Curr Genet, 1989 Oct, 16(4), 247 - 52 A hot-spot for transposition of various Ty elements on chromosome V in Saccharomyces cerevisiae; Lochmuller H et al.; Ty4 is a novel transposable element in the yeast, Saccharomyces cerevisiae, which is present in only a few copies in the genome (Stucka et al . 1989) . In strain C836 one of the three copies (Ty4-90) is contained in cosmid clone c90, where it resides on chromosome V . Analysis of this region reveals a "hot-spot" of transposition: in addition to Ty4-90, the locus contains a complete Ty3 element and seven singular delta, sigma and tau elements . Three tRNA genes (for His, Lys, and Ile) are located in this region, and these are closely associated with one or the other of the elements, a phenomenon commonly observed in yeast . A comparison of c90 with corresponding regions from other strains shows that the locus is highly polymorphic and that this polymorphism is explicitly associated with Ty transposition and recombination events. Curr Genet, 1989 Oct, 16(4), 241 - 6 Synthesis and function of the mitochondrial intron--encoded bI4 RNA maturase from Saccharomyces cerevisiae . Effects of upstream frame-shift mutations; Goguel V et al.; We have analyzed the expression and function of the intron-encoded bI4 maturase when frame-shift mutations in the upstream exon alter the translational process . By constructing secondary cis-acting mