutant unable to maintain {B} (mab) and found that it is allelic to MAK10, one of the three chromosomal MAK genes required for the maintenance of L-A . The heat curing of {B} always coincided with the loss of L-A . To confirm that {B} is located on L-A, we purified viral particles containing either L-A or M1 from strains with or without {B} activity and transfected these purified particles into a strain which did not have either L-A or M1 . The transfectants harboring L-A and M1 from a {B} strain showed the {B} phenotype, but the transfectants with L-A and M1 from a {B-o} strain did not show the {B} phenotype . Furthermore, the transfectants having L-A from a {B} strain and M1 from a {B-o} strain also showed the {B} phenotype . Therefore, we concluded that {B} is a property of a variant of L-A . In the transfection experiment, we also proved that the superkiller phenotype of the {B} strain is a property of L-A and that L-A with {B} activity can maintain a higher copy number of M1 regardless of the source of M1 viruslike particles . These data suggest that MAK genes whose mutations are suppressed by {B} are concerned with the protection of M1 (+) single-stranded RNA or the formation of M1 viruslike particles and that an L-A with more efficient production of M1 viruslike particles can completely dispense with the requirement for those MAK genes. Mol Cell Biol, 1988 Feb, 8(2), 912 - 22 The SPS100 gene of Saccharomyces cerevisiae is activated late in the sporulation process and contributes to spore wall maturation; Law DT et al.; We previously described the use of a differential hybridization screen of a genomic DNA library of Saccharomyces cerevisiae to identify sporulation-specific (SPS) genes (A . Percival-Smith and J . Segall, Mol . Cell . Biol . 4:142-150, 1984) . This initial screen identified 14 SPS genes that are first expressed 6 to 8 h after transfer of cells to sporulation medium . Accumulation of transcripts corresponding to these genes becomes maximal at 8 to 12 h of sporulation, the time at which meiotic events are nearing completion, and by 15 h of sporulation, transcript levels are beginning to decrease . In the present study two additional SPS genes, first expressed at 12 h of sporulation, were isolated . The steady-state level of transcripts corresponding to these two genes, termed SPS100 and SPS101, remains unchanged from 15 to 35 h, a time coincident with spore wall maturation . The nature of the putative 34.2-kilodalton protein encoded by the SPS100 gene is consistent with its being a component of the glycoprotein matrix of the spore wall; the protein contains a potential signal sequence and cleavage site and numerous sites for potential glycosylation . A MATa sps100/MAT alpha sps100 strain was found to be indistinguishable from the wild-type strain when assessed for efficiency of ascus formation and spore viability . However, a more detailed analysis of the mutant strain revealed that the SPS100 gene product serves a protective role during the early stages of spore wall formation . The time at which resistance to ether could first be detected in developing spores was delayed by 5 h in the mutant strain relative to the wild-type strain . This phenotype is presumably a reflection of a defect in spore wall maturation . This study has confirmed that temporally distinct classes of sporulation-specific genes are sequentially activated during the process of meiosis and spore formation and has shown that the SPS100 gene, identified on the basis of its developmental-specific expression pattern, contributes to spore development. Mol Cell Biol, 1988 Feb, 8(2), 664 - 73 ATR1, a Saccharomyces cerevisiae gene encoding a transmembrane protein required for aminotriazole resistance; Kanazawa S et al.; In Saccharomyces cerevisiae, 3-amino-1,2,4-triazole (aminotriazole) competitively inhibits the activity of imidazoleglycerolphosphate dehydratase, the product of the HIS3 gene . Wild-type strains are able to grow in the presence of 10 mM aminotriazole because they induce the level of imidazoleglycerolphosphate dehydratase . However, strains containing gcn4 mutations are unable to grow in medium containing aminotriazole because they lack the GCN4 transcriptional activator protein necessary for the coordinate induction of HIS3 and other amino acid biosynthetic genes . Here, we isolated a new gene, designated ATR1, which when present in multiple copies per cell allowed gcn4 mutant strains to grow in the presence of aminotriazole . In wild-type strains, multiple copies of ATR1 permitted growth at extremely high concentrations of aminotriazole (80 mM), whereas a chromosomal deletion of ATR1 caused growth inhibition at very low concentrations (5 mM) . When radioactive aminotriazole was added exogenously, cells with multiple copies of ATR1 accumulated less aminotriazole than wild-type cells, whereas cells with the atr1 deletion mutation retained more aminotriazole . Unlike the mammalian mdr or yeast PDR genes that confer resistance to many drugs, ATR1 appears to confer resistance only to aminotriazole . Genetic analysis, mRNA mapping, and DNA sequencing revealed that (i) the primary translation product of ATR1 contains 547 amino acids, (ii) ATR1 transcription is induced by aminotriazole, and (iii) the ATR1 promoter region contains a binding site for the GCN4 activator protein . The deduced amino acid sequence suggests that ATR1 protein is very hydrophobic with many membrane-spanning regions, has several potential glycosylation sites, and may contain an ATP-binding site . We suggest that ATR1 encodes a membrane-associated component of the machinery responsible for pumping aminotriazole (and possibly other toxic compounds) out of the cell. Mutat Res, 1988 Feb, 204(2), 239 - 49 Mitotic crossing-over by anticancer drugs in Saccharomyces cerevisiae strain D5; Ferguson LR et al.; Treatment with an anticancer drug causing mitotic crossing-over could lead to expression of recessive genes, previously masked in a heterozygote . Used clinically, such drugs might cause an increased risk of cancer in cases of familial tumours, such as Wilm's tumour or retinoblastoma . Potentially, novel forms of drug resistance could also be unmasked by such a recombinogenic event . We have estimated the extent of this potential problem in current clinical drugs by comparing a range of antitumour agents for ability to cause mitotic crossing-over in Saccharomyces cerevisiae strain D5 . We have compared these data with ability to cause an increase in total aberrant colonies in the same experiments . Although many of the agents known to cause point mutation also have some ability for mitotic crossing-over, there are also point mutagens which have little recombinogenic potential . Conversely, some effective recombinogens appear to be either very specific or rather ineffective point mutagens . Although the most generally effective agents in the present experiments were alkylating agents, several other types of drug including DNA-cutting agents, topoisomerase inhibitors, other DNA-binding drugs and antimetabolites may stimulate mitotic crossing-over . None of the mitotic inhibitors or the DNA minor groove binding drugs tested caused recombinogenic events . It would seem that the ability to induce mitotic crossing-over is an important endpoint in its own right . Assays for this event might provide an important complement to other assays commonly required for registration of new pharmaceuticals. Proc Natl Acad Sci U S A, 1988 Feb, 85(3), 743 - 6 Specific interaction between a Saccharomyces cerevisiae protein and a DNA element associated with certain autonomously replicating sequences; Eisenberg S et al.; We have isolated a protein from Saccharomyces cerevisiae that binds specifically to a nucleotide sequence associated with the autonomously replicating sequence (ARS) ARS120, located in the telomeric region of a yeast chromosome . "Footprinting" analysis revealed that a 26-base-pair DNA sequence, 5'-CAAGTGCCGTGCATAATGATGTGGGT-3', was protected by this protein from DNase I digestion . A plasmid containing 48 direct tandem repeats of this oligonucleotide was constructed and used to affinity-purify the binding activity . The purified protein, OBF1 (origin binding factor), showed specific binding to ARS120 . The 26-base-pair OBF1-protected sequence was sufficient for the recognition and binding of the protein, since the mobility of a DNA fragment containing the synthetic binding site was retarded in agarose gels when incubated with OBF1 . By performing competition experiments with a number of different ARSs, we showed that OBF1 binds tightly to some but not all ARSs . Interestingly, OBF1 does not appear to have a discernible affinity for ARS1 or the ARSs associated with mating type loci, HML alpha and HMRa, which are substrates for a DNA-binding activity reported by others . Since OBF1 appears to bind to DNA associated with a number of ARSs, we suggest that this protein may have a function related to ARS activity, perhaps in the initiation of DNA replication at selected ARSs. Mol Cell Biol, 1988 Feb, 8(2), 822 - 7 SPT3 is required for normal levels of a-factor and alpha-factor expression in Saccharomyces cerevisiae; Hirschhorn JN et al.; Mutations in the Saccharomyces cerevisiae SPT3 gene were previously found to cause suppression of Ty and delta insertion mutations in 5'-noncoding regions of genes . This suppression likely results from the fact that SPT3 is required for transcription initiation in delta sequences . Other additional phenotypes of spt3 mutants, including a mating defect, suggest that SPT3 is required for normal levels of expression of other genes . We analyzed the mating defect in spt3 mutants and showed that the levels of transcripts of the three major mating pheromone genes, MF alpha 1, MFa1, MFa2, were all reduced . The reduction in expression of these genes in spt3 mutants was not due to expression of a silent mating type cassette . Furthermore, we showed that the spt3 mating defect was manifest at the levels of both cellular fusion and nuclear fusion. Mol Gen Genet, 1988 Feb, 211(2), 260 - 5 Genetic characterization of hyperresistance to formaldehyde and 4-nitroquinoline-N-oxide in the yeast Saccharomyces cerevisiae; Mack M et al.; The hyperresistance to 4-nitroquinoline-N-oxide (4-NQO) and formaldehyde (FA) of yeast strains transformed with the multi-copy plasmids pAR172 and pAR184, respectively, is due to the two genes, SNQ and SFA, which are present on these plasmids . Restriction analysis revealed the maximal size of SFA as 2.7 kb and of SNQ as 2.2 kb, including transcription control elements . The presence of the smallest 2.7 kb subclone carrying SFA increased hyperresistance to formaldehyde fivefold over that of the original pAR184 isolate . No such increase in hyperresistance to 4-NQO was seen with the smaller subclones of the pAR172 isolate . Disruption of the SFA gene led to a threefold increase in sensitivity to FA as compared with the wild type . Expression of gene SNQ introduced on a multi-copy vector into haploid yeast mutants rad2, rad3, and snm1 did not complement these mutations that block excision repair. Microbiologia, 1988 Feb, 4(1), 61 - 4 {Distribution of potassium and sodium in the vacuole and cytoplasm of Saccharomyces cerevisiae}; Ortega MD; In Na+ grown yeast cells the vacuole is the main Na+ reservoir, maintaining higher Na+/K+ ratios than those in the cytoplasm . This asymmetric distribution may enhance Na+ tolerance . However, in growing cells the effect is of low significance. J Gen Microbiol, 1988 Feb, 134 ( Pt 2), 333 - 7 A study of the role of the hexose monophosphate pathway with respect to fatty acid biosynthesis in sporulation of Saccharomyces cerevisiae; Dickinson JR et al.; 13C NMR was used to study the pattern of label incorporation from {2-13C}acetate into trehalose during sporulation in Saccharomyces cerevisiae . A wild-type strain and a strain homozygous for the zwf1 mutation (which affects glucose-6-phosphate dehydrogenase) were used . In the wild-type it was possible to deduce the cycling of glucose 6-phosphate around the hexose monophosphate pathway whilst in the mutant strain this did not occur . The requirement of the hexose monophosphate pathway for providing NADPH for fatty acid biosynthesis was examined using 13C NMR and GC/MS . The wild-type strain produced a typical profile of fatty acids with palmitoleic acid being the most abundant whereas the mutant contained only one-quarter the amount of total fatty acid . As zwf1 homozygous diploids are able to sporulate this indicates that the large amount of fatty acid biosynthesis observed in sporulation of wild-type strains is not essential to the process. Curr Genet, 1988 Feb, 13(2), 113 - 24 Arginine repression of the Saccharomyces cerevisiae ARG1 gene . Comparison of the ARG1 and ARG3 control regions; Crabeel M et al.; The Saccharomyces cerevisiae ARG1 gene coding for argininosuccinate synthetase has been isolated and the nucleotide sequence of both its control region and of its amino terminal end coding region determined . The startpoint of transcription was established by S1-mapping and reverse transcriptase procedures . Northern blot hybridizations showed that whereas arginine-specific repression reduced the enzyme activity fivefold, it did not reduce the steady state level of the corresponding messenger in proportion; by analogy with the coregulated ARG3 gene, this result suggests a post-transcriptional regulatory mechanism . In contrast, proportionally between enzyme activity and mRNA content was observed under conditions where general amino acid control (known to be transcriptional) was operating . Comparing the 5' untranscribed domains of ARG1 and ARG3 revealed a first region of homology between the TATA box and the transcription startpoint . In this region a 10 bp (ARG3) or 11 bp (ARG1) central box is flanked by two segments which, by mutation, have been shown to be part of the ARG operator (Crabeel et al . 1985) . The repressor is assumed to bind at this primary target site prior to establishing contacts with the proximal part of the nascent mRNA molecule (Crabeel et al . 1985) . By in vitro directed deletion mutagenesis we show that the central conserved box of ARG3 is not essential for arginine-specific repression to occur . Another region of homology was found in the leader part of the messenger RNA; deletion of this region causes a small reduction in ARG3 expression but also does not alter regulation . Neither of these two regions are thus part of the primary repressor target site . In addition, in terms of post-transcriptional regulation, the latter result indicates that no sequence specificity is required in the RNA recognition step. J Bacteriol, 1988 Feb, 170(2), 708 - 13 Regulation of nitrogen assimilation in Saccharomyces cerevisiae: roles of the URE2 and GLN3 genes; Courchesne WE et al.; Mutations in the GLN3 gene prevented a normal increase in the NAD-glutamate dehydrogenase and glutamine synthetase levels in glutamate-grown Saccharomyces cerevisiae cells, whereas mutations in the URE2 gene resulted in high levels of these enzymes in glumate- and glutamine-grown cells . A ure2 gln3 double mutant had low levels of glutamate dehydrogenase and glutamine synthetase in cells grown on glutamate and glutamine; thus, gln3 mutations were epistatic to the ure2 mutations . The results suggest that the GLN3 product is capable of promoting increases in enzyme levels in the absence of a functional URE2 product and that the URE2 product antagonizes the GLN3 product . The URE2 and GLN3 genes were also found to regulate the level of arginase activity . This regulation is completely independent of the regulation of arginase by substrate induction . The activities of glutamate dehydrogenase, glutamine synthetase, and arginase were higher in cells grown on glutamate as the nitrogen source than they were in cells grown under a nitrogen-limiting condition . It had previously been shown that the levels of these enzymes can be increased by glutamine deprivation . We propose that the URE2-GLN3 system regulates enzyme synthesis, in response to glutamine and glutamate, to adjust the intracellular concentration of ammonia so as to maintain glutamine at the level required for optimal growth. Genetics, 1988 Feb, 118(2), 203 - 12 Isolation and analysis of a novel class of suppressor of Ty insertion mutations in Saccharomyces cerevisiae; Fassler JS et al.; Using a new scheme for the isolation of suppressor of Ty insertion mutations (spt mutations) in yeast, we have identified six new SPT genes . Mutations in two of these genes, SPT13 and SPT14, exhibit a novel suppression pattern: suppression of complete Ty insertion mutations, but not of solo delta insertion mutations . Transcriptional analysis shows that spt13- and spt14-mediated suppression of Ty insertion mutations is the result of an elevation in the levels of adjacent gene transcription . In spite of the failure of these mutations to suppress solo delta insertion mutations, they do cause changes in transcription of at least one solo delta insertion mutation . In addition, spt13 and spt14 mutations are epistatic to mutations in certain other SPT genes that do suppress solo delta insertion mutations . These results suggest that the SPT13 and SPT14 gene products may act via sequences in both the delta and epsilon regions of Ty elements . Finally, mutations in SPT13 cause sporulation and mating defects and SPT14 is essential for growth, suggesting that these two genes have important roles in general cellular functions. Mol Cell Biol, 1988 Feb, 8(2), 875 - 83 Nematode repetitive DNA with ARS and segregation function in Saccharomyces cerevisiae; Felsenstein KM et al.; Several members of a repetitive DNA family in the nematode Caenorhabditis elegans have been shown to express ARS and centromeric function in Saccharomyces cerevisiae . The repetitive family, denoted CeRep3, consists of dispersed repeated elements about 1 kilobase in length, present 50 to 100 times in the nematode genome . Three elements were sequenced and found to contain DNA sequences homologous to yeast ARS and CEN consensus sequences . Nematode DNA segments containing these repeats were tested for ARS and CEN (or SEG) function after ligation to shuttle vectors and introduction into yeast cells . Such nematode segments conferred ARS function to the plasmid, as judged by an increased frequency of transformation compared with control plasmids without ARS function . Some, but not all, also conferred to the plasmid increased mitotic stability, increased frequency of 2+:2- segregation in meiosis, and decreased plasmid copy number . These effects are similar to those of yeast centromeric DNA . In view of these results, we suggest that the CeRep3 repetitive family may have replication and centromeric functions in C . elegans. Mol Cell Biol, 1988 Feb, 8(2), 655 - 63 The HAP3 regulatory locus of Saccharomyces cerevisiae encodes divergent overlapping transcripts; Hahn S et al.; Activation of the CYC1 upstream activation site, UAS2, and transcription of several other genes encoding respiratory functions requires the product of the regulatory gene HAP2 . We report here the isolation and characterization of a second UAS2 regulatory gene, HAP3 . Like mutations in HAP2, a mutation in HAP3 abolishes the activity of UAS2 and prevents growth on nonfermentable carbon sources . The HAP3 gene was cloned and, surprisingly, was found to encode two divergently transcribed, overlapping transcripts: a 570-base RNA and a 3-kilobase (kb) RNA . Chromosomal disruption experiments defined the critical region for HAP3 function to a 1.3-kb segment in which the two transcripts overlap . Analysis of the HAP3 DNA sequence showed that the 570-base transcript could encode a protein of 144 amino acids . Synthesis of the 144-amino-acid protein under regulatory control in vivo demonstrated that this protein is essential for activity of UAS2 as well as for growth on nonfermentable carbon sources . The largest open reading frame in the critical region of the 3-kb transcript is only 86 amino acids . Using site-directed mutagenesis, we demonstrated that the 86-amino-acid open reading frame was not involved in UAS2 activity . The possible role of this 3-kb antisense RNA in HAP3 expression or function is discussed. Mol Cell Biol, 1988 Feb, 8(2), 647 - 54 Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site; Forsburg SL et al.; We analyzed upstream activation sequence 2 (UAS2), one of two independent UAS elements in the CYC1 gene of Saccharomyces cerevisiae . Deletions and linker scanning mutations across the 87 base pairs previously defined as UAS2 showed two separate functional elements required for full activity . Region 1, from -230 to -200, contains the principal activation site and responds to the trans-acting regulatory loci HAP2 and HAP3 . A portion of region 1 is homologous to two other HAP2-HAP3-responsive UASs and includes the G----A transition mutation UP1, which increases UAS2 activity . This consensus sequence TNATTGGT bears striking similarity to several CAAT box sequences of higher cells . Region 2, from -192 to -178, substantially enhances the activity of region 1, yet has little activity by itself . These regions bind distinct proteins found in crudely fractionated yeast extracts. J Bacteriol, 1988 Feb, 170(2), 828 - 33 Regulation of phosphatidylinositol kinase activity in Saccharomyces cerevisiae; Holland KM et al.; The effects of growth phase and carbon source on membrane-associated phosphatidylinositol kinase in cell extracts of Saccharomyces cerevisiae were examined . Phosphatidylinositol kinase activity increased 2- and 2.5-fold in glucose- and glycerol-grown cells, respectively, in the stationary phase as compared with the exponential phase of growth . The increase in phosphatidylinositol kinase activity in the stationary phase of growth correlated with an increase in the relative amounts of phosphatidylinositol 4-phosphate, the product of the reaction . The increase in phosphatidylinositol kinase activity was not due to the presence of water-soluble effector molecules in cell extracts as indicated by mixing experiments . Phosphatidylinositol kinase activity decreased in cell extracts of exponential-phase cells preincubated under phosphorylation conditions which favor cyclic AMP-dependent protein kinase activity . Phosphatidylinositol kinase activity was not affected in cell extracts of stationary-phase cells preincubated under phosphorylation conditions. Biophys J, 1988 Feb, 53(2), 193 - 203 Ionophore properties of a synthetic alpha-helical transmembrane fragment of the mitochondrial H+ ATP synthetase of Saccharomyces cerevisiae . Comparison with alamethicin; Molle G et al.; A 22-amino acid polypeptide was synthesized to model the central transmembrane segment of subunit 8 of the H+ ATP synthetase of Saccharomyces cerevisiae and to test ionophore properties . Solid-phase synthesis was conducted on benzhydrilamino resin, and purification followed by high pressure liquid chromatography allowed the isolation of the pure product whose NH2 terminal was acetylated and whose molecular weight determined by Fast Atomic Bombardment was the expected 2,666 . The infrared spectrum of this peptide in the solid state reveals a fully alpha-helical conformation, whereas in low dielectric constant solvents the alpha-helical content is 60%, as determined by circular dichroism studies . Macroscopic current-voltage curves displayed by different planar lipid bilayers (monomyristoleoyl-glycerol and phosphatidylethanolamine) doped with this peptide suggest a weakly voltage-dependent conductance . Only one conductance level is observed in any given single-channel conductance experiment . However, a series of experiments shows a distribution of conductance states, most often 440 or 3,000 pS, and occasionally 80, 1,200, or 6,500 pS . This behavior contrasts with the usual behavior of alamethicin, chosen as a model of "aggregating-helices" ionophore and whose conductance fluctuates continually between substates, through uptake and release of monomers . Nevertheless, alamethicin too can display, under certain conditions, long-lived and mono-level conductance states similar to those reported here for the newly synthesized peptide . These properties could possibly be explained by the formation of large domains of helical rods with a set of allowed and independent ionic pathways. Biochim Biophys Acta, 1988 Jan 29, 952(2), 238 - 43 The binding of glucose and nucleotides to hexokinase from Saccharomyces cerevisiae; Woolfitt AR et al.; The binding of glucose, ADP and AdoPP{NH}P, to the native PII dimer and PII monomer and the proteolytically-modified SII monomer of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from Saccharomyces cerevisiae was monitored at pH 6.7 by the concomitant quenching of protein fluorescence . The data were analysed in terms of Qmax, the maximal quenching of fluorescence at saturating concentrations of ligand, and {L}0.5, the concentration of ligand at half-maximal quenching . No changes in fluorescence were observed with free enzyme and nucleotide alone . In the presence of saturating levels of glucose, Qmax induced by nucleotide was between 2 and 7%, and {L}0.5 was between 0.12 and 0.56 mM, depending on the nucleotide and enzyme species . Qmax induced by glucose alone was between 22 and 25%, while {L}0.5 was approx . 0.4 mM for either of the monomeric hexokinase forms and 3.4 for PII dimer . In the presence of 6 mM ADP or 2 mM AdoPP{NH}P, Qmax for glucose was increased by up to 4% and {L}0.5 was diminished 3-fold for hexokinase PII monomer, 6-fold for SII monomer, and 15-fold for PII dimer . The results are interpreted in terms of nucleotide-induced conformational change of hexokinase in the presence of glucose and synergistic binding interactions between glucose and nucleotide. Biochem Biophys Res Commun, 1988 Jan 29, 150(2), 794 - 801 Human lysozyme: sequencing of a cDNA, and expression and secretion by Saccharomyces cerevisiae; Yoshimura K et al.; A cDNA encoding human lysozyme was isolated from a human placenta cDNA library . The cDNA was 1.5 kb in size and coded for a signal peptide consisting of 18 amino acids and mature lysozyme . The amino acid sequence of the mature lysozyme, deduced from the nucleotide sequence, was identical with the published sequence . In the 3'-noncoding region of the cDNA, an Alu sequence was found in the reverse orientation . In a protein coding region, the human lysozyme cDNA shows 60.1% and 51.3% similarity with chicken lysozyme and human alpha-lactalbumin cDNAs, respectively . When the cDNA was expressed in Saccharomyces cerevisiae, an active and correctly processed human lysozyme was secreted efficiently into the culture medium. Eur J Biochem, 1988 Jan 15, 171(1-2), 417 - 24 Primary structure of the uracil transport protein of Saccharomyces cerevisiae; Jund R et al.; We present in this paper the nucleotidic sequence of the FUR4 gene encoding the uracil permease in the yeast Saccharomyces cerevisiae . The deduced amino acid sequence of the permease has 633 residues; it consists of many hydrophobic stretches, only the N-terminal and C-terminal ends of the protein (about 100 and 50 amino acids respectively) being mostly hydrophilic . No N-terminal hydrophobic signal peptide is present, although it is shown in this work that the biosynthesis of the uracil permease goes through the secretion/glycosylation pathway . Using the results of three different methods, allowing the prediction of transmembrane alpha helices in proteic sequences, we drew a model of folding of the permease in the membrane. J Biol Chem, 1988 Jan 15, 263(2), 917 - 24 DNA polymerase III from Saccharomyces cerevisiae . I . Purification and characterization; Bauer GA et al.; Yeast cells from a wild type or protease-deficient strain were lysed in the absence or presence of protease inhibitors and the extracts analyzed by analytical high pressure liquid chromatography on diethylaminoethyl silica gel . Conditions that inhibited protease action caused elution of a novel DNA polymerase activity at a position in the gradient distinct from the elution positions of both DNA polymerase I and II . In large scale purifications, this DNA polymerase, called DNA polymerase III, copurified with a single-stranded DNA dependent 3'-5' exonuclease activity, exonuclease III, to near homogeneity . Glycerol gradient centrifugation partially dissociated the complex to yield two peaks of exonuclease III activity, one at 7.7 S together with the DNA polymerase, and one at 4.0 S without polymerase activity . Gel filtration indicated that the complex has a molecular mass greater than 400 kDa . Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the complex consists of several subunits: 140, 62, 55, and 53 kilodaltons, some of which may be proteolysis products . The exonuclease component of the complex can excise single nucleotide mismatches providing a base-paired primer-template which can be elongated by the DNA polymerase . Under replication conditions, the complex exhibits a measurable turnover rate of dTTP to dTMP and it contains no primase activity . The enzymatic activities of the 3'-5' exonuclease are consistent with a proofreading function during in vivo DNA replication . A second exonuclease activity, exonuclease IV, separated from the complex late in the purification scheme . It degrades both single-stranded and double-stranded DNA in the 5'----3' direction. Eur J Biochem, 1988 Jan 15, 171(1-2), 171 - 6 Control of enzyme synthesis in the lysine biosynthetic pathway of Saccharomyces cerevisiae . Evidence for a regulatory role of gene LYS14; Ramos F et al.; Several enzymes of the lysine pathway of Saccharomyces cerevisiae were found to respond to an induction mechanism mediated by the product of gene LYS14 in the presence of 2-aminoadipate semialdehyde, an intermediate of this pathway . This novel regulatory mechanism appears independent of the specific repression by lysine and of the general control of amino acid biosynthesis . Genes LYS1, LYS9 and LYS14 have been cloned and their DNAs used to assay the corresponding messenger RNAs . The results suggest that the induction mechanism, as well as the specific and general regulations, operate at the transcriptional level . The synthesis of saccharopine dehydrogenase (glutamate-forming), previously shown to require the unlinked genes LYS9 and LYS14, is also affected by the induction mechanism . The leaky auxotrophic behaviour of lys14 mutants is explained by the low basal level of expression of LYS9, the structural gene of this enzyme, in the absence of induction by 2-aminoadipate semialdehyde. J Biol Chem, 1988 Jan 15, 263(2), 925 - 30 DNA polymerase III from Saccharomyces cerevisiae . II . Inhibitor studies and comparison with DNA polymerases I and II; Burgers PM et al.; The newly identified yeast DNA polymerase III was compared to DNA polymerases I and II and the mitochondrial DNA polymerase . Inhibition by aphidicolin (I50) of DNA polymerases I, II, and III was 4, 6, and 0.6 micrograms/ml, respectively . The mitochondrial enzyme was insensitive to the drug . N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate strongly inhibited DNA polymerase I (I50 = 0.3 microM), whereas DNA polymerase III was less sensitive (I50 = 80 microM) . Conditions that allowed proteolysis to proceed during the preparation of extracts converted DNA polymerase II from a sensitive form (I50 = 2.4 microM) to a resistant form (I50 = 2 mM) . The mitochondrial DNA polymerase is insensitive (I50 greater than 5 mM) . With most other inhibitors tested (N-ethylmaleimide, heparin, salt) only small differences were observed between the three nuclear DNA polymerases . Polyclonal antibodies to DNA polymerase III did not inhibit DNA polymerases I and II, nor were those polymerases recognized by Western blotting . Monoclonal antibodies to DNA polymerase I did not crossreact with DNA polymerases II and III . The results show that DNA polymerase III is distinct from DNA polymerase I and II. Biochim Biophys Acta, 1988 Jan 13, 937(1), 81 - 7 The influence of ATP on sugar uptake mediated by the constitutive glucose carrier of Saccharomyces cerevisiae; Schuddemat J et al.; The glucose carrier of Saccharomyces cerevisiae transports the phosphorylatable sugars glucose, mannose, fructose and 2-deoxy-D-glucose (2-dGlc) and the non-phosphorylatable sugar 6-deoxy-D-glucose (6-dGlc) . Reduction of the ATP concentration by, for example, incubating cells with antimycin A, results in a decrease in uptake of 2-dGlc and fructose . These uptake velocities can be increased again by raising the ATP level . These results establish a role of ATP in sugar transport . Transport of glucose and mannose is less affected by changes in the ATP concentration than 2-dGlc and fructose uptake, while the 6-dGlc transport is independent of the amount of ATP in the cells . Also, reduction of the kinase activity by incubation with xylose diminished transport of 2-dGlc and fructose, while the uptake of glucose and mannose remained unchanged . It is discussed that these results are due to transport-associated phosphorylation with ATP as substrate and the hexokinases and the glucokinase as phosphorylating enzymes. J Biol Chem, 1988 Jan 5, 263(1), 454 - 60 Replicase of L-A virus-like particles of Saccharomyces cerevisiae . In vitro conversion of exogenous L-A and M1 single-stranded RNAs to double-stranded form; Fujimura T et al.; Virus-like particles that contain L-A double-stranded RNA are known to have transcriptase activity whose product is L-A single-stranded plus RNA . In low salt conditions, these particles release their double-stranded RNA and can then use added plus L-A or plus M1 single-stranded RNAs as templates to synthesize their respective double-stranded RNAs . The reaction requires dialyzed L-A virus-like particles as the source of the enzyme, a partially purified cell extract (host factor(s)), added single-stranded RNA as a template, and polyethylene glycol 6000, along with four NTPs . Crude host factor extracts prepared from mak3 or mak10ta mutants also support the reaction as effectively as that from a wild type strain, while a crude extract prepared from a pet18 mutant grown under the nonpermissive conditions is less effective . Template specificity of the in vitro reaction is the same as that expected for the enzyme reaction in vivo . Plus L-A and plus M1 single-stranded RNAs, but not 18 S rRNA, are converted to their respective double-stranded RNAs with net RNA synthesis . The newly synthesized strand of M1 double-stranded RNA is a full-length minus strand . This demonstration of replicase activity in the mature L-A virus-like particles which contain L-A double-stranded RNA is consistent with our previous L-A double-stranded RNA replication model; the difference between the mature L-A virus-like particles and L-A double-stranded RNA-synthesizing particles (expected to be replication intermediates in vivo) is just that the former contain L-A double-stranded RNA, while the latter contain L-A plus single-stranded RNA. J Biol Chem, 1988 Jan 5, 263(1), 45 - 51 Characterization and function of catalytic subunit alpha of H+-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae . A study with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole; Uchida E et al.; Subunit alpha (Mr 89,000) from vacuolar membrane H+-translocating adenosine triphosphatase of the yeast Saccharomyces cerevisiae was found to bind 8-azido{alpha-32P}adenosine triphosphate . Labeling by this photosensitive ATP derivative was saturable with an apparent dissociation constant of 10(-6) to 10(-5) M and decreased in the presence of ATP and ADP . The enzyme was inactivated by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), with about 1 microM causing half-maximal inactivation in the neutral pH range . This inactivation was prevented by the presence of ATP, ADP, or adenosyl-5'-yl imidodiphosphate (AMP-PNP) . The original activity was restored by treating the inactivated enzyme with 2-mercaptoethanol . Kinetic and chemical studies of the inactivation showed that the activity was lost on chemical modification of a single tyrosine residue per molecule of the enzyme . When the enzyme was inactivated with {14C}NBD-Cl, subunit alpha was specifically labeled, and this labeling was completely prevented by the presence of ATP, GTP, ADP, or AMP-PNP . From these results, it was concluded that subunit alpha of yeast vacuolar H+-ATPase has a catalytic site that contains a single, essential tyrosine residue . The kinetics of single site hydrolysis of {gamma-32P}ATP (Grubmeyer, C., Cross, R . L., and Penefsky, H . S . (1982) J . Biol . Chem . 