Biochemistry, 1987 Aug 25, 26(17), 5377 - 82 Yeast DNA primase is encoded by a 59-kilodalton polypeptide: purification and immunochemical characterization; Biswas EE et al.; The DNA primase from the yeast Saccharomyces cerevisiae has been purified 9200-fold to homogeneity . The yeast DNA primase is a monomeric protein of molecular weight 59,000, and under conditions described in this report, it is stable at 4 or -80 degrees C . The primase does not bind to DEAE-cellulose, is not inhibited by a high concentration of alpha-amanitin (4 mg/mL), and is capable of synthesizing small (up to 15 nucleotides in length) ribo or ribo-deoxy mixed initiator RNA primers . The primer synthesis is stimulated by ATP; however, other ribonucleotides could be replaced by deoxynucleotides without any measurable effect on the overall DNA synthesis . Thus, the purified primase is distinct from the RNA polymerases of S . cerevisiae . Immunoblot analysis of the polypeptides in a crude cell extract using a mouse polyclonal antibody prepared against the highly purified primase indicates that the 59-kilodalton polypeptide is the native form and not a degraded form of a larger polypeptide; however, primase is degraded rapidly to smaller polypeptides by yeast proteases especially in the absence of protease inhibitors. Nucleic Acids Res, 1987 Aug 25, 15(16), 6387 - 403 Analysis of transcripts of the major cluster of tRNA genes in the mitochondrial genome of S . cerevisiae; Francisci S et al.; The transcripts of a 6Kbp region of the mitochondrial genome of S . cerevisiae, localized in the 21S rRNA-OXI1 span and including 12 tRNA genes (from tRNA(thr) to tRNA(ala)) and several G+C clusters, have been studied by analysis of in vitro capped primary transcripts and by fine mapping of the 5' ends of transcripts . The study was performed in the w.t . strain D273-10B and in several rho- mutants retaining different, partially overlapping portions of the studied region; the mutants accumulate incompletely-processed precursors of tRNAs due to the absence of the tRNA synthesis locus . Results show the presence in the region of four sites at which initiation occurs at a consensus nonanucleotide ATTATAAGTA (or a minor variant of the same); however different initiation sites are used in different strains, and several differences as compared to initiation in vitro can also be observed . Termini arising by processing are often localized at AATATAA or AATATATTTT sequences localized immediately adjacent to a G+C cluster or a tRNA sequence. Experientia, 1987 Aug 15, 43(8), 886 - 8 Protective effect of vitamins against trichothecene toxicity towards Saccharomyces cerevisiae; Yagen B et al.; Several trichothecene mycotoxins were shown to inhibit the growth of Saccharomyces cerevisiae . This effect was most pronounced with the macrocyclic trichothecenes, especially verrucarin A . Much less growth inhibition was observed with T-2 toxin . Verrucarol, diacetoxyscirpenol, acetyl T-2 toxin, HT-2 toxin, T-2 tetraol and neosolaniol were inactive at a concentration of 75 micrograms of toxin per disc . Incubation of S . cerevisiae with verrucarin A together with vitamins resulted in a decrease in toxicity . Pyridoxine-HCl, Ca-pantothenate, thiamine-HCl and alpha-tocopheryl acetate were amongst the most potent of the vitamins tested which reversed growth inhibition, overcoming the inhibitory potential of the toxins. Biochem Biophys Res Commun, 1987 Aug 14, 146(3), 1093 - 101 Reversion of 7-methylguanosine 5'-phosphate inhibition of mRNA translation by polysomal and soluble factors isolated from Saccharomyces cerevisiae; Parets Suler A et al.; Protein fractions that overcome m7GMP inhibition of mRNA translation have been purified from the yeast S . cerevisiae . An active fraction isolated from polysomes contains two polypeptides of 220- and 190-kDa . The active fraction isolated from postribosomal supernatant contains a major polypeptide of 28-kDa and other species of 32-, 24-, 22- and 21-kDa, and sediments in sucrose gradients as a high molecular weight complex of about 200,000 . This fraction restored yeast mRNA translation in reticulocyte lysates under conditions of yeast and globin mRNA competition; however, this effect was not observed with the 220- and 190-kDa polypeptides from polysomes . Nevertheless, translation of yeast mRNA was stimulated by a partially purified fraction containing a 28-kDa polypeptide from polysomes. Cell, 1987 Aug 14, 50(4), 593 - 602 S . cerevisiae U1 RNA is large and has limited primary sequence homology to metazoan U1 snRNA; Kretzner L et al.; We have cloned and sequenced the yeast SNR19 gene and show here that snR19 is the yeast homolog of metazoan U1 snRNA . sn R19 is 569 nucleotides long, strikingly larger than its metazoan counterpart . The two molecules resemble each other closely in the predicted secondary structure of their first 50 nucleotides . Primary sequence homology is restricted to some of their single-stranded regions, including 11 consecutive nucleotides at the 5' end of the two molecules, the region that interacts with pre-mRNA 5' splice junctions . snR19 is spliceosome-associated and required for in vitro pre-mRNA splicing . We also note that 8 sequences in snR19 have extensive complementarity to snR20, the large yeast U2 RNA, suggesting that yeast U1 may interact with yeast U2 by base-pairing. Cell, 1987 Aug 14, 50(4), 585 - 92 An essential snRNA from S . cerevisiae has properties predicted for U4, including interaction with a U6-like snRNA; Siliciano PG et al.; Three yeast snRNAs (snR20, snR7, and snR14) have been implicated in pre-mRNA splicing . snR20 and snR7 contain domains of homology to U2 and U5, respectively, and each is required for viability . These RNAs are found associated with the spliceosome, as is snR14 . We show here that snR14 is also an essential gene product . Sequence analysis reveals that, like snR7 and snR20, snR14 contains a consensus binding site for the Sm antigen, a feature common to all mammalian snRNAs involved in splicing . Moreover, snR14 exhibits several blocks of sequence and structural homology to U4, which in metazoans is found in association with U6 . Native gel electrophoresis demonstrates that snR14 is in fact base-paired with another yeast snRNA, designated snR6, which has primary sequence homology to U6 . We conclude that snR14 is the yeast analog of U4. J Biol Chem, 1987 Aug 5, 262(22), 10426 - 9 Identification of guanine nucleotides bound to ras-encoded proteins in growing yeast cells; Gibbs JB et al.; We have analyzed the guanine nucleotides bound to mammalian ras and yeast RAS proteins overexpressed in {32P}orthophosphate-labeled cultures of exponentially growing Saccharomyces cerevisiae cells . Whereas S . cerevisiae RAS1 and RAS2 proteins were immunoprecipitated bound entirely to GDP, mammalian Harvey ras was isolated with GTP and GDP bound in near-equimolar proportions . In a strain overexpressing a RAS2 variant where the RAS unique C-terminal domain was deleted, both GTP and GDP were detected in a ratio of 3:97 . Increased amounts of GTP (16-75% of total guanine nucleotide) were observed bound to all ras proteins containing mutations that inhibit GTP hydrolytic activity . Increasing proportions of GTP bound to the various ras proteins correlated with increasing biological potency to bypass cdc25 lethality in yeast. Virology, 1987 Aug, 159(2), 450 - 3 Expression and glycosylation of the respiratory syncytial virus G protein in Saccharomyces cerevisiae; Ding MX et al.; A cDNA encoding the entire amino acid sequence of the G glycoprotein of respiratory syncytial virus (RSV) was inserted into a yeast-Escherichia coli shuttle vector such that expression of the virus gene was regulated by the yeast GAL1 promoter . Transformation of Saccharomyces cerevisiae with the vector led to the formation of the G protein when cells were grown in the presence of galactose . Under these conditions the RSV G appeared as a 60- to 65-kDa glycosylated protein . Expression of the G cDNA in secretory mutants of S . cerevisiae yielded a protein of 35 kDa in a mutant unable to glycosylate secreted proteins and a 65-kDa polypeptide in a mutant unable to transport proteins beyond the endoplasmic reticulum . The RSV protein formed in the latter mutant was converted to a 60-kDa protein by endoglycosidase H . Our results show that yeast can recognize the internal signal sequence of RSV G protein and add glycosyl groups to the polypeptide in the endoplasmic reticulum . Evidence is presented for both N- and O-linked glycosylation of the virus glycoprotein. Mol Cell Biol, 1987 Aug, 7(8), 2914 - 24 Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression; Hoekema A et al.; The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets . The degree of this biased codon usage in each gene is positively correlated to its expression level . Highly expressed genes use these 25 major codons almost exclusively . As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene . The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome . Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level . The PGK protein levels dropped 10-fold . The steady-state mRNA levels also declined, but to a lesser extent (threefold) . Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons . By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically . We conclude that efficient mRNA translation is required for maintaining mRNA stability in S . cerevisiae . These findings have important implications for the study of the expression of heterologous genes in yeast cells. Cell, 1987 Jul 17, 50(2), 277 - 87 Three different genes in S . cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase; Toda T et al.; We have isolated three genes (TPK1, TPK2, and TPK3) from the yeast S . cerevisiae that encode the catalytic subunits of the cAMP-dependent protein kinase . Gene disruption experiments demonstrated that no two of the three genes are essential by themselves but at least one TPK gene is required for a cell to grow normally . Comparison of the predicted amino acid sequences of the TPK genes indicates conserved and variable domains . The carboxy-terminal 320 amino acid residues have more than 75% homology to each other and more than 50% homology to the bovine catalytic subunit . The amino-terminal regions show no homology to each other and are heterogeneous in length . The TPK1 gene carried on a multicopy plasmid can suppress both a temperature-sensitive ras2 gene and adenylate cyclase gene. J Biol Chem, 1987 Jul 5, 262(19), 9160 - 5 Construction of a synthetic Holliday junction analog and characterization of its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions; Evans DH et al.; We describe the construction and characterization of an oligonucleotide Holliday junction analog and characterize its interaction with a Saccharomyces cerevisiae endonuclease that cleaves Holliday junctions . A Holliday junction analog containing four duplex arms and 54 base pairs was constructed by annealing four unique synthetic oligonucleotides . Mixing curve analysis showed that the complex contained a 1:1:1:1 mol ratio of the four unique sequence strands . In addition, a linear duplex with a sequence identical to two of the junction arms was also constructed for use as a control fragment . High resolution gel exclusion chromatography was used to purify and characterize the synthetic junction . The synthetic Holliday junction was found to be a specific inhibitor of a S . cerevisiae enzyme that catalyzes the cleavage of Holliday junctions . Under standard cleavage conditions, 50% inhibition was observed at a synthetic Holliday junction to substrate ratio of 7/1, whereas no inhibition by linear duplex was observed at molar ratios in excess of 150/1 . Kinetic analysis showed that Holliday junction was a competitive inhibitor of the reaction and had an apparent Ki = 2.5 nM, although the mode of inhibition was complex . The synthetic Holliday junction was not a substrate for the enzyme, but was found to form a specific complex with the enzyme as evidenced by polyacrylamide gel electrophoresis DNA binding assays. Cell, 1987 Jul 3, 50(1), 17 - 29 Multiple exon-binding sites in class II self-splicing introns; Jacquier A et al.; Partial deletion of the exon 5' to S . cerevisiae intron a5, a self-splicing mitochondrial class II intron, reveals the existence of several sites of intron-exon interaction . We have identified two of the corresponding exon-binding sites in intron a5 by comparative sequence analysis and RNAase H digestion of the intron complexed to a DNA version of its 5' exon . Introduction of mutations in either the intronic sites or the complementary exonic sequences affects splicing in vitro, whereas double mutants in which intron-exon pairings have been restored show normal activity . Some of the mutants accumulate a product that was shown to be the intron-3' exon lariat, a postulated splicing intermediate . The possible role of one of the intronic sites in aligning exons for the ligation step is discussed. Rev Argent Microbiol, 1987 Jul-Sep, 19(3), 109 - 19 {Saccharomyces cerevisiae: porphobilinogenase activity in a wild-type strain and its heme-deficient mutant}; Araujo LS et al.; Properties of Porphobilinogenase (PBGase), the enzyme complex converting porphobilinogen (PBG) into uroporphyrinogens, were comparatively studied in a wild strain D273-10B and its mutant B231 of Saccharomyces cerevisiae, Figure 1 shows the growth curves for both strains . The basic pattern of growth was observed but, although S . cerevisiae is a facultative aerobe and was grown on dextrose, a diauxic growth curve was not observed . The beginning of the exponential phase was slightly delayed for the mutant, so, its generation time (G = 3.20 h) was greater than that for the wild strain (G = 1.26 h) . Optimum conditions for extracting the enzyme from both strains were found to be sonication at 10 mu for 3 min (Table 1) . Table 2 shows the effect of centrifugation at 24,000 xg for 30 min on activity . For both strains the amount of porphyrins formed was the same either in the absence or presence of air . It was found (Figure 2) that urogen formation was linear with protein over a wide range of concentrations and with incubation time up to 2h in agreement with previous results for the enzyme of different sources . Figure 3 shows the effect of pH on PBGase activity . An optimum pH of 7.4 was found for both strains employing sodium phosphate buffer pH 8.0 . The shape of the pH curve as well as optimum pH were the same in both Tris-HCl and phosphate buffer, however PBGase was 15% less active in the former . When plots of velocity against PBG concentration were analyzed for PBGase, it was found that measuring the rate of the reaction on the basis of total urogen formation, saturation curves for wild and mutant strains harvested at the exponential phase, followed classical Michaelis-Menten kinetics . Saturation was reached at PBG concentration of about 70-90 microM . Therefore, double reciprocal plots (Figure 4) were linear and from these plots apparent Km's values of 20 and 14 microM were obtained for the wild and mutant strain respectively . It is known that in some organisms, the activity of the enzyme of heme synthesis is significantly influenced by the days of growing; therefore the effect of time growing on PBGase activity was studied (Figure 5) . A well defined maximum of enzyme activity was observed for the mutant strain after 20h of growing; while activity of wild strain did not significantly vary during growth. Mol Cell Biol, 1987 Jul, 7(7), 2397 - 405 Genetic manipulation of centromere function; Hill A et al.; A conditional centromere was constructed in Saccharomyces cerevisiae by placing the centromere of chromosome III immediately downstream from the inducible GAL1 promoter from S . cerevisiae . By utilizing growth conditions that favor either transcriptional induction (galactose-carbon source) or repression (glucose-carbon source) from the GAL1 promoter, centromere function can be switched off or on, respectively . With the conditional centromere we were able to radically alter the mitotic transmission pattern of both monocentric and dicentric plasmids . Moreover, it was possible to selectively induce the loss of a single chromosome from a mitotically dividing population of cells . We observed that the induction of chromosome III aneuploidy resulted in a dramatic change in cell morphology . The construction of a conditional centromere represents a novel way to create conditional mutations of cis-acting DNA elements and will be useful for further analysis of this important stabilizing element. Mol Cell Biol, 1987 Jul, 7(7), 2329 - 34 Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae; Simon JR et al.; Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication . These constructs transform S . cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene . Single-stranded DNA was found to transform S . cerevisiae cells at efficiencies greater than that of double-stranded DNA . No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences . Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events . Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA. J Biol Chem, 1987 Jun 15, 262(17), 8159 - 64 Carbon isotope effects on the pyruvate dehydrogenase reaction and their importance for relative carbon-13 depletion in lipids; Melzer E et al.; A method has been developed for the positional 13C isotope analysis of pyruvate and acetate by stepwise quantitative degradation . On its base, the kinetic isotope effects on the pyruvate dehydrogenase reaction (enzymes from Escherichia coli and Saccharomyces cerevisiae) for both of the carbon atoms involved in the bond scission (double isotope effect determination) and on C-3 of pyruvate have been determined . The experimental k12/k13 values with the enzyme from E . coli on C-1 and C-2 of pyruvate are 1.0093 +/- 0.0007 and 1.0213 +/- 0.0017, respectively, and, with the enzyme from S . cerevisiae, the values are 1.0238 +/- 0.0013 and 1.0254 +/- 0.0016, respectively . A secondary isotope effect of 1.0031 +/- 0.0009 on C-3 (CH3-group) was found with both enzymes . The size of the isotope on C-1 indicates that decarboxylation is more rate-determining with the yeast enzyme than with the enzyme from E . coli, although it is not the entirely rate-limiting step in the overall reaction sequence . Assuming appropriate values for the intrinsic isotope effect on the decarboxylation step (k3) and the equilibrium isotope effect on the reversible substrate binding (k1, k2), one can calculate values for the partitioning factor R (k3/k2: E . coli enzyme 4.67, S . cerevisiae enzyme 1.14) and the intrinsic isotope effects related to the carbonyl-C (k1/k'1 = 1.019; k3/k'3 = 1.033) . The isotope fractionation at C-2 of pyruvate gives strong evidence that the well known relative carbon-13 depletion in lipids from biological material is mainly caused by the isotope effect on the pyruvate dehydrogenase reaction . In addition, our results indicate an alternating 13C abundance in fatty acids, that has already been verified in some cases. Yeast, 1987 Jun, 3(2), 77 - 84 Proliferation of microbodies in Saccharomyces cerevisiae; Veenhuis M et al.; The development of microbodies in the yeast Saccharomyces cerevisiae was studied in response to different conditions of growth . Various strains of S . cerevisiae were investigated, using cells from the exponential growth phase on glucose as an inoculum in all transfer experiments . Electron microscopy, including serial sectioning, revealed that these cells generally contained one to four small microbodies which were localized in the vicinity of the cell wall and characterized by the presence of catalase . Transfer of these glucose-grown cells into media supplemented with various compounds known to induce microbody proliferation in other yeasts--i.e . uric acid, alkylated amines, amino acids, C2-compounds such as ethanol or acetate, in the presence or absence of compounds that induce oxygen radical formation--did not result in a significant change in the number of microbody profiles observed . Marked microbody proliferation was, however, observed after a shift of cells into media containing oleic acid and was associated with the induction of activities of beta-oxidation enzymes . In addition, catalase and isocitrate lyase were present in enhanced levels . Kinetic experiments suggested that these microbodies developed from those originally present in the inoculum cells . In thin sections up to 14 microbody profiles were occasionally observed, often present in small clusters . Their ultimate volume fraction amounted to 8-10% of the cytoplasmic volume. Yeast, 1987 Jun, 3(2), 63 - 70 Artifactual immunofluorescent labelling in yeast, demonstrated by affinity purification of antibody; Lillie SH et al.; In the course of making antibodies against various yeast (S . cerevisiae) proteins, we have noted that it is common to observe reactivity of rabbit sera with a number of extraneous bands on Western transfers of yeast proteins . The pattern of reactive bands can change within a period of weeks, even when the rabbit has not been injected with antigen . A simple method of affinity purification, using antigen bound to nitrocellulose, is employed to remove the reactivity with these extraneous bands from immune sera . The importance of affinity purification is demonstrated by our attempts to immunolocalize a 55 kd yeast protein (p55) . Immune serum stains yeast cells to give a striking pattern of spots and blotches not seen with preimmune serum . However, affinity purification of anti-p55 antibody shows that this pattern is not due to staining by anti-p55 antibody; rather the pattern is due to staining left in the serum depleted of anti-p55 antibody. Yeast, 1987 Jun, 3(2), 131 - 7 Specificity of DNA uptake during whole cell transformation of S . cerevisiae; Bruschi CV et al.; We have studied the mechanism of DNA transformation of whole yeast cells in Saccharomyces cerevisiae with particular emphasis on the role of the cell wall complex in DNA uptake . Two new aspects of the process have been investigated in order to evaluate its specificity . Such aspects are: (i) effect of monovalent vs . divalent cations during incubation with the transforming DNA and (ii) timing of DNA adsorption and uptake . We found that the specificity for cation requirement is a strain-dependent characteristic influenced by the presence of transforming DNA in the cell suspension . This finding is supported by reports from several laboratories that some yeast strains show mutually exclusive transformability with monovalent vs . divalent cations . While irreversible adsorption of plasmid DNA molecules is induced by both heat shock and polyethylene-glycol (PEG), DNA uptake seems to occur only after the removal of PEG . In the course of this study we have developed a new, alternative method of whole cell DNA transformation with CaCl2 able to transform strains that do not respond to other methods. J Gen Microbiol, 1987 Jun, 133 ( Pt 6), 1583 - 8 Novel genetic components controlling invertase production in Saccharomyces cerevisiae; del Castillo Agudo L et al.; Low levels of invertase (EC 3.2.1.26) activity were observed in most diploid strains of S . cerevisiae used in this work . There was no effect of mating type on invertase levels, and cell surface was not a limiting factor, because an increase in ploidy did not cause further decrease in specific invertase activity . Finally, some diploids showed the activity expected from the additive effects of different SUC genes, and haploid strains possessing two SUC genes expressed very variable invertase activities depending on the strain . This suggested the existence of one or more additional genes which control the levels of invertase . Genetic analysis of SUC5 strains provided evidence of the existence of a new gene, RPS5, which drastically reduced the specific invertase activity in strains possessing active SUC alleles . The recessive allele of this gene (rps5) allows expression of higher levels of invertase . We suggest that genes similar RPS5 are responsible for the low levels of invertase activity observed in diploid strains of S . cerevisiae. Mol Gen Genet, 1987 Jun, 208(1-2), 159 - 67 Structure of the yeast HIS5 gene responsive to general control of amino acid biosynthesis; Nishiwaki K et al.; The nucleotide sequence of a 2.1 kb DNA fragment bearing the HIS5 gene of Saccharomyces cerevisiae, which encodes histidinol-phosphate aminotransferase (EC 2.6.1.9), has been determined . An open reading frame of 1,152 bp was found . S1 nuclease mapping indicated that the major transcription starts at position -37 from the ATG codon and the minor (approximately 20%) at -34 in both repressive and derepressive conditions . Northern analysis indicated that transcription of the HIS5 gene is under the general control of amino acid biosynthesis . The 5' noncoding region of the gene, thus far examined up to position -616, contains three copies of sequences homologous to the short repeats of the consensus sequence, 5'-AATGTGACTC-3', suggested for general amino acid control in the HIS1, HIS3, HIS4, and TRP5 at positions -336, -275 and -205 . The consensus sequence closest to the open reading frame was shown to be necessary but not sufficient for general amino acid control, by examination of beta-galactosidase appearance in S . cerevisiae cells carrying various mutant HIS5 promoter regions fused to the lac'Z gene and inserted at the leu2 locus of chromosome III. Proc Natl Acad Sci U S A, 1987 Jun, 84(12), 4063 - 7 Import and processing of human ornithine transcarbamoylase precursor by mitochondria from Saccharomyces cerevisiae; Cheng MY et al.; Expression of the subunit precursor of the human mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; EC 2.1.3.3) was programmed in Saccharomyces cerevisiae from a 2-micron plasmid by using an inducible galactose operon promoter . In the presence of the inducing sugar (galactose), two polypeptides were specifically precipitable with anti-OTCase antiserum: the human OTCase precursor (40 kDa); and the mature OTCase subunit (36 kDa) . When yeast cells containing these species were lysed and fractionated, the OTCase precursor was found to be associated with mitochondrial membranes, while the mature subunit was found partly with mitochondrial membranes and partly in the soluble mitochondrial matrix-containing fraction . When OTCase enzymatic activity was assayed in fractions similarly derived from an S . cerevisiae strain devoid of yeast OTCase activity (an arg3 mutant) but expressing human OTCase, activity was detected specifically in the mitochondrial matrix fraction . A mutant human OTCase precursor containing an artificial mutation in the NH2-terminal leader peptide (arginine-23 to glycine) was similarly examined . As was previously observed with mammalian mitochondria, this precursor failed both to reach the matrix compartment and to be proteolytically processed; it also failed to exhibit OTCase enzymatic activity . Presence of OTCase enzymatic activity in an arg3 strain expressing wild-type precursor was utilized to obtain selective growth in a medium devoid of arginine but supplemented with the OTCase substrate ornithine . We conclude that, during evolution, the pathway of mitochondrial import utilized by the human OTCase precursor is conserved between yeast and humans, and that, by using selective growth conditions, it may be possible to examine genetically this pathway in S . cerevisiae. Mol Cell Biol, 1987 Jun, 7(6), 2087 - 96 Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae; Sauer B; The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae . The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GALI promoter . lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations . Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose . These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S . cerevisiae is unimpaired by chromatin structure. Arch Biochem Biophys, 1987 May 1, 254(2), 568 - 78 In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae . 2 . Reaction mechanism of aspartate transcarbamylase dissociated from carbamylphosphate synthetase by genetic alteration; Belkaid M et al.; The reaction mechanism of Saccharomyces cerevisiae aspartate transcarbamylase was studied in permeabilized cells of a mutant in which this enzyme is not associated to carbamylphosphate synthetase . The results obtained indicate an ordered mechanism in which carbamylphosphate binds first, followed by aspartate, with dissociation of the products in the order phosphate then carbamylaspartate . Interestingly, this clear-cut mechanism differs from the more complex behavior shown by aspartate transcarbamylase when this enzyme is associated to carbamylphosphate synthetase in wild-type S . cerevisiae (B . Penverne and G . Herve, Arch . Biochem . Biophys . (1983) 225, 562-575) . This difference indicates that the association of the two enzymes within the multienzymatic complex alters the apparent kinetic properties of aspartate transcarbamylase . Such an enzyme-enzyme interaction might be related to the channeling of carbamylphosphate from one catalytic site to the other one. J Bacteriol, 1987 May, 169(5), 2132 - 6 Regulation of dipeptide transport in Saccharomyces cerevisiae by micromolar amino acid concentrations; Island MD et al.; Prototrophic Saccharomyces cerevisiae X2180, when grown on unsupplemented minimal medium, displayed little sensitivity to ethionine- and m-fluorophenylalanine-containing toxic dipeptides . We examined the influence of the 20 naturally occurring amino acids on sensitivity to toxic dipeptides . A number of these amino acids, at concentrations as low as 1 microM (leucine and tryptophan), produced large increases in sensitivity to leucyl-ethionine, alanyl-ethionine, and leucyl-m-fluorophenylalanine . Sensitivity to ethionine and m-fluorophenylalanine remained high under either set of conditions . The addition of 0.15 mM tryptophan to a growing culture resulted in the induction of dipeptide transport, as indicated by a 25-fold increase in the initial rate of L-leucyl-L-{3H}leucine accumulation . This increase, which was prevented by the addition of cycloheximide, began within 30 min and peaked approximately 240 min after a shift to medium containing tryptophan . Comparable increases in peptidase activity were not apparent in crude cell extracts from tryptophan-induced cultures . We concluded that S . cerevisiae possesses a specific mechanism for the induction of dipeptide transport that can respond to very low concentrations of amino acids. Exp Cell Res, 1987 May, 170(1), 64 - 79 The cytoplasmic pH, ATP content and total protein synthesis rate during heat-shock protein inducing treatments in yeast; Weitzel G et al.; In S . cerevisiae the induction of heat-shock protein (HSP) synthesis is accompanied by a decrease in the cytoplasmic and vacuolar pH as determined by means of {31P}NMR spectroscopy . The relationship of HSP synthesis and acidification of the cytoplasmic pH is dose-dependent under a variety of treatments (temperature increases (23-32 degrees C), addition of 2,4-dinitrophenol (greater than 1 mM), sodium arsenite (greater than 3.75 X 10(-5) M) or sodium cyanide (greater than 10 mM} . Changes in the intracellular pH occur within 5 min after treatment, attain a maximum within 30 min and are subsequently stable . HSPs 98, 85 and 70 show maximum synthesis rates 1-2 h after a 40 degrees C heat shock . The synthesis rates then decline . HSPs 56, 44 and 33 reveal a smaller and slower increase and almost no decrease in the synthesis rate within 4 h at 40 degrees C . The similar dose dependencies of HSP synthesis and cytoplasmic pH . as well as the immediate response of the pH, can also be demonstrated in the mitochondrial mutant of S . cerevisiae (Q0) . This result indicates that the heat-shock response is mainly independent of intact oxidative phosphorylation . No correlation was observed between HSP synthesis rate and total intracellular ATP content. Biochem Int, 1987 May, 14(5), 963 - 70 Status of calcium influx in cell cycle of S . cerevisiae; Anand S et al.; The transport of calcium was assayed in exponentially growing and G1 arrested temperature sensitive cdc mutants of Saccharomyces cerevisiae . There was no statistically significant difference in the rate of Ca2+ influx in cdc 28, cdc 37 and cdc 4 arrested cells, as well as in wild type cells arrested in G1 phase in comparison to exponentially growing cells . There was however a significant increase in Ca2+ uptake in cdc 7 and cdc 24 arrested cells . The former is known to arrest before bud emergence and initiation of DNA synthesis; arrest of the latter affects bud formation while DNA synthesis continues . The results suggest that Ca2+ may have a role in bud formation and growth. Arch Microbiol, 1987 May, 147(4), 358 - 63 Glycerol production in relation to the ATP pool and heat production rate of the yeasts Debaryomyces hansenii and Saccharomyces cerevisiae during salt stress; Larsson C et al.; Changes in glycerol production and two parameters related to energy metabolism i.e . the heat production rate and the ATP pool, were assayed during growth of Saccharomyces cerevisiae and Debaryomyces hansenii in 4 mM and 1.35 M NaCl media . For both of the yeasts, the specific ATP pool changed during the growth cycle and reached maximum values around 10 nmol per mg dry weight in both types of media . The levels of glycerol were markedly enhanced by high salinity . In the presence of 1.35 M NaCl, D . hansenii retained most of its glycerol produced intracellularly, while S . cerevisiae extruded most of the glycerol to the environment . The intracellular glycerol level of S . cerevisiae equalled or exceeded that of D . hansenii, however, with values never lower than 3 mumol per mg dry weight at all phases of growth . When D . hansenii was grown at this high salinity the intracellular level of glycerol was found to correlate with the specific heat production rate . No such correlation was found for S . cerevisiae . We concluded that during salt stress, D . hansenii possesses the capacity to regulate the metabolism of glycerol to optimize growth, while S . cerevisiae may not be able to regulate when exposed to different demands on the glycerol metabolism. Mol Gen Genet, 1987 May, 207(2-3), 342 - 8 Phleomycin resistance encoded by the ble gene from transposon Tn 5 as a dominant selectable marker in Saccharomyces cerevisiae; Gatignol A et al.; Phleomycin, a water-soluble antibiotic of the bleomycin family is as effective against Saccharomyces cerevisiae cells as against Escherichia coli cells . The ble gene of transposon Tn5, which confers resistance to phleomycin, was inserted in place of the iso-1-cytochrome C (CYC1) gene on an autonomously replicative multicopy E . coli-yeast shuttle plasmid . Higher resistance levels are obtained in S . cerevisiae when the region immediately upstream from the initiation codon conforms to the nucleotide sequence stringencies observed in almost every yeast gene . The expected regulation pattern of the whole CYC1 promoter confers different phleomycin resistance levels to the cell under varying physiological conditions . Partial deletions in the CYC1 promoter lead to changes in the resistance level of cells which are mostly accounted for by the removal of known positive and negative regulatory elements . Some of the vector constructions allow direct selection of phleomycin-resistant transformants on rich media. Biochem Biophys Res Commun, 1987 Apr 29, 144(2), 613 - 9 A novel yeast secretion vector utilizing secretion signal of killer toxin encoded on the yeast linear DNA plasmid pGKL1; Tokunaga M et al.; The NH2-terminal signal region comprising of approximately 70% length of the prepro-sequence of the pGKL killer precursor protein was found to direct an efficient secretion of the mouse alpha-amylase into the culture medium of Saccharomyces cerevisiae . The alpha-amylase molecule secreted into the culture medium was identified by both immuno-blotting and assay of the enzyme activity . The amount of alpha-amylase secreted via the killer toxin signal was comparable to that directed by the leader sequence of mating factor alpha . The secretion of alpha-amylase using the killer toxin signal was blocked at 37C but not at 25C in sec18-1 host, indicating that alpha-amylase is exported through the normal secretion pathway of S . cerevisiae. Biochim Biophys Acta, 1987 Apr 27, 906(1), 81 - 99 Protein glycosylation in yeast; Tanner W et al.; S . cerevisiae contains many mannose-rich glycoproteins that possess N- and O-linked carbohydrate chains, and both types may even occur on one and the same protein . The steps in the synthesis of asparagine-linked chains begin with assembly and transfer of the lipid-linked precursor to protein in a way common to all eucaryotes . Subsequent modifications lead to mannosyl extensions of various lengths, but complex type carbohydrate structures are not formed . Oligosaccharides O-linked to serine/threonine consist exclusively of mannose in S . cerevisiae . The mannose residue attached directly to the protein is transferred from Dol-P-Man in a unique way, which has been observed so far for fungal cells only . The cellular localization of the glycosylation reactions is summarized and the problem of transmembrane translocation of the sugar precursors at the ER and the Golgi is discussed . Some aspects of secretory (sec) and asparagine linked glycosylation (alg) mutants have been covered, and the various hypotheses related to the possible functions of this costly protein modification process are discussed . The article may also be helpful for those, who want to exploit the yeast's protein synthesizing machinery by genetically manipulating the cells. FEBS Lett, 1987 Apr 6, 214(1), 158 - 62 Fatty acid synthesis in mitochondria from Saccharomyces cerevisiae; Bessoule JJ et al.; The ability of purified mitochondria isolated from S . cerevisiae to synthesize fatty acids and especially very long chain fatty acids (VLCFA) has been investigated . The VLCFA synthesis requires malonyl-CoA as the C2 unit donor and NADPH as the reducing agent . Moreover the yeast mitochondrial elongase is able to accept either exogenous long chain fatty acyl-CoAs as substrates or elongate endogenous substrates . In the latter case, ATP is required for full activity . Besides this important VLCFA formation, the mitochondria from S . cerevisiae were also able to synthesize C16 and C18. Mol Cell Biol, 1987 Apr, 7(4), 1338 - 45 Phosphorylation of the Saccharomyces cerevisiae equivalent of ribosomal protein S6 has no detectable effect on growth; Johnson SP et al.; The phosphorylation of mammalian ribosomal protein S6 is affected by a variety of agents, including growth factors and tumor promoters, as well as by expressed oncogenes . Its potential role in the regulation of protein synthesis has been the object of much study . We have developed strains of Saccharomyces cerevisiae in which the phosphorylatable serines of the equivalent ribosomal protein (S10) were converted to alanines by site-directed mutagenesis . The S10 of such cells is not phosphorylated . Comparison of these cells with the parental cells, whose genomes differ by only six nucleotides, revealed no differences in the lag phase or logarithmic phase of a growth cycle, in growth on different carbon sources, in sporulation, or in sensitivity to heat shock . We conclude that in S . cerevisiae the phosphorylation of ribosomal protein S10 may play no role in regulating the synthesis of proteins . This conclusion leads one to ask whether certain protein phosphorylations are simply the adventitious, if easily observable, result of the imperfect specificity of one or another protein kinase. Mol Cell Biol, 1987 Apr, 7(4), 1476 - 85 Signal processing, glycosylation, and secretion of mutant hemagglutinins of a human influenza virus by Saccharomyces cerevisiae; Abdul Jabbar M et al.; We investigated the nature of signal recognition, transport, and secretion of mutant hemagglutinins (HAs) of a human influenza virus by the yeast Saccharomyces cerevisiae . The cDNA sequences encoding variant forms of influenza HA were expressed in S . cerevisiae . The HA polypeptides (HA500 and HA325) that were synthesized with their N-terminal signal peptides were correctly targeted to the membrane compartment where they were glycosylated . In contrast, the HA polypeptides (HA484 and HA308) lacking the signal peptide were expressed in the cytoplasm and did not undergo any glycosidic modification, demonstrating the importance of the heterologous signal sequence in the early steps of translocation in S . cerevisiae . The analysis of the N-terminal amino acid sequence of HA500 and HA325 polypeptides demonstrated the correct cleavage of the signal peptide, indicating the structural compatibility of a heterologous signal peptide for efficient recognition and processing by the yeast translocation machinery . The membrane-sequestered and glycosylated HA polypeptides were relatively stable in S . cerevisiae compared with the signal-minus, nonglycosylated HA molecules . Although both the anchor-minus HA (HA500) and HA1 (HA325) polypeptides were targeted efficiently to the membrane, their glycosylation and transport patterns were shown to be different . During pulse-chase, the HA500 remained cell-associated with no detectable secretion into the extracellular medium, whereas the HA325 secreted into the medium . Furthermore, only the cell-associated and secreted forms of HA325 and not HA500 appeared to have undergone hyperglycosylation with the extensive addition of high-molecular-weight outer-chain mannans . Possible reasons for the observed phenotypic behavior of these two mutant HAs are discussed. Mol Cell Biol, 1987 Apr, 7(4), 1371 - 7 Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae; Toda T et al.; We have cloned a gene (BCY1) from the yeast Saccharomyces cerevisiae that encodes a regulatory subunit of the cyclic AMP-dependent protein kinase . The encoded protein has a structural organization similar to that of the RI and RII regulatory subunits of the mammalian cyclic AMP-dependent protein kinase . Strains of S . cerevisiae with disrupted BCY1 genes do not display a cyclic AMP-dependent protein kinase in vitro, fail to grow on many carbon sources, and are exquisitely sensitive to heat shock and starvation. J Biochem (Tokyo), 1987 Apr, 101(4), 949 - 56 Possible involvement of acetyl coenzyme A carboxylase as well as fatty acid synthetase in the temperature-controlled synthesis of fatty acids in Saccharomyces cerevisiae; Hori T et al.; Fatty acid synthetase (FAS) preparations from Saccharomyces cerevisiae cells grown at either 35 or 10 degrees C produced the same products at different temperatures and showed quite similar temperature-dependencies in Arrhenius plots, with break points at 25 degrees C . This break point does not appear to reflect a phase transition of phospholipids present in the purified FAS preparations but rather is associated with protein conformational changes . S . cerevisiae cells grown at 35 degrees C and then shifted to 10 degrees C produced fatty acids with a shorter average chain length than those fatty acids synthesized at 10 degrees C by cells already adapted to 10 degrees C (hyper response) . Acetyl-CoA carboxylase activity was relatively higher in the cells grown at 35 degrees C than in the cells grown at 10 degrees C; moreover, fatty acids with longer average chain lengths were synthesized in vitro at higher malonyl-CoA concentrations, which was consistent with the difference in the average chain lengths of newly synthesized fatty acids in cells grown at 35 and 10 degrees C . However, the activity levels of acetyl-CoA carboxylase and fatty acid synthetase alone did not account for the hyper response phenomena. Cell, 1987 Mar 13, 48(5), 801 - 12 A genetic analysis of dicentric minichromosomes in Saccharomyces cerevisiae; Koshland D et al.; We have developed an assay in S . cerevisiae in which clones of cells that contain intact dicentric minichromosomes are visually distinct from those that have rearranged to monocentric minichromosomes . We find that the instability of dicentric minichromosomes is apparently due to mitotic nondisjunction accompanied by occasional structural rearrangements . Monocentric minichromosomes arising by rearrangement of the plasmid are rapidly selected in the population since dicentric minichromosomes depress the rate of cell division . We show that the ability of one centromere to compete with another in dicentric minichromosomes requires the presence of both of the conserved structural elements, CDE II and CDE III . Dicentric minichromosomes can be stabilized if one of the centromeres on the molecule is functionally hypomorphic because of mutations in CDE II even though these mutant centromeres are highly efficient in monocentric molecules . Stable dicentric molecules can also be produced by decreasing the space between two wild-type centromeres on the same molecule . These results suggest plausible pathways for changes in chromosome number that accompany evolution. Cell, 1987 Mar 13, 48(5), 789 - 99 The S . cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway; Broek D et al.; The gene corresponding to the S . cerevisiae cell division cycle mutant cdc25 has been cloned and sequenced, revealing an open reading frame encoding a protein of 1589 amino acids that contains no significant homologies with other known proteins . Cells lacking CDC25 have low levels of cyclic AMP and decreased levels of Mg2+-dependent adenylate cyclase activity . The lethality resulting from disruption of the CDC25 gene can be suppressed by the presence of the activated RAS2val19 gene, but not by high copy plasmids expressing a normal RAS2 or RAS1 gene . These results suggest that normal RAS is dependent on CDC25 function . Furthermore, mutationally activated alleles of CDC25 are capable of inducing a set of phenotypes similar to those observed in strains containing a genetically activated RAS/adenylate cyclase pathway, suggesting that CDC25 encodes a regulatory protein . We propose that CDC25 regulates adenylate cyclase by regulating the guanine nucleotide bound to RAS proteins. Yeast, 1987 Mar, 3(1), 51 - 7 Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures; Wickner RB et al.; MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16-1 mutation . MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth . As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M1 replication defects of mak16-1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library . The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I . Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade 1, confirming that this clone was derived from the appropriate region of chromosome I . An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis . 185 amino acids of this open reading frame were replaced with a 1.2 kb fragment carrying the S . cerevisiae URA3 gene by a one-step gene disruption . The resulting strains grew at a rate indistinguishable from the wild type at 20 degrees C, 30 degrees C, or 37 degrees C, but could not grow at 8 degrees C . The deleted region is thus essential only at 8 degrees C, and we name this gene LTE1 (low temperature essential). Cell, 1987 Feb 13, 48(3), 507 - 15 The Saccharomyces and Drosophila heat shock transcription factors are identical in size and DNA binding properties; Wiederrecht G et al.; The heat shock transcription factor (HSTF) has been purified to apparent homogeneity from S . cerevisiae and D . melanogaster by sequence-specific DNA-affinity chromatography . A synthetic oligonucleotide containing an hsp83-like heat shock element (HSE) was prepared and ligated into concatamers and covalently coupled to Sepharose . This DNA-affinity resin allowed the rapid isolation of a yeast and a Drosophila protein with the same apparent molecular weight (70 kd) . The yeast HSTF will bind to both its own and the Drosophila HSEs . Similarly, the Drosophila HSTF will bind to both its own and the yeast HSEs . The yeast and Drosophila HSTFs were subjected to preparative SDS gel electrophoresis, and the 70 kd polypeptides were eluted, renatured, and observed to generate the identical footprint pattern as the native HSTFs . Affinity-purified Drosophila HSTF was further shown to stimulate specific HSE-dependent transcription from a Drosophila hsp70 gene in vitro. J Bacteriol, 1987 Feb, 169(2), 779 - 84 Effect of cell cycle position on thermotolerance in Saccharomyces cerevisiae; Plesset J et al.; We showed that the heat killing curve for exponentially growing Saccharomyces cerevisiae was biphasic . This suggests two populations of cells with different thermal killing characteristics . When exponentially growing cells separated into cell cycle-specific fractions via centrifugal elutriation were heat shocked, the fractions enriched in small unbudded cells showed greater resistance to heat killing than did other cell cycle fractions . Cells arrested as unbudded cells fell into two groups on the basis of thermotolerance . Sulfur-starved cells and the temperature-sensitive mutants cdc25, cdc33, and cdc35 arrested as unbudded cells were in a thermotolerant state . Alpha-factor-treated cells arrested in a thermosensitive state, as did the temperature-sensitive mutant cdc36 when grown at the restrictive temperature . cdc7, which arrested at the G1-S boundary, arrested in a thermosensitive state . Our results suggest that there is a subpopulation of unbudded cells in exponentially growing cultures that is in G0 and not in G1 and that some but not all methods which cause arrest as unbudded cells lead to arrest in G0 as opposed to G1 . It has been shown previously that yeast cells acquire thermotolerance to a subsequent challenge at an otherwise lethal temperature during a preincubation at 36 degrees C . We showed that this acquisition of thermotolerance was corrected temporally with a transient increase in the percentage of unbudded cells during the preincubation at 36 degrees C . The results suggest a relationship between the heat shock phenomenon and the cell cycle in S . cerevisiae and relate thermotolerance to transient as well as to more prolonged residence in the G0 state. Mol Cell Biol, 1987 Feb, 7(2), 632 - 8 Physical analysis of the COR region: a cluster of six genes in Saccharomyces cerevisiae; Barry K et al.; Six genes, CYC1, UTR1, UTR3, OSM1, tRNAGly, and RAD7, have been localized within an 8-kilobase region on chromosome X of the yeast Saccharomyces cerevisiae . The physical structures and the transcripts of these genes were identified by analyzing a normal strain and six deletion mutants by genomic blotting, transcriptional analysis, and gene disruption procedures . The well-studied CYC1 gene encodes iso-1-cytochrome c; the tRNAGly gene encodes a tRNA; deletion of OSM1 and RAD7 causes sensitivity to hypertonic medium and UV irradiation, respectively . There were no observable phenotypes in strains having deletions of the UTR1, UTR3, and tRNAGly gene . The high density of transcripts, with little or almost no intragenic regions, indicates that the chromosomal organization of S . cerevisiae resembles the chromosomal organization of procaryotes rather than higher eucaryotes. Antonie Van Leeuwenhoek, 1987, 53(2), 77 - 84 Three newly delimited species of Saccharomyces sensu stricto; Martini AV et al.; Deoxyribonucleic acid reassociation studies of 24 different wine and beer-associated strains of Saccharomyces confirmed the presence of three separate species . S . cerevisiae and S . bayanus strains had only 22% of their genomes in common . S . pastorianus, with intermediate hybridization values between S . cerevisiae and S . bayanus (52 and 72%, respectively) could possibly be a natural hybrid of the two species . S . pastorianus replaces S . carlsbergensis with which it is homologous for 93% of its genome, since the former species was described first by Hansen in 1904 . These data do not agree with the results of traditional physiological tests. Mol Cell Biol, 1987 Jan, 7(1), 68 - 75 Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae; Gaudet A et al.; Centromere DNA from 11 of the 16 chromosomes of the yeast Saccharomyces cerevisiae have been analyzed and reveal three sequence elements common to each centromere, referred to as conserved centromere DNA elements (CDE) . The adenine-plus-thymine (A + T)-rich central core element, CDE II, is flanked by two short conserved sequences, CDE I (8 base pairs {bp}) and CDE III (25 bp) . Although no consensus sequence exists among the different CDE II regions, they do have three common features of sequence organization . First, the CDE II regions are similar in length, ranging from 78 to 86 bp measured from CDE I to the left boundary of CDE III . Second, the base composition is always greater than 90% A + T . Finally, the A and T residues in these segments are often arranged in runs of A and runs of T residues, sometimes with six or seven bases in a stretch . We constructed insertion, deletion, and replacement mutations in the CDE II region of the centromere from chromosome III, CEN3, designed to investigate the length and sequence requirements for function of the CDE II region of the centromere . We analyzed the effect of these altered centromeres on plasmid and chromosome segregation in S . cerevisiae . Our results show that increasing the length of CDE II from 84 to 154 bp causes a 100-fold increase in chromosome nondisjunction . Deletion mutations removing segments of the A + T-rich CDE II DNA also cause aberrant segregation . In some cases partial function could be restored by replacing the deleted DNA with fragments whose primary sequence or base composition is very different from that of the wild-type CDE II DNA . In addition, we found that identical mutations introduced into different positions in CDE II have very similar effects. Mol Cell Biol, 1987 Jan, 7(1), 403 - 9 Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human homolog, and its possible role as a transcription factor; Bram RJ et al.; A protein that binds specifically to Saccharomyces cerevisiae centromere DNA element I was purified on the basis of a nitrocellulose filter-binding assay . This protein, termed centromere-binding protein 1 (CP1), was heat stable and renaturable from sodium dodecyl sulfate (SDS), and assays of eluates from SDS gels indicated a molecular weight of 57,000 to 64,000 . An activity with similar specificity and stability was detected in human lymphocyte extracts, and analysis in SDS gels revealed a molecular weight of 39,000 to 49,000 . CP1-binding sites occurred not only at centromeres but also near many transcription units, for example, adjacent to binding sites for the GAL4-positive regulatory protein upstream of the GAL2 gene in S . cerevisiae and adjacent to the TATA element of the adenovirus major late promoter . A factor (termed USF) that binds to the latter site and stimulates transcription has been isolated from HeLa cells by others. Curr Genet, 1987, 11(6-7), 483 - 90 Gene-protein assignments within the yeast Yarrowia lipolytica dsRNA viral genome; el-Sherbeini M et al.; Some strains of the yeast Yarrowia lipolytica possess virus-like particles (VLPs) which encapsidate a double-stranded RNA (dsRNA) genome designated Ly . We report here that these VLPs have two associated polypeptides of molecular weights 83 kd (VLy-P1) and 77 kd (VLy-P2) . Denatured Ly-dsRNA was used to program a cell-free rabbit reticulocyte translation system, resulting in the appearance of four major products, viz . Ly-P1 (83 kd); Ly-P2 (77 kd); Ly-P3 (74 kd) and Ly-P4 (68 kd) . The in vivo viral-associated protein VLy-P1 co-migrated on SDS-polyacrylamide gels with the in vitro product Ly-P1 and, similarly, VLy-P2 co-migrated with Ly-P2 . Peptide mapping data confirm the identity of the in vivo products (VLy-P1 and VLy-P2) and their in vitro counterparts . The conclusion made is that VLy-P1 and VLy-P2 are almost identical primary translation products of the Ly genome, derived from a single or multiple species of Ly-dsRNA . RNA blot hybridizations using L1A M1 and separately, L2A M2 probes prepared from appropriate K1 and K2 Saccharomyces cerevisiae killer strains, failed to show any detectable homology to Ly-dsRNA, substantiating the uniqueness of the Ly genome with respect to the K1 and K2 S . cerevisiae dsRNA killer systems. Curr Genet, 1987, 11(6-7), 445 - 50 Genetic mapping of two pairs of linked ribosomal protein genes in Saccharomyces cerevisiae; Papciak SM et al.; We have used the 2 mu mapping method described by Falco and Botstein (1983) and tetrad analysis to map four ribosomal protein genes (two linked pairs) in S . cerevisiae . One pair (rp28-rp55 copy 1) is on chromosome XV, 14 cM proximal to ARG8 . The other pair (rp55-rp28 copy 2) is 19 cM from the centromere on the left arm of chromosome XIV . To map copy 1 we used the E . coli beta-galactosidase gene rather than a yeast gene to mark the ribosomal protein chromosomal locus . This provided a more sensitive color screening assay for chromosome loss in the 2 mu method . It also removed the restriction that the mapping tester strains must be mutant for the plasmid marker. Curr Genet, 1987, 11(6-7), 429 - 34 Mutations suppressing the effects of a deletion of the phosphoglucose isomerase gene PGI1 in Saccharomyces cerevisiae; Aguilera A; A mutant with a deletion covering the phosphoglucose isomerase gene PGI1, allele pgil delta, can only grow on a medium containing fructose and low concentrations of glucose whereas growth is completely inhibited by glucose concentrations higher than 0.4% . This was used to select suppressor mutants restoring growth on synthetic media with 2% glucose as the sole carbon source . One complementation group, SPG1, was defined by recessive mutations . The ability to grow on glucose media was strictly dependent on functional mitochondria . The generation time of the selected mutants on YEP glucose was 6-8 h . No ethanol was formed from glucose and the levels of respiration were very high . These phenotypes were also observed in single pgil delta mutants when growing on fructose media supplemented with 0.4% glucose . The other glycolytic enzymes, the enzymes of the glucose-6-phosphate oxidation pathway as well as catabolite repression were normal in suppressed pgil delta mutants . The suppressor mutation alone caused no abnormal phenotype . The results suggest that the spg1 suppressor mutations allow S . cerevisiae pgil delta mutant strains to grow on glucose by using the Pentose-P cycle in combination with unusual strong respiration. Curr Genet, 1987, 11(4), 295 - 301 A mitochondrial frameshift-suppressor (+1) {corrected} of the yeast S . cerevisiae maps in the mitochondrial 15S rRNA locus; Weiss-Brummer B et al.; The first case of a +1 "extrageneic" frameshift suppressor (MF1), mapping in the yeast mitochondrial 15S rRNA gene is reported . The suppressor was identified by genetic analyses in a leaky mitochondrial oxil frameshift mutant and the respective wild-type strain 777-3A of the yeast S . cerevisiae . This is in accordance with the finding that all mitochondrial frameshift mutants isolated from this strain tend to be leaky to a variable degree . MF1 does not suppress known nonsense mutations created by a direct basepair exchange in strain 777-3A . These mutants exhibit a non-leaky phenotype (Weiss-Brummer et al . 1984). Gene, 1987, 55(2-3), 303 - 17 Expression of a wheat alpha-gliadin gene in Saccharomyces cerevisiae; Neill JD et al.; A vector was constructed that directs the expression of foreign genes in the yeast Saccharomyces cerevisiae . This vector contains an expression site that was constructed by in vitro modification of the iso-1-cytochrome c (CYC1) gene of S . cerevisiae . The expression of heterologous sequences can be experimentally controlled by catabolite control sequences, promoter and transcription initiation sequences and termination sequence derived from the CYC1 gene . A portion of a genomic wheat alpha-gliadin gene consisting of the entire 861 bp of protein-coding sequence, 18 bp of 5' leader sequence and 54 bp of 3'-noncoding sequence was inserted into the expression site . A CYC1::alpha-gliadin transcript of approx . 1050 nucleotides was synthesized in transformed yeast under the control of the CYC1 regulatory region . The transcripts terminated within the alpha-gliadin 3'-noncoding region, near a nucleotide sequence similar to the yeast transcription termination consensus sequence . The alpha-gliadin was immunochemically detected in total protein extracts from transformed cells and accounted for approx . 0.1% of the total cellular protein . The size of alpha-gliadin synthesized in yeast is the same as that of mature wheat alpha-gliadin . This is consistent with recognition and cleavage of the signal peptide by yeast . Due to the amino acid composition of alpha-gliadin, the availability of glutamine tRNA is a potential translational limitation to high-level synthesis in yeast. Microbios, 1987, 51(206), 37 - 42 Analysis of fatty acid composition of Candida species by gas-liquid chromatography using a polar column; Kobayashi K et al.; The fatty acid composition of representative Candida species was examined by gas-liquid chromatography (GLC) using a polar column . The major fatty acids were C14:0, C16:0, C18:0 saturated, C16:1 and C18:1 monoenoic series, with or without C18 polyunsaturated acids (C18:2 and C18:3) . In Torulopsis glabrata and Saccharomyces cerevisiae the C18:2 and C18:3 acids were not found, but the C10:0 and C12:0 acids were detected in S . cerevisiae . These results indicated that the Candida genus could be distinguished from Torulopsis and Saccharomyces genera by GLC analysis of fatty acids . Quantitative differences in the fatty acid composition between cells grown at high temperature (37 degrees C) and low temperature (25 degrees C) were found generally in Candida species, and the amounts of C18 polyunsaturated acids (C18:2 and C18:3) increased in the cells grown at 25 degrees C . Each Candida species showed a characteristic profile in fatty acid composition . Determination of the cellular fatty acid composition in Candida species is likely to be useful for the grouping or chemotaxonomy of newer isolates of Candida species. Gene, 1987, 54(1), 1 - 22 The AT spacers and the var1 genes from the mitochondrial genomes of Saccharomyces cerevisiae and Torulopsis glabrata: evolutionary origin and mechanism of formation; de Zamaroczy M et al.; Intergenic sequences represent 63% of the mitochondrial 'long' (85 kb) genome of Saccharomyces cerevisiae . They comprise 170-200 AT spacers that correspond to 47% of the genome and are separated from each other by GC clusters, ORFs, ori sequences, as well as by protein-coding genes . Intergenic AT spacers have an average size of 190 bp, and a GC level of 5%; they are formed by short (20-30 nt on the average) A/T stretches separated by C/G mono- to trinucleotides . An analysis of the primary structures of all intergenic AT spacers already sequenced (32 kb; 80% of the total) has shown that they are characterized by an extremely high level of short sequence repetitiveness and by a characteristic sequence pattern; the frequencies of A/T isostichs conspicuously deviate from statistical expectations, and exponentially decrease when their (AT + TA)/(AA + TT) ratio, R, decreases . A situation basically identical was found in the AT spacers of the mitochondrial genome (19 kb) of Torulopsis glabrata . The sequence features of the AT spacers indicate that they were built in evolution by an expansion process mainly involving rounds of duplication, inversion and translocation events which affected an initial oligodeoxynucleotide (endowed with a particular R ratio) and the sequences derived from it . In turn, the initial oligodeoxynucleotide appears to have arisen from an ancestral promoter-replicator sequence which was at the origin of the nonanucleotide promoters present in the mitochondrial genomes of several yeasts . Common sequence patterns indicate that the AT spacers so formed gave rise to the var1 gene (by linking and phasing of short ORFs), to the DNA stretches corresponding to the untranslated mRNA sequences and to the central stretches of ori sequences from S . cerevisiae. J Mol Evol, 1987, 24(3), 252 - 9 Molecular evolution of the Saccharomyces cerevisiae histone gene loci; Smith MM; The core histone genes of Saccharomyces cerevisiae are arranged as duplicate nonallelic sets of specifically paired genes . The identity of structural organization between the duplicated gene pairs would have its simplest evolutionary origin in the duplication of a complete locus in a single event . In such a case, the time since the duplication of one of the genes should be identical to that since duplication of the gene adjacent to it on the chromosome . A calculation of the evolutionary distances between the coding DNA sequences of the histone genes leads to a duplication paradox: The extents of sequence divergence in the silent component of third-base positions for adjacent pairs of genes are not identical . Estimates of the evolutionary distance between the two H3-H4 noncoding intergene DNA sequences are large; the divergence between the two separate sequences is indistinguishable from the divergence between either of the regions and a randomly generated permutation of itself . These results suggest that the duplication event may have occurred much earlier than previously estimated . The potential age of the duplication, and the attractive simplicity of the duplication of both the H3-H4 and the H2A-H2B gene pairs having taken place in a single event, leads to the hypothesis that modern haploid S . cerevisiae may have evolved by diploidization or fusion of two ancient fungi. Gene, 1987, 54(1), 113 - 23 Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus; Schultz LD et al.; The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein . This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast . Since gp350 proved highly toxic to S . cerevisiae, initial cellular growth required repression of the expression of gp350 . Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced . After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium . Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion . The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350 . N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350 . Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation . The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein. Mol Cell Biol, 1987 Jan, 7(1), 410 - 9 Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and analysis of the CEN1-ADE1-CDC15 region; Steensma HY et al.; To continue the systematic examination of the physical and genetic organization of an entire Saccharomyces cerevisiae chromosome, the DNA from the CEN1-ADE1-CDC15 region from chromosome I was isolated and characterized . Starting with the previously cloned ADE1 gene (J . C . Crowley and D . B . Kaback, J . Bacteriol . 159:413-417, 1984), a series of recombinant lambda bacteriophages containing 82 kilobases of contiguous DNA from chromosome I were obtained by overlap hybridization . The cloned sequences were mapped with restriction endonucleases and oriented with respect to the genetic map by determining the physical positions of the CDC15 gene and the centromeric DNA (CEN1) . The CDC15 gene was located by isolating plasmids from a YCp50 S . cerevisiae genomic library that complemented the cdc15-1 mutation . S . cerevisiae sequences from these plasmids were found to be represented among those already obtained by overlap hybridization . The cdc15-1-complementing plasmids all shared only one intact transcribed region that was shown to contain the bona fide CDC15 gene by in vitro gene disruption and one-step replacement to delete the chromosomal copy of this gene . This deletion produced a recessive lethal phenotype that was also recessive to cdc15-1 . CEN1 was located by finding a sequence from the appropriate part of the cloned region that stabilized the inheritance of autonomously replicating S . cerevisiae plasmid vectors . Finally, RNA blot hybridization and electron microscopy of R-loop-containing DNA were used to map transcribed regions in the 23 kilobases of DNA that went from CEN1 to CDC15 . In addition to the transcribed regions corresponding to the ADE1 and ADC15 genes, this DNA contained five regions that gave rise to polyadenylated RNA, at least two regions complementary to 4S RNA species, and a Ty1 transposable element . Notably, a higher than average proportion of the DNA examined was transcribed into RNA. Mol Cell Biol, 1987 Jan, 7(1), 258 - 65 Ty1 sequence with enhancer and mating-type-dependent regulatory activities; Errede B et al.; Some insertion mutations in Saccharomyces cerevisiae activate the expression of adjacent structural genes . The CYC7-H2 mutation is a Ty1 insertion 5' to the iso-2-cytochrome c coding region of CYC7 . The Ty1 insertion causes a 20-fold increase in CYC7 expression in a and alpha haploid cell types of S . cerevisiae . This activation is repressed in the a/alpha diploid cell type . Previous computer analysis of the CYC7-H2 Ty1 activator region identified two related sequences with homology both to mammalian enhancers and to a yeast a/alpha control site . A 112-base-pair (bp) DNA fragment encompassing one of these blocks of homology functioned as one component of the Ty1 activator . A 28-bp synthetic oligonucleotide with the wild-type homology block sequence was also functional . A single base pair mutation within the enhancer core of the synthetic 28-bp regulatory element reduced its activation ability to near background amounts . In addition, the 112-bp Ty1 fragment by itself functioned as a target for repression of adjacent gene expression in a/alpha diploid cells. Mol Cell Biol, 1987 Jan, 7(1), 121 - 8 Signal peptide specificity in posttranslational processing of the plant protein phaseolin in Saccharomyces cerevisiae; Cramer JH et al.; We linked the cDNA coding region for the bean storage protein phaseolin to the promoter and regulatory region of the Saccharomyces cerevisiae repressible acid phosphatase gene (PHO5) in multicopy expression plasmids . Yeast transformants containing these plasmids expressed phaseolin at levels up to 3% of the total soluble cellular protein . Phaseolin polypeptides in S . cerevisiae were glycosylated, and their molecular weights suggested that the signal peptide had been processed . We also constructed a series of plasmids in which the phaseolin signal-peptide-coding region was either removed or replaced with increasing amounts of the amino-terminal coding region for acid phosphatase . Phaseolin polypeptides with no signal peptide were not posttranslationally modified in S . cerevisiae . Partial or complete substitution of the phaseolin signal peptide with that from acid phosphatase dramatically inhibited both signal peptide processing and glycosylation, suggesting that some specific feature of the phaseolin signal amino acid sequence was required for these modifications to occur . Larger hybrid proteins that included approximately one-half of the acid phosphatase sequence linked to the amino terminus of the mature phaseolin polypeptide did undergo proteolytic processing and glycosylation . However, these polypeptides were cleaved at several sites that are not normally used in the unaltered acid phosphatase protein. Biochimie, 1987 Jan, 69(1), 53 - 62 Sensitivity of yeast cells to reactive oxygen species generated in the extracellular space; Chaput M et al.; Even when cytoplasmic scavenging activities are plentiful, yeast cells (S . cerevisiae) remain particularly sensitive towards reactive oxygen species generated in the extracellular space (either by the xanthine/xanthine oxidase reaction or by the redox cycling of menadione) . A sharp reduction of the extent of cellular alterations when SOD and/or catalase were supplemented in the incubation buffer, points to a contribution of both O-.2 and H2O2 in the toxic process . Although oxygen metabolites as well as t-butylhydroperoxide (tBH), a highly toxic organic peroxide, may be directly responsible for cellular damage, their toxicity is largely reduced in the presence of Desferal . A role of metal ions in potentiating the toxicity points to the involvement of OH . radicals, actually produced in the medium . With tBH, metal cations would be rather active in promoting peroxidative chain reactions . In the case of an extracellular oxidative attack, it may be foreseen that the plasma membrane will form a preferential target . An increased permeability of the plasma membrane towards ionized molecules and uncharged polycarboxylic acids is indeed observed after an oxidative treatment . The loss of selective permeability is, as a rule, correlated with a drop in viability . Early alterations, disrupting the functional organization of the plasma membrane have been sought . The permease involved in the active transport of purine(s) has appeared to be an appropriate marker for checking its functional integrity . This transport function appears to be very sensitive to damage induced by O-.2 generators, particularly under conditions in which the resulting lethality is still kept low and in which the energization of active transport processes remains unimpaired. Biol Cell, 1987, 61(3), 171 - 5 The energetic growth yields of the yeast Candida parapsilosis; Camougrand N et al.; The energetic growth yields of Candida parapsilosis were compared with those of Saccharomyces cerevisiae as a function of the energy source in the presence or absence of antimycin A, an inhibitor of the second phosphorylation site . When glycerol was used as energy source, the energetic growth yields were quite similar in C . parapsilosis and S . cerevisiae . On the other hand, when experiments were carried out with glucose as energy source, although three phosphorylation sites were available, glucose was found to be a poor energy source for C . parapsilosis . When C . parapsilosis was grown in the presence of antimycin A, on glucose: YGluS = 3YGlu + AS and on glycerol: YGlyS = 2 YGly + AS . It was concluded that growth in the presence of antimycin A could occur due to the functioning of the third phosphorylation site . This result agrees with previous works indicating that in C . parapsilosis the alternative pathway merges into the main respiratory chain at the cytochrome c level . Although the doubling time of C . parapsilosis was much less temperature-sensitive than that of S . cerevisiae, the energetic growth yield was the same at 13 degrees C and 28 degrees C, and consequently, the secondary pathway did not seem to be thermogenic. Experientia Suppl, 1987, 52, 431 - 7 Synaptic relations in meiotic gene conversion at the iterated CUP1r locus of S . cerevisiae; Welch JW et al.; This study concerns a comparative molecular analysis of copy number changes in two hybrids that differ in the extent of homologies at the CUP1r locus . Hybrid JW1020 is a diploid wherein each parent contributed nine identical, tandemly arrayed 2.0 kb repeat units . Genomic DNA was isolated from each of the spore colonies in a sample of 200 unselected tetrads . About 15% displayed copy number changes, i.e., increases or decreases of one or more complete units . Changes on a per tetrad basis occurred as often in a single spore colony as changes in each of two spores . Such double changes are rarely reciprocal in character . To account for the observed qualitative and quantitative copy number shifts, we propose a molecular recombination model that posits partial, incomplete synaptic pairing and gene conversion of the unpaired regions with or without associated crossing over . A second contrasting study centers on the copy number alterations and recombinational events uncovered in a molecular analysis of 50 unselected tetrads generated by hybrid EB8 . Unlike the hybrid JW1020, the EB8 diploid strain carried a six copy tandem array of 1.1 kb units at the CUP1r locus in one parental homologue and a five copy array of 1.6 kb units at the corresponding chromosome VIII locus . These natural alleles were recovered from industrial yeast strains by conventional genetic procedures and characterized by restrictional analysis . Twelve tetrads exhibit evidence for several different types of recombination events . However,ordinary crossover exchanges are conspicuously absent . We suppose that the repetitious nonhomologies generate DNA configurations sufficient to disrupt the effective synapsis over the entire locus.(ABSTRACT TRUNCATED AT 250 WORDS) Curr Genet, 1987, 11(6-7), 451 - 7 Nucleotide sequencing analysis of a LEU gene of Candida maltosa which complements leuB mutation of Escherichia coli and leu2 mutation of Saccharomyces cerevisiae; Takagi M et al.; The expression of a LEU gene from Candida maltosa (designated as C-LEU2) isolated previously (Kawamura et al . 1983) was shown to be regulated, when transferred into Saccharomyces cerevisiae, by leucine and threonine in the medium, as in the case of LEU2 gene of S . cerevisiae . The coding region together with the regulatory region was subcloned and the nucleotide sequence was determined . When the sequence of the coding region was compared with that of LEU2, the homology was 72% for base pairs and 76% for deduced amino acids . Comparison of the regulatory region of C-LEU2 with those of LEU1 and LEU2 suggested a few short consensus sequences which are involved in regulation of gene expression by leucine and threonine in the medium. Curr Genet, 1987, 11(5), 377 - 83 The Yarrowia lipolytica LEU2 gene; Davidow LS et al.; A 2810 bp DNA fragment containing the beta-isopropylmalate dehydrogenase gene of the dimorphic yeast Yarrowia lipolytica has been sequenced . The sequence contains an open reading frame of 405 codons, predicting a protein of 43,366 molecular weight . Protein sequence homology with the polypeptide encoded by the LEU2 gene of Saccharomyces cerevisiae is 64%, whereas DNA sequence homology is 61% . The 5'- and 3'-flanking regions of the Y . lipolytica LEU2 gene share only some general structural features common to genes of S . cerevisiae such as the presence and location of TATA boxes, CAAT boxes, CACACA repeats, the lack of G residues in the 5'-untranslated region and 3'-transcription terminators . Transcription of a 1.4 kb mRNA begins at a small cluster of sites approximately 40 base pairs before the initial ATG. Curr Genet, 1987, 12(5), 337 - 48 Efficient integrative transformation of Cephalosporium acremonium; Skatrud PL et al.; A hybrid gene, IPNSp/HPTorf, was constructed by placing an 850 bp sequence of Cephalosporium acremonium DNA next to the 5' end of a bacterial open reading frame, HPTorf . The sequence was obtained as an 850 bp NcoI restriction fragment from the 5' non-coding region of the C . acremonium isopenicillin N synthetase (IPNS) gene . The HPTorf was obtained from a bacterial gene that coded for a hygromycin B phosphotransferase (HPT) . Plasmids that contained IPNSp/HPTorf transformed C . acremonium to a stably maintained hygromycin B resistant phenotype . Southern analysis of total DNA from transformants demonstrated multiple integrations of the transforming DNA in the high molecular weight DNA of most transformants, but single integrations were observed in a few transformants . The number of transformants per microgram of DNA was about 100 times greater than for plasmids that contained the HPTorf without any juxtaposed eucaryotic promoter sequence . Plasmids with the promoterless HPTorf and plasmids with a truncated S . cerevisiae phosphoglycerate kinase promoter juxtaposed to the HPTorf transformed C . acremonium at equivalent low frequencies . Transformation of C . acremonium with linearized plasmid DNA produced at least 2-3 fold more transformants than the corresponding circular molecule . Several observations were made concerning protoplast formation and handling which made the transformation procedure more efficient and allowed a greater proportion of protoplasts to regenerate to viable walled cells . Plasmids were constructed that contained both the IPNSp/HPTorf and additional elements: fragments of C . acremonium ribosomal DNA (rDNA), or a fragment of C . acremonium mitochondrial DNA possessing activity as an autonomous replication sequence (ARS) in S . cerevisiae, or putative transcriptional termination/polyadenylation signals from the IPNS gene . These plasmids transformed C . acremonium at frequencies experimentally equivalent to those containing IPNSp/HPTorf without any of these additional elements. Gene, 1987, 58(2-3), 189 - 99 Complete nucleotide sequence of the Rhodosporidium toruloides gene coding for phenylalanine ammonia-lyase; Anson JG et al.; The complete nucleotide sequence of the Rhodosporidium toruloides gene coding for the enzyme phenylalanine ammonia-lyase (PAL) has been determined . The primary structure of PAL was deduced from the nucleotide sequence of the two cDNA clones, pPAL1 and pPAL2, which covered the entire amino acid-coding sequence . Comparison of cDNA and genomic sequences of pal revealed the presence of six introns . The nucleotide sequences of these introns were compared to those from other fungi . The primary amino acid sequence of the enzyme exhibits only 30.8% identity with the determined primary sequence of PAL from Phaseolus vulgaris . Upstream from the structural gene there is a stretch of C + T-rich DNA similar to that found upstream from a number of Neurospora and Saccharomyces cerevisiae genes . In the case of S . cerevisiae, these C + T-rich sequences are thought to be involved in the transcription of highly expressed genes. Curr Genet, 1987, 12(8), 561 - 7 The secretion and post translational modification of interferons from Saccharomyces cerevisiae; Piggott JR et al.; Studies with three interferon molecules, IFN-alpha 2, IFN-beta 1, and a "hybrid" interferon, IFNX-430 are described which illustrate that both the expression and secretion characteristics of heterologous proteins in yeast cells reflect properties of the proteins themselves . Recombinant DNA techniques have also been used to demonstrate that the efficient processing of mature heterologous proteins from the yeast alpha factor secretion leader can be affected by sequences on the carboxyl side of the initial cleavage site . Secretion studies with heterologous proteins in S . cerevisiae are aimed at maximising yield, the percentage of extracellular product and correct amino terminus sequence . The results presented here show that all three factors are susceptible to currently unpredictable properties of the foreign sequence . This situation, in turn, means that heterologous proteins can be used as tools in the biochemical dissection of the yeast secretion process. Dev Biol Stand, 1987, 67, 185 - 99 Construction of expression vectors for the production of interferons in yeast; Devenish RJ et al.; Expression vectors designed for the production of foreign proteins in yeast are constructed as a composite of functional DNA segments brought together in a single plasmid DNA molecule . Such a composite DNA molecule constitutes a complex entity in terms of its replication and selection functions (in both E . coli and S . cerevisiae) and its gene expression properties . The latter depend critically upon transcriptional control signals (the promoter and terminator regions from a highly expressed yeast gene) that are used to direct the expression of foreign genes in yeast cells . This paper describes the construction of such an expression vector based on the transcription control signals of the yeast phosphoglycerate kinase gene and its application to the production of human interferon-alpha's in yeast cells. J Biol Chem, 1986 Dec 25, 261(36), 17183 - 91 Characterization of COX9, the nuclear gene encoding the yeast mitochondrial protein cytochrome c oxidase subunit VIIa . Subunit VIIa lacks a leader peptide and is an essential component of the holoenzyme; Wright RM et al.; The gene COX9 for subunit VIIa of cytochrome c oxidase from Saccharomyces cerevisiae has been cloned with the aid of an oligonucleotide probe . From the nucleotide sequence of COX9, we deduce that subunit VIIa is derived from a precursor that is 59 amino acids in length (Mr = 6963) . This precursor is longer than mature subunit VIIa by one amino acid at its NH2 terminus and four amino acids at its COOH terminus . COX9 exists as a single copy in the haploid genome of S . cerevisiae and produces one major transcript . When the genomic copy of COX9 is removed, cells lack a functional cytochrome c oxidase holoenzyme . From the predicted secondary structure of subunit VIIa, previous data concerning its relationship to the lipid bilayer of the inner membrane and the location of its hydrophobic domains (Power, S.D., Lochrie, M.A., and Poyton, R.O . (1986) J . Biol . Chem . 261, 9206-9209) and the finding that it is essential for the holoenzyme, we propose a model for subunit VIIa which suggests that this small integral protein plays a role in holoenzyme assembly or stability. Mol Cell Biol, 1986 Dec, 6(12), 4516 - 25 Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome; Coleman KG et al.; Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae . A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector . Plasmids containing pyk1-complementing DNA were obtained from other investigators . Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes . These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector . A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined . Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3) . Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome . The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans . As previous studies with S . cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency. J Bacteriol, 1986 Dec, 168(3), 1468 - 71 Biological activity of the Asn-5,Arg-7 tridecapeptide encoded by MF alpha 2 of Saccharomyces cerevisiae; Raths S et al.; The precursor predicted by the nucleotide sequence of the MF alpha 2 gene of Saccharomyces cerevisiae contains one copy of the tridecapeptide alpha-factor previously characterized (H2N-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-COOH) and one copy of a peptide that contains two conservative amino acid substitutions (H2N-Trp-His-Trp-Leu-Asn-Leu-Arg-Pro-Gly-Gln-Pro-Met-Tyr-COOH) . To determine whether the novel molecule possesses biological activity, the Asn-5,Arg-7 tridecapeptide was prepared chemically by solid-phase peptide synthesis . Growth arrest and morphogenesis assays gave identical activity profiles for the Asn-5,Arg-7 peptide and the other gene product, the Gln-5,Lys-7 peptide . The activities of the two peptides were additive and indistinguishable for S . cerevisiae X2180-1A . When present in fourfold molar excess, the biologically inactive desTrp-1,Ala-3 dodecapeptide reversed activity of the Asn-5,Arg-7 and Gln-5,Lys-7 tridecapeptides . Furthermore, neither peptide caused growth arrest of a MATa ste2(Ts) mutant when assayed at the restrictive temperature . These studies suggest that both pheromones interact with the alpha-factor receptor in a similar manner. Mol Cell Biol, 1986 Dec, 6(12), 4763 - 6 DNA binding is not sufficient for nuclear localization of regulatory proteins in Saccharomyces cerevisiae; Silver PA et al.; We showed by immunofluorescence that the procaryotic DNA-binding protein LexA and a chimeric protein that contains the DNA-binding portion of LexA (amino acids 1 to 87) and a large portion (amino acids 74 to 881) of the Saccharomyces cerevisiae positive regulatory GAL4 protein (GAL4 gene product) are not preferentially localized in the nucleus in S . cerevisiae. Mol Cell Biol, 1986 Dec, 6(12), 4281 - 94 Structure of the Saccharomyces cerevisiae HO gene and analysis of its upstream regulatory region; Russell DW et al.; The HO gene product of Saccharomyces cerevisiae is a site-specific endonuclease that initiates mating type interconversion . We have determined the nucleotide sequence of a 3,129-base-pair (bp) segment containing HO . The segment contains a single long open reading frame encoding a polypeptide of 586 amino acids, which has unusual (unbiased) codon usage and is preceded by 762 bp of upstream region . The predicted HO protein is basic (16% lysine and arginine) and is calculated to have a secondary structure that is 30% helical . The corresponding transcript is initiated approximately 50 nucleotides prior to the presumed initiation codon . Insertion of an Escherichia coli lacZ gene fragment into the putative HO coding segment inactivated HO and formed a hybrid HO-lacZ gene whose beta-galactosidase activity was regulated by the mating type locus in the same manner as HO (repressed by a 1-alpha 2) . Upstream regions of 1,360 and 762 bp conferred strong repression; 436 bp led to partial constitutivity and 301 bp to full constitutivity . Thus, DNA sequences that confer repression of HO by a1-alpha 2 are at least 250 nucleotides upstream of the transcription start point and are within 436 nucleotides of the HO initiation codon . The progressive loss of repression suggests that both the -762 to -436 and the -436 to -301 intervals contain sites for regulation by a1-alpha 2 . The HO gene contains two distinct regions that promote autonomous replication of plasmids in S . cerevisiae . These regions contain sequences that are homologous to the two conserved sequences that are associated with ARS activity. Mol Cell Biol, 1986 Dec, 6(12), 4425 - 32 Highly mutable sites for ICR-170-induced frameshift mutations are associated with potential DNA hairpin structures: studies with SUP4 and other Saccharomyces cerevisiae genes; Hampsey DM et al.; The majority of the mutations induced by ICR-170 in both the CYC1 gene (J . F . Ernst et al . Genetics 111:233-241, 1985) and the HIS4 gene (L . Mathison and M . R . Culbertson, Mol . Cell . Biol . 5:2247-2256, 1985) of the yeast Saccharomyces cerevisiae were recently shown to be single G . C base-pair insertions at monotonous runs of two or more G . C base pairs . However, not all sites were equally mutable; in both the CYC1 and HIS4 genes there is a single highly mutable site where a G . C base pair is preferentially inserted at a {sequence in text} . Here we report the ICR-170 mutagen specificity at the SUP4-o tyrosine tRNA gene of yeast . Genetic fine structure analysis and representative DNA sequence determination of ICR-170-induced mutations revealed that there is also a single highly mutable site in SUP4-o and that the mutation is a G . C base-pair insertion at a monotonous run of G . C base pairs . Analysis of DNA sequences encompassing the regions of highly mutable sites for all three genes indicated that the mutable sites are at the bases of potential hairpin structures; this type of structure could not be found at any of the other, less mutable G . C runs in SUP4, CYC1, and HIS4 . Based on these results and recent information regarding novel DNA structural conformations, we present a mechanism for ICR-170-induced mutagenesis . (i) ICR-170 preferentially binds to DNA in the beta conformation; factors that increase the temporal stability of this structure, such as adjacent stem-and-loop formation, increase the frequency of ICR-170 binding; (ii) the observed mutagen specificity reflects formation of a preferred ICR-170 intercalative geometry at {sequence in text} sites; (iii) during replication or repair, ICR-170 remains associated with the single-stranded template; (iv) stuttering or strand slippage by the polymerization complex as it encounters the mutagen results in nucleotide duplication; (v) subsequent replication or mismatch repair fixes the insertion into the genome . This mechanism accounts for both the IRC-170 mutagenic specificity and the molecular basis of the highly mutable sites in S . cerevisiae. J Biol Chem, 1986 Nov 25, 261(33), 15572 - 6 Reconstitution of Saccharomyces cerevisiae phosphatidylserine synthase into phospholipid vesicles . Modulation of activity by phospholipids; Hromy JM et al.; Membrane-associated phosphatidylserine synthase was purified from Saccharomyces cerevisiae (Bae-Lee, M., and Carman, G . M . (1984) J . Biol . Chem . 259, 10857-10862) and reconstituted into phospholipid vesicles containing phosphatidylcholine/phosphatidylethanolamine/ phosphatidylinositol/phosphatidylserine . Reconstitution was performed by removing detergent from an octyl glucoside/phospholipid/Triton X-100/enzyme mixed micelle by Sephadex G-50 super-fine chromatography . The average diameter of the vesicles was 90 nm, and the enzyme was reconstituted asymmetrically with the active site facing outward . The enzymological properties of reconstituted phosphatidylserine synthase were determined in the absence of detergent . The enzyme was reconstituted into vesicles with phospholipid compositions approximating those of wild type and mutant strains of S . cerevisiae . Reconstituted activity was modulated by the phosphatidylinositol/phosphatidylserine ratio in the vesicles . The modulation of activity observed in the vesicles is enough to account for some of the fluctuations in the phosphatidylserine content in vivo. Nucleic Acids Res, 1986 Nov 11, 14(21), 8347 - 60 The relationship between mRNA stability and length in Saccharomyces cerevisiae; Santiago TC et al.; A rapid and convenient procedure has been developed for the measurement of mRNA half-life in S.cerevisiae using the transcriptional inhibitor, 1,10-phenanthroline . A range of half-lives from 6.6 +/- 0.67 minutes to over 100 minutes, relative to the stability of the 18S rRNA control, has been obtained for fifteen mRNAs . They include the pyruvate kinase and actin mRNAs, as well as 13 randomly picked mRNAs of unknown function . The mRNAs clearly fall into two populations when their lengths and half-lives are analysed; one population is considerably more stable than the other when mRNAs of similar length are compared . Also, within each population, there is an inverse relationship between mRNA length and half-life . These results suggest that mRNA length and at least one additional factor strongly influence mRNA stability in yeast. Cell, 1986 Nov 7, 47(3), 413 - 22 RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor; Powers S et al.; We have identified a gene (SUPH) of S . cerevisiae that is required for both RAS function and mating by cells of a mating type . supH is allelic to ste16, a gene required for the production of the mating pheromone a-factor . Both RAS and a-factor coding sequences terminate with the potential acyltransferase recognition sequence Cys-A-A-X, where A is an aliphatic amino acid . Mutations in SUPH-STE16 prevent the membrane localization and maturation of RAS protein, as well as the fatty acid acylation of it and other membrane proteins . We propose the designation RAM (RAS protein and a-factor maturation function) for SUPH and STE16 . RAM may encode an enzyme responsible for the modification and membrane localization of proteins with this C-terminal sequence. Cell, 1986 Nov 7, 47(3), 401 - 12 The ras-related YPT1 gene product in yeast: a GTP-binding protein that might be involved in microtubule organization; Schmitt HD et al.; The 23.5 kd protein product of the ras-related YPT1 gene of S . cerevisiae was found to be essential for cell growth . The loss of YPT1 function, studied in cells with the YPT1 gene on chromosome VI regulated by the galactose-inducible GAL10 promoter, led to arrested cells that were multibudded and exhibited a complete disorganization of microtubules and an apparent loss of nuclear integrity . The YPT protein binds GTP specifically . GTP binding of the protein is essential for its intracellular function . The Asn121----IIe substitution, generated by site-directed mutagenesis, had a dominant lethal phenotype, the expression of the mutant protein led to binucleated cells and abnormal spindles . In contrast to the S . cerevisiae RAS1 and RAS2 gene products, the YPT protein seems to be involved, directly or indirectly, in microtubule organization and function. Environ Health Perspect, 1986 Nov, 69, 183 - 202 Toxicology of haloacetonitriles; Hayes JR et al.; Haloacetonitriles are by-products of water chlorination and may form in vivo from the reaction of residual chlorine with endogenous compounds such as amino acids . Dibromoacetonitrile (DBAN) was negative in selected mutagenic assays; dichloroacetonitrile (DCAN) was mutagenic in S . typhimurium, but not in S . cerevisiae . Both DBAN and DCAN may be carcinogenic . There is a paucity of basic toxicological data for these compounds . The studies described were conducted to determine the acute, subacute, and subchronic toxicity of DBAN and DCAN . The acute oral LD50 values (mg/kg) in mice and rats are: DBAN, mice: 289 (M), 303 (F); DBAN, rats: 245 (M), 361 (F); DCAN, mice: 270 (M), 279 (F); DCAN, rats: 339 (M), 330 (F) . Death was preceded by slowed respiration, depressed activity, prostration, and coma . There were no apparent compound-related gross pathological effects . DBAN (in corn oil) was administered by gavage to male and female CD rats for 14 or 90 days at levels of 23, 45, 90, and 180 mg/kg/day or 6, 23, and 45 mg/kg/day, respectively . Mortality was 100% at 180 mg/kg and 40% (M) and 20% (F) at 90 mg/kg/day . Compound-related mortality was 10% (M) and 5% (F) at 45 mg/kg and 0% (M) and 10% (F) at 23 mg/kg during the 90-day study . No consistent, significant, adverse compound-related effects on any of the parameters evaluated were evident . Possible target organs might be spleen, thymus, and liver . The no-observed adverse-effect level (NOAEL) for 14 days was 45 mg/kg/day and for 90 days was 23 mg/kg/day . DCAN (in corn oil) was administered by gavage to male and female CD rats for 14 or 90 days at levels of 12, 23, 45, and 90 mg/kg/day or 8, 33, and 65 mg/kg/day, respectively . There were no deaths during the 14-day study . Compound-related mortality was 50% (M) and 25% (F) at 65 mg/kg, 10% (M) and 5% (F) at 33 mg/kg, and 5% (M) and 0% (F) at 8 mg/kg during the 90-day study . Body weights were significantly lower at 90 and 65 mg/kg/day; weight and ratios of spleen and gonads and cholesterol levels were significantly lower at 90 mg/kg/day . No consistent, significant adverse compound-related effects on any of the parameters evaluated were evident . The NOAEL for 14 days was 45 mg/kg/day and for 90 days was 8 mg/kg/day. Mol Cell Biol, 1986 Nov, 6(11), 3847 - 53 Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms; Struhl K; his3 and pet56 are adjacent Saccharomyces cerevisiae genes that are transcribed in opposite directions from initiation sites that are separated by 200 base pairs . Under normal growth conditions, in which his3 and pet56 are transcribed at similar basal levels, a poly(dA-dT) sequence located between the genes serves as the upstream promoter element for both . In contrast, his3 but not pet56 transcription is induced during conditions of amino acid starvation, even though the critical regulatory site is located upstream of both respective TATA regions . Moreover, only one of the two normal his3 initiation sites is subject to induction . From genetic and biochemical evidence, I suggest that the his3-pet56 intergenic region contains constitutive and inducible promoters with different properties . In particular, two classes of TATA elements, constitutive (Tc) and regulatory (Tr), can be distinguished by their ability to respond to upstream regulatory elements, by their effects on the selection of initiation sites, and by their physical structure in nuclear chromatin . Constitutive and inducible his3 transcription is mediated by distinct promoters representing each class, whereas pet56 transcription is mediated by a constitutive promoter . Molecular mechanisms for these different kinds of S . cerevisiae promoters are proposed. Mol Cell Biol, 1986 Nov, 6(11), 3774 - 84 Glycolytic gene expression in Saccharomyces cerevisiae: nucleotide sequence of GCR1, null mutants, and evidence for expression; Baker HV; In Saccharomyces cerevisiae, the gcr mutation is known to have a profound effect on the levels of most glycolytic enzymes, reducing them to 5% of normal or less in growth on noncarbohydrates . Here I report the preparation of chromosomal gcr insertion and deletion mutations . The null mutations were recessive, were not lethal, and caused a pattern of glycolytic enzyme deficiency similar to that seen earlier for the gcr1-1 allele, including the partial inducibility by glucose of the residual enzyme activities . DNA sequence analysis showed that GCR1 encoded a protein of molecular weight 94,414, with a very low codon bias index, characteristic of several S . cerevisiae regulatory genes; adjacent 5' and 3' sequences contained elements suggesting that it was transcribed, polyadenylated, and translated . RNA gel transfer hybridization experiments with purified polyadenylated RNA and a probe complementary to the 5' portion of the open reading frame showed that Ger was expressed as a polyadenylated transcript . Together with previous work, the present results suggest that the Gcr product may be a transcriptional factor necessary specifically for the high-level transcription of a limited set of genes whose products, the enzymes of glycolysis, constitute a substantial fraction of cell proteins and are responsible for the primary metabolic flux in many cells. Genetics, 1986 Nov, 114(3), 753 - 67 Thymidine utilization by tut mutants and facile cloning of mutant alleles by plasmid conversion in S . cerevisiae; Sclafani RA et al.; Plasmid pJM81 contains a Herpes simplex virus thymidine kinase (TK) gene that is expressed in yeast . Cells containing the plasmid utilize thymidine (TdR) and the analogue 5-bromodeoxyuridine (BUdR) for specific incorporation into DNA . TdR auxotrophs, harboring plasmid pJM81 and a mutation in the yeast gene TMP1 require high concentrations of TdR (300 micrograms/ml) to support normal growth rates and the wild-type mitochondrial genome (rho+) cannot be maintained . We have identified a yeast gene, TUT1, in which recessive mutations allow efficient utilization of lower concentrations of TdR . Strains containing the mutations tmp1 and tut1, as well as plasmid pJM81, form colonies at 2 micrograms/ml TdR, grow at nearly normal rates and maintain the rho+ genome at 50 micrograms/ml TdR . These strains can be used to radiolabel DNA specifically and to synchronize DNA replication by TdR starvation . In addition, the substitution of BUdR for TdR allows the selective killing of DNA-synthesizing cells by 310-nm irradiation and allows the separation of replicated and unreplicated forms of DNA by CsCl equilibrium density banding . We also describe a unique, generally applicable system for cloning mutant alleles that exploits the fact that Tk+ yeast cells are sensitive to 5-fluorodeoxyuridine (FUdR) and that gene conversions can occur between a yeast chromosome and a TK-containing plasmid. J Bacteriol, 1986 Oct, 168(1), 467 - 9 Mediation, by Saccharomyces cerevisiae translocation signals, of beta-lactamase transport through the Escherichia coli inner membrane and sensitive method for detection of signal sequences; Roggenkamp R et al.; Signal sequences of Saccharomyces cerevisiae invertase and alpha-factor pheromone were tested for the ability to mediate protein transport through the inner membrane of Escherichia coli by fusion to bacterial beta-lactamase lacking the signal sequence (blaS0) . Both types of transformants exhibited ampicillin resistance in accordance with the transport of the fused protein to the periplasmic compartment . This compartment contained most of the beta-lactamase activity present in the cell . Therefore, the tested yeast signal sequences, which conferred translocation of their proteins across the membrane of the endoplasmic reticulum in S . cerevisiae, can provide the same function in E . coli . The screening for ampicillin resistance among blaS0 fusions provides a convenient method for the isolation of functional yeast and possibly higher eucaryotic signal sequences. Mol Cell Biol, 1986 Oct, 6(10), 3502 - 12 Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae; Brandriss MC et al.; delta 1-Pyrroline-5-carboxylate (P5C) dehydrogenase, the second enzyme in the proline utilization (Put) pathway of Saccharomyces cerevisiae and the product of the PUT2 gene, was localized to the matrix compartment by a mitochondrial fractionation procedure . This result was confirmed by demonstrating that the enzyme had limited activity toward an externally added substrate that could not penetrate the inner mitochondrial membrane (latency) . To learn more about the nature of the import of this enzyme, three gene fusions were constructed that carried 5'-regulatory sequences through codons 14, 124, or 366 of the PUT2 gene ligated to the lacZ gene of Escherichia coli . When these fusions were introduced into S . cerevisiae either on multicopy plasmids or stably integrated into the genome, proline-inducible beta-galactosidase was made . The shortest gene fusion, PUT2-lacZ14, caused the production of a high level of beta-galactosidase that was found exclusively in the cytoplasm . The PUT2-lacZ124 and PUT2-lacZ366 fusions made lower levels of beta-galactosidases that were mitochondrially localized . Mitochondrial fractionation and protease-protection experiments showed that the PUT2-lacZ124 hybrid protein was located exclusively in the matrix, while the PUT2-lacZ366 hybrid was found in the matrix as well as the inner membrane . Thus, the amino-terminal 124 amino acids of P5C dehydrogenase carries sufficient information to target and deliver beta-galactosidase to the matrix compartment . The expression of the longer hybrids had deleterious effects on cell growth; PUT2-lacZ366-containing strains failed to grow on proline as the sole source of nitrogen . In the presence of the longest hybrid beta-galactosidase, the wild-type P5C dehydrogenase was still properly localized in the matrix compartment, but its activity was reduced . The nature of the effects of these hybrid proteins on cell growth is discussed. Mol Cell Biol, 1986 Oct, 6(10), 3401 - 9 Repair of heteroduplex plasmid DNA after transformation into Saccharomyces cerevisiae; Bishop DK et al.; Purified heteroduplex plasmid DNAs containing 8- or 12-base-pair insertion mismatches or AC or CT substitution mismatches were used to transform Saccharomyces cerevisiae . Two insertion mismatches, separated by 943 base pairs, were repaired independently of each other at least 55% of the time . This suggested that repair tracts were frequently shorter than 1 kilobase . The two insertion mismatches were repaired with different efficiencies . Comparison of the repair efficiency of one mismatched site with or without an adjacent mismatch suggests that mismatches promote their own repair and can influence the repair of neighboring mismatches . When two different plasmids containing single-insertion mismatches were transformed into S . cerevisiae cells, a slight preference towards insertion was detected among repair products of one of the two plasmids, while no repair preference was detected among transformants with the second plasmid. Biochemistry, 1986 Sep 9, 25(18), 5117 - 25 Identification an