257, 12092-12100) indicated the formation of an enzyme-ATP complex and subsequent hydrolysis of bound ATP to ADP and Pi at the NBD-Cl-sensitive catalytic site . NBD-Cl inactivated the single site hydrolysis and inhibited the formation of an enzyme-ATP complex . Dicyclohexylcarbodiimide did not affect the single site hydrolysis, but inhibited the enzyme activity under steady-state conditions. Genetics, 1988 Jan, 118(1), 41 - 7 UGA suppressors in Saccharomyces cerevisiae: allelism, action spectra and map positions; Ono BI et al.; Sixty independent UGA suppressors of Saccharomyces cerevisiae have been studied . They are dominant and are divided into 16 groups (loci) by recombination . Suppressors representing these loci are divided into two classes by action spectra; four in class 1 (a broad action spectrum) and 12 in class 2 (a narrow action spectrum) . Class 1 suppressors are less frequent in terms of not only total number but also number per locus than class 2 suppressors, indicating difference in either or both mutation frequency and selective pressure between suppressors of the two classes . Two of the class 1 suppressors, SUP152 and SUP161, do not recombine with SUP28 and SUP33, leucine-inserting UAA suppressors, respectively, indicating that they are mutations in genes coding for tRNA(Leu)UUA . Of the remaining two class 1 suppressors, SUP160 which causes lethality in the psi+ cytoplasm is mapped on chromosome XV very close to the centromere, and SUP165 on the right arm of chromosome XIV 44 cM distal to lys9 . Of the class 2 suppressors, ten do not recombine with one or another of previously known UGA suppressors . The remaining two class 2 suppressors, SUP154 and SUP155, are mapped on the left and right arms of chromosome VII, respectively. Mol Biol (Mosk), 1988 Jan-Feb, 22(1), 224 - 30 {Comparative analysis of the structure of double-stranded RNA from killer strains of Saccharomyces cerevisiae}; Amosova OA et al.; Double-stranded RNAs (M and L molecules) of two strains of the killer system Saccharomyces cerevisiae M437 (wild type) and ski-5 (superkiller mutant) were studied by means of electron microscopy and high resolution thermal melting . The M molecules of the ski-5 mutant were by 100 b.p . shorter than those of M437 . L molecules were of the same length for both strains . Analysis of the differential melting curves of L molecules showed that L molecules differ significantly in their nucleotide sequences, whereas M molecules were practically identical . It was found that M molecules contained a long AU region: that of M molecules of M 437 was 170-180 b.p . long and contained almost no GC pairs, whereas the AU region of M molecules of the ski-5 mutant was three times shorter and contained GC pairs. Environ Mol Mutagen, 1988, 11(4), 497 - 508 Induction of mitotic chromosome loss in the diploid yeast Saccharomyces cerevisiae D61.M by genotoxic carcinogens and tumor promoters; Albertini S et al.; Three genotoxic carcinogens and eight tumor promoters were tested for induction of aneuploidy, specifically chromosome loss, in Saccharomyces cerevisiae D61.M . This is a heterozygous diploid yeast strain that permits the scoring of segregants expressing three linked recessive markers (cyhR2, ade6, and leu1), two of which (ade6 and leu1) are located close to the centromere on opposite arms of chromosome VII . The centromere marker leu was routinely checked, and a positive control (bavistan) was run with every experiment . The three genotoxic carcinogens aflatoxin B1, benzo(a)pyrene, and 7,12-dimethylbenz(a)anthracene did not induce aneuploidy, independent of the presence or absence of an exogenous metabolic activation system (rat liver homogenate; S9) . Four of the eight tumor promoters tested induced chromosome loss but not mitotic recombination or mutation: cholic acid, lithocholic acid, phenobarbital, and saccharin . Diethylstilbestrol (DES) led to positive as well as to negative results in several independent experiments . In the case of the positive experiment, DES also induced putative recombinants . Three tumor promoters induced neither chromosome loss nor mitotic recombination: anthralin, 4,4'-dichloro-diphenyl-ethane (DDT) and gamma-hexachlorcyclohexane (lindane) . From our experiments it can be concluded that the hypothesis put forward by Parry et al . {Nature; 294:263-265}, according to which tumor promoters induce chromosome loss in yeast, is not correct in a general sense . In our set of eight tumor promoters, only one half distinctly induced chromosome loss. Folia Microbiol (Praha), 1988, 33(1), 34 - 7 A rapid preparation of plasmid DNA from Saccharomyces cerevisiae; Brozmanova J et al.; A procedure for extraction of plasmid DNA from Saccharomyces cerevisiae is described . The plasmid DNA of interest is extracted together with 2-micron circular DNA naturally occurring in many yeast strains . Spheroplasts are lyzed at alkaline pH which denatures linear but not covalently closed circular (CCC) DNA . The CCC DNA is recovered by ethanol precipitation and can be detected by gel electrophoresis or used for routine bacterial transformation. Curr Genet, 1988, 13(1), 37 - 40 Allelism between pso1-1 and rev3-1 mutants and between pso2-1 and snm1 mutants in Saccharomyces cerevisiae; Cassier-Chauvat C et al.; In the yeast Saccharomyces cerevisiae, allelism between the pso1-1 and the rev3-1 mutants on the one hand and the pso2-1 and snm1 mutants on the other, is demonstrated by the comparison of phenotypes, complementation tests and meiotic segregation analysis. Curr Genet, 1988, 13(1), 21 - 3 A colony procedure for transformation of Saccharomyces cerevisiae; Keszenman-Pereyra D et al.; A rapid and simple yeast transformation procedure has been developed using colonies on agar plates . Saccharomyces cerevisiae SHY3 cells were picked up from colonies on YPD plates grown freshly or stored at 4 degrees C and incubated with M13RK9-T DNA at 30 degrees C for 1-2 h in a solution of Li+, Ca2+, Mg2+, triacetin and polyethylene glycol . About 3,500 transformants were obtained per microgram of double stranded M13RK9-T DNA . Unlike the existing spheroplast techniques, single stranded M13RK9-T DNA transformed intact cells below one-hundredth frequency of the duplex form. Environ Mol Mutagen, 1988, 11(3), 323 - 31 Effect of treatment medium on induction of aneuploidy by nocodazole in Saccharomyces cerevisiae; Taylor-Mayer RE et al.; While studying ways to improve responsiveness of Saccharomyces cerevisiae strain D61.M to agents that induce aneuploidy, we noted that nocodazole, which strongly induces aneuploidy when yeast cells are treated in yeast extract-peptone-dextrose (YEPD) medium, had no effect when a synthetic complete (SC) medium was used . Further study revealed that the presence of peptone was necessary for induction . Other aneuploidy-inducing agents, including ethyl acetate, acetone, and methyl benzimidazole-2-yl-carbamate (MBC), were equally active in either medium . Benomyl, which degrades to MBC, was less active in SC than in YEPD medium. Radiobiologiia, 1988 Jan-Feb, 28(1), 134 - 7 {Genetic effects of the decay of radionuclide products of nuclear fission in Saccharomyces cerevisiae cells . Lethal effects of the decay of 89Sr incorporated in cells of different ploidies and radiosensitivity}; Korolev VG et al.; Decay of 89Sr incorporated in yeast cells produces a pronounced inactivating effect . The transmutation mainly contributes (about 80%) to cell inactivation . Haploid cells are more sensitive to 89Sr disintegration than diploid and tetraploid ones . A radiosensitive mutant XRS2, that is particularly sensitive to the transmutation effect of radionuclides, has proved to be sensitive to 89Sr transmutation as well . At the same time, another radiosensitive mutant, rad 54, does not virtually differ from the wild-type strain by its sensitivity to 89Sr decay. Mol Gen Genet, 1988 Jan, 211(1), 168 - 75 Regulation of the TRP4 gene of Saccharomyces cerevisiae at the transcriptional level and functional analysis of its promotor; Furter R et al.; The TRP4 gene of Saccharomyces cerevisiae, which encodes anthranilate phosphoribosyl transferase (E.C . 2.4.2.18), is subject to the general control of amino acid biosynthesis . The regulation takes place at the transcriptional level by increasing the amount of initiation and not by changing the stability of mRNA . We have observed a change in the utilization of TRP4 mRNA start sites, depending on whether cells were grown under repressing or derepressing conditions . The function of promoter elements has been tested by deletion analysis with a plasmid-encoded TRP4 gene . A routinely practicable method was used for copy-number calibration of plasmids based on 2 micron DNA . Promoter structures and spacing problems in the TRP4 promoter region are discussed. Proc Natl Acad Sci U S A, 1988 Jan, 85(2), 534 - 8 Generation of telomere-length heterogeneity in Saccharomyces cerevisiae; Shampay J et al.; Chromosome ends in the lower eukaryotes terminate in variable numbers of tandem, simple DNA repeats . We tested predictions of a model in which these telomeric repeats provide a substrate for the addition of more repeats by a terminal transferase-like mechanism that, in concert with DNA polymerase and primase, effectively counterbalances the loss of DNA due to degradation or incomplete replication . For individual chromosome ends in yeast, the mean length of any given telomere was shown to vary between different clonal populations of the same strain and to be determined by the initial length of that telomere in the single cell giving rise to the clone . This type of variation was independent of the major yeast recombination pathway . The length heterogeneity at each telomeric end increased with additional rounds of cell division or DNA replication . Lengths of individual telomeres within a single clone varied independently of each other . Thus, this clonal variability is distinct from genetic regulation of chromosome length, which acts on all chromosome ends coordinately . These in vivo phenomena suggest that lengthening and shortening activities act on yeast telomeres during each round of replication. Environ Mol Mutagen, 1988, 11(1), 31 - 40 Aneuploidy induction in Saccharomyces cerevisiae by two solvent compounds, 1-methyl-2-pyrrolidinone and 2-pyrrolidinone; Mayer VW et al.; A number of solvent compounds that were tested in Saccharomyces cerevisiae were potent inducers of aneuploidy, although they did not induce any other genetic effects . As an extention of these earlier findings, 1-methyl-2-pyrrolidinone was tested and was found to induce aneuploidy . Several structurally related compounds were also tested; 2-pyrrolidinone induced aneuploidy, but succinimide, pyrrolidine, 1-methylpyrrolidine, 1-methyl-3-pyrrolidinol, and 2-pyrrolidineethanol did not . Maleimide and its N-hydroxy, N-methyl, and N-ethyl derivatives were also negative for aneuploidy induction. Mutat Res, 1988 Jan, 197(1), 59 - 66 Effects of excess thymidylate on thymidylate low-requiring strains of Saccharomyces cerevisiae: high mutagenicity and absence of DNA strand breaks; Bender E et al.; dTMP exposure concentrations of 0.1 mM or higher are genotoxic in exponentially growing cells of thymidylate low-requiring mutants of Saccharomyces cerevisiae . Mutagenicity of excess dTMP is highest in an exposure concentration 10-fold of that needed for external supplementation of endogenously blocked thymidylate synthesis . Still higher dTMP concentrations are primarily cytotoxic . The canavanine forward-mutation system shows excess dTMP to be as potent a mutagen as irradiation by ultraviolet light . Mutagenicity of excess dTMP, however, differs from that of direct DNA-attacking mutagens in that it is highest in the absence of significant toxicity . Alkaline sucrose gradient centrifugation shows that excess dTMP does not induce significant numbers of DNA single- or double-strand breaks, while conditions of thymidylate deprivation lead to DNA-strand breaks and thymineless death. Mol Cell Biol, 1988 Jan, 8(1), 361 - 70 Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo; Ganguly S et al.; We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNA(Leu) gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro . Parallel studies were carried out with the wild-type (Su-) tRNA(Leu) gene . Extracts from HeLa or CV1 cells transcribed both tRNA(Leu) genes . The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNA(Leu) . Surprisingly, when the same tRNA(Leu) genes were introduced into CV1 cells, only pre-tRNAs(Leu) were produced . The pre-tRNAs(Leu) made in vivo were of the same size and contained the 5'-leader and 3'-trailer sequences as did pre-tRNAs(Leu) made in vitro . Furthermore, the pre-tRNAs(Leu) made in vivo were processed to mature tRNA(Leu) when incubated with HeLa cell extracts . A tRNA(Leu) gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini . Thus, processing of pre-tRNA(Leu) in CV1 cells is blocked at the level of 5'- and 3'-end maturation . One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAs(Leu) synthesized in CV1 cells are not transported to the appropriate site. Mol Cell Biol, 1988 Jan, 8(1), 309 - 20 Identification of a DNA segment that is necessary and sufficient for alpha-specific gene control in Saccharomyces cerevisiae: implications for regulation of alpha-specific and a-specific genes; Jarvis EE et al.; STE3 mRNA is present only in Saccharomyces cerevisiae alpha cells, not in a or a/alpha cells, and the transcript level increases about fivefold when cells are treated with a-factor mating pheromone . Deletions in the 5' noncoding region of STE3 defined a 43-base-pair (bp) upstream activation sequence (UAS) that can impart both modes of regulation to a CYC1-lacZ fusion when substituted for the native CYC1 UAS . UAS activity required the alpha 1 product of MAT alpha, which is known to be required for transcription of alpha-specific genes . A chromosomal deletion that removed only 14 bp of the STE3 UAS reduced STE3 transcript levels 50- to 100-fold, indicating that the UAS is essential for expression . The STE3 UAS shares a 26-bp homology with the 5' noncoding sequences of the only other known alpha-specific genes, MF alpha 1 and MF alpha 2 . We view the homology as having two components--a nearly palindromic 16-bp "P box" and an adjacent 10-bp "Q box." A synthetic STE3 P box was inactive as a UAS; a perfect palindrome P box was active in all three cell types . We propose that the P box is the binding site for a transcription activator, but that alpha 1 acting via the Q box is required for this activator to bind to the imperfect P boxes of alpha-specific genes . Versions of the P box are also found upstream of a-specific genes, within the binding sites of the repressor alpha 2 encoded by MAT alpha . Thus, the products of MAT alpha may render gene expression alpha or a-specific by controlling access of the same transcription activator to its binding site, the P box. Mol Cell Biol, 1988 Jan, 8(1), 210 - 25 Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae; Buchman AR et al.; Two DNA-binding factors from Saccharomyces cerevisiae have been characterized, GRFI (general regulatory factor I) and ABFI (ARS-binding factor I), that recognize specific sequences within diverse genetic elements . GRFI bound to sequences at the negative regulatory elements (silencers) of the silent mating type loci HML E and HMR E and to the upstream activating sequence (UAS) required for transcription of the MAT alpha genes . A putative conserved UAS located at genes involved in translation (RPG box) was also recognized by GRFI . In addition, GRFI bound with high affinity to sequences with the (C1-3A)-repeat region at yeast telomeres . Binding sites for GRFI with the highest affinity appeared to be of the form 5'-(A/G)(A/C)ACCCANNCA(T/C)(T/C)-3', where N is any nucleotide . ABFI-binding sites were located next to autonomously replicating sequences (ARSs) at controlling elements of the silent mating type loci HMR E, HMR I, and HML I and were associated with ARS1, ARS2, and the 2 micron plasmid ARS . Two tandem ABFI binding sites were found between the HIS3 and DED1 genes, several kilobase pairs from any ARS, indicating that ABFI-binding sites are not restricted to ARSs . The sequences recognized by ABFI showed partial dyad-symmetry and appeared to be variations of the consensus 5'-TATCATTNNNNACGA-3' . GRFI and ABFI were both abundant DNA-binding factors and did not appear to be encoded by the SIR genes, whose products are required for repression of the silent mating type loci . Together, these results indicate that both GRFI and ABFI play multiple roles within the cell. J Bacteriol, 1988 Jan, 170(1), 266 - 71 Structure and transcription of the allantoate permease gene (DAL5) from Saccharomyces cerevisiae; Rai R et al.; We determined the nucleotide sequence of the DAL5 gene, which encodes a component of the allantoate transport system . Translation of the sequence revealed that the DAL5 gene product is highly hydrophobic . It possesses an alternating motif of hydrophilic sequences that can potentially be folded into alpha-helices and hydrophobic sequences that can potentially be folded into beta-pleated sheets . These are expected characteristics of an integral membrane protein, which correlate well with DAL5 gene function . S1 protection fragments generated by DAL5 transcripts exhibited high heterogeneity over a 30-base-pair range . This pattern of fragments was not affected by growth conditions of the cells or the conditions of the assay. J Mol Evol, 1988, 27(4), 341 - 50 Evolutionary relationship and secondary structure predictions in four transport proteins of Saccharomyces cerevisiae; Weber E et al.; The comparison of the amino acid sequences of four yeast transport proteins indicates that there is a questionable relatedness between the uracil permease (FUR4) and the purine-cytosine permease (FCY2), whereas the arginine permease (CAN1) and the histidine permease (HIP1) clearly originated from a common molecular ancestor . The analysis of the primary structure of these transport proteins by two methods of secondary structure predictions suggests the presence of 9-12 membrane-spanning alpha-helices in each polypeptide chain . These results are concordant in that 90% of the alpha-helices were determined by both methods to be at the same positions . In the aligned sequences HIP1 and CAN1, the postulated membrane-spanning alpha-helices often start at corresponding sites, even though the overall sequence similarity of the two proteins is only 30% . In the aligned DNA coding sequences of CAN1 and HIP1, synonymous nucleotide substitutions occur with very similar frequencies in regions where the replacement substitution (changing the amino acids) frequencies are widely different . Moreover, our data suggest that the replacement substitutions can be considered as neutral in the N-terminal segment, whereas the other regions are subject to a conservative selective pressure because, if compared to a random drift, the replacement substitutions are underrepresented. Folia Microbiol (Praha), 1988, 33(4), 285 - 91 Uptake of L-lysine by a double mutant of Saccharomyces cerevisiae; Garcia JC et al.; A gap1 can1 mutant of Saccharomyces cerevisiae with a single lysine transport system remaining was used to study detailed kinetics of this transport . Its half-saturation constant was 78 mumol per litre, its maximum rate of transport was 0.29 mumol L-lysine per g dry matter per minute, both parameters being lower by more than an order of magnitude in comparison with the GAP system . The pH optimum lay at very acid values of about 3, the temperature dependence without any transition point showed an activation energy of 48 kJ/mol . The transport was inhibited by common metabolic inhibitors (3'-chlorophenylhydrazonomalononitrile, antimycin, 2-deoxy-D-glucose, sodium arsenate) as well as by a membrane-active one (uranyl nitrate) . The specificity of the system was extremely high, none of the natural amino acids acting as competitor to L-lysine . The maximum accumulation ratio attained (at about 5 mg dry matter per mL) was 100: 1-120: 1, in agreement with the measured protonmotive force under the assumption of 1 H+ ion being transported with 1 lysine molecule . The ratio decreased with increasing external concentration of lysine to as little as 4: 1 at 1 mmol lysine per litre . It also decreased with increasing suspension density and it was at extremely low suspension densities (0.2 mg dry matter per mL) that ratios of as much as 500: 1 were reached . Application of group-specific inhibitors showed that the active site of the carrier contains an essential histidine residue. Folia Microbiol (Praha), 1988, 33(4), 281 - 4 Effect of ethanol on the specific transport system for L-lysine in Saccharomyces cerevisiae; Garcia JC et al.; Preincubation of resting cells of Saccharomyces cerevisiae double mutant can1 gap1 (with a single transport system for L-lysine) with metabolic substrates stimulated subsequent uptake of lysine . While in the wild type the stimulation is connected primarily with carrier protein synthesis (delayed, cycloheximide-inhibitable effect) in the mutant an immediate tapping of an energy source (antimycin-inhibited) is practically solely involved. Biochem J, 1988 Jan 1, 249(1), 163 - 70 Purification and properties of an extracellular glucoamylase from a diastatic strain of Saccharomyces cerevisiae; Kleinman MJ et al.; The extracellular glucoamylase from certain strains of Saccharomyces cerevisiae can be purified from culture medium by a simple chromatographic procedure . The native enzyme is heavily glycosylated and has an Mr of about 250,000, but gel filtration indicates the existence of oligomers of larger size . Dissociation yields a form of Mr about 70,000 . The glucoamylase is rich in serine and threonine and in aspartic acid plus asparagine, and has a pI of 4.62 and a pH optimum of 4.5-6.5 . The thermostability and resistance to denaturants of the yeast enzyme is compared with those of two other fungal glucoamylases . Kinetic data for the yeast enzyme and a variety of substrates is presented; the enzyme is particularly ineffective in cleaving alpha-(1----6)-glycosidic bonds. Proc Natl Acad Sci U S A, 1988 Jan, 85(1), 55 - 9 The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin; MacKay VL et al.; Saccharomyces cerevisiae a cells secrete an extracellular protein, called "barrier" activity, that acts as an antagonist of alpha factor, the peptide mating pheromone produced by mating-type alpha cells . We report here the DNA sequence of BAR1, the structural gene for barrier activity . The deduced primary translation product of 587 amino acids has a putative signal peptide, nine potential asparagine-linked glycosylation sites, and marked sequence similarity of the first two-thirds of the protein with pepsin-like proteases . Barrier activity was abolished by in vitro mutation of an aspartic acid predicted from this sequence homology to be in the active site . Therefore, barrier protein is probably a protease that cleaves alpha factor . The sequence similarity suggests that the first two-thirds of the barrier protein is organized into two distinct structural domains like those of the pepsin-like proteases . However, the BAR1 gene product has a third carboxyl-terminal domain of unknown function; deletion of at least 166 of the 191 amino acids of this region has no significant effect on barrier activity. Folia Microbiol (Praha), 1988, 33(6), 447 - 52 Ethanol tolerance of Saccharomyces cerevisiae and its relationship to lipid content and composition; Ghareib M et al.; Saccharomyces cerevisiae strain 14-12 is a highly ethanol-tolerant organism . It can grow in the presence of 13% ethanol but growth is completely prevented at 14% ethanol . A relationship was detected between yeast lipids and ethanol tolerance . A gradual decrease of lipid content was recorded as the concentration of supplemented ethanol increased . Moreover, free fatty acids were comparatively decreased in these lipid extracts . When separately added to media with 14% ethanol different lipids produced varied stimulatory effects on yeast growth . Maximum yield of yeast growth was obtained at 14% ethanol in the presence of lecithin, palmitic acid and cholesterol . Yeast lipids produced in the presence of these fractions are characterized by a relatively high percentage of free fatty acids . The change in the percentage of free fatty acids was shown to be the controlling factor in ethanol tolerance. Acta Biochim Pol, 1988, 35(2), 105 - 18 Protein composition of Saccharomyces cerevisiae mitochondrial ribosomes; Mieszczak M et al.; Protein composition of mitochondrial ribosomes of the yeast Saccharomyces cerevisiae was analysed by two-dimensional electrophoresis . The small (37S) mitoribosomal subunit contains 36 different polypeptides with molecular weights ranging from 10,000 to 60,000 . The large (50S) subunit is composed of 41 proteins with molecular weights from 10,000 to 43,000 . The molecular weights of mitoribosomal small and large subunits are 1.85 MDa and 2.35 MDa, respectively . Proteins represent 60-62% and 42-45% of the total mass of 37S and 50S subunits respectively . On the basis of the protein content and molecular weights of individual proteins we conclude that all mitoribosomal proteins are present in the mitoribosome in equimolar proportions. J Basic Microbiol, 1988, 28(5), 335 - 42 Construction of promoter-probe vectors for Candida maltosa, a n-alkane-assimilating yeast, using the LEU2 gene of Saccharomyces cerevisiae; Takagi M et al.; For the purpose of isolation of promoter regions which are regulated by a carbon source in the medium in an n-alkane-assimilating yeast, Candida maltosa, two promoter-probe vectors were constructed . Each of them consists of the LEU2 gene of Saccharomyces cerevisiae whose 5'-noncoding region was trimmed with BAL31, an autonomously replicating sequence isolated from C . maltosa genome (the TRA region) which we have previously isolated, and the pBR322 sequence . One of them, pPLC2, having the TATA box, lacks the regulatory sequence ("sequence L") of the LEU2 gene, and the other, pPLC1, lacks both the TATA box and sequence L . Using pPLC1 as a short-gun cloning vector in C . maltosa, many promoter regions which were active when glucose was present in the medium as a carbon source were obtained from the genome of C . maltosa . The sizes of the inserted fragments of two of them were determined . (In this paper, a promoter region refers to a promoter which includes a TATA box, plus a regulatory sequence such as an UAS (upstream activating sequence)-like sequence). Folia Microbiol (Praha), 1988, 33(5), 372 - 6 The irreversibility of thiamin transport in Saccharomyces cerevisiae; Ruml T et al.; Neither exit nor counterflow efflux of thiamin, taken up previously by an active transport, were found in Saccharomyces cerevisiae, in either the wild type or a mutant with a lower rate of thiamin phosphorylation . Complete inhibition of thiamin phosphorylation by oxythiamin did not lead to any release of thiamin taken up by the cell. Folia Microbiol (Praha), 1988, 33(4), 292 - 7 Increased sterol formation in Saccharomyces cerevisiae . Analysis of cell components and ultrastructure of vacuoles; Behalova B et al.; In Saccharomyces cerevisiae nitrogen limitation under aerobic conditions (low specific growth rate) provokes an enhanced synthesis of sterols . Analysis of east cultures during the enhanced sterol biosynthesis showed a temporary decrease of protein content and a simultaneous increase in polysaccharide and lipid levels . This was reflected in the ultrastructure of cells where numerous lipid globules (spherosomes, oleosomes) appeared around extensive membrane-bound compartments containing membrane vesicles and lipoprotein material . Electronograms showed that such compartments were formed between the layers of endoplasmic reticulum and belonged to the vacuome phase of the yeast cell . It appears that vacuoles formed in yeast during enhanced synthesis of sterols have a storage rather than a lysosomal function. Nahrung, 1988, 32(4), 311 - 8 Structure of protein solutions . Part 2 . Solutions of total protein of yeast Saccharomyces cerevisiae; Soloshenko VM et al.; Water systems formed by total protein of yeast Saccharomyces cerevisiae and model systems on the basis of unfractionated casein were studied by viscosimetry and electron microscopy . Methodologically, both methods offered a possibility of distinguishing several quantitatively different concentration intervals in protein solutions under different conditions . Viscosimetry gave the most informative results at the concentration intervals where the solutions approached the true ones, while electron microscopy did so when supermolecular formations prevailed . In comparing the data obtained by these two methods the following conclusions were drawn: At low temperatures (4-10 degrees C) yeast protein was in molecular-dissociated condition . Association in the system starts at 10 degrees C, and becomes enhanced with a rise in temperature . A temperature rise to 25-30 degrees C promotes a more compact packing of polypeptide chains; further heating leads to high-temperature denaturation . Lipids induce covering of polypeptide chains and prevent associations, thus leading to an increase in the system's thermostability. Prog Clin Biol Res, 1988, 268B, 85 - 90 Development of a heterologous gene expression system for the Na,K-ATPase subunits in the yeast Saccharomyces cerevisiae; Horowitz B et al.; cDNA fragments coding for the alpha and beta subunits of the Na,K-ATPase were separately ligated into the yeast expression vector YEp1PT in both the sense (YEpNKA(+)) and anti-sense (YEpNKA(-)) orientations with respect to the promoter . The recombinant plasmids were introduced into Saccharomyces cerevisiae strain UT4 by transformation . Total RNA from the transformed strains was isolated and analyzed by Northern hybridization . The resulting autoradiogram revealed strong signals indicative of a high level of transcriptional expression of both subunits in both orientations of the cDNA . 35S-Methionine labeled extracts were immunoprecipitated with antibodies specific for the beta subunit . A beta subunit translation product was produced from YEp beta NKA(+) but not from YEp beta NKA(-) . Experiments to detect an alpha specific translation product are in progress. Mol Microbiol, 1988 Jan, 2(1), 89 - 99 Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae; Britton P et al.; Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA which was sequenced . Two non-overlapping open reading frames (ORFs) were identified . The largest, encoding a polypeptide of 382 amino acids (relative molecular mass (Mr) 43,483), was shown to be the viral nucleoprotein gene . The second ORF, found 3' to the larger ORF, encodes a polypeptide of 78 amino acids (Mr 9068) which has yet to be assigned to a viral product . The nucleoprotein gene was expressed in yeast cells under the control of two types of yeast promoters: the constitutive PGK promoter, and the inducible GAL1 promoter . Yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of Mr 47,000, identical to the viral product, that reacted with a specific monoclonal antibody. Mol Gen Genet, 1988 Jan, 211(1), 155 - 9 Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae; Zealey GR et al.; A 2 micron circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed . Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell . Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity . The mitotic stability of the plasmid in "high-copy" and "low-copy" number cells was determined . "High-copy" number cells showed a greater mitotic stability . The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression. Mol Cell Biol, 1988 Jan, 8(1), 505 - 10 SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae; Wilson RB et al.; sra5 mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km cyclic AMP (cAMP) phosphodiesterase activity . We have cloned SRA5 by complementation . It maps to the right arm of chromosome XV, tightly linked to PRT1, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase . Disruptions of SRA5 allowed ras1 ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP . sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP. Acta Microbiol Pol, 1988, 37(3-4), 271 - 80 Biological effect of alkoxymethylene trimethylammonium chlorides on yeast Saccharomyces cerevisiae; Skala J et al.; Six compounds of the group of quaternary ammonium salts have been tested for their biological activity using yeasts as a biological system . They have an inhibitory effect on respiration, cell growth and amino acid transport . A destroying action on protoplast regeneration and respiration has been also observed . The studied chemicals appear to have very pleiotropic action, focused on a damage of mitochondrial and cell plasma membranes. Eur J Biochem, 1987 Dec 30, 170(1-2), 241 - 6 Secretion of biologically active porcine prophospholipase A2 by Saccharomyces cerevisiae . Use of the prepro sequence of the alpha-mating factor; van den Bergh CJ et al.; The cDNA coding for porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in Saccharomyces cerevisiae . Expression and secretion of proPLA could only be obtained after fusing the proPLA to the prepro sequence of the yeast alpha-mating factor . Upon secretion, the fusion protein was cleaved by the KEX2 protease yielding a 140-amino-acid zymogen-like form of the phospholipase A2 . This protein was purified in high yield by ion-exchange chromatography . Limited proteolysis with trypsin cleaved the 'zymogen' to yield active phospholipase A2, which was indistinguishable from the authentic porcine pancreatic enzyme . These results show that a protein with a disulphide bridge content as high as 7 per 124 amino acid residues can be correctly processed by the yeast secretory apparatus. Eur J Biochem, 1987 Dec 30, 170(1-2), 217 - 24 Influence of different 5'-flanking sequences of tRNA genes on their in vivo transcription efficiencies in Saccharomyces cerevisiae; Dingermann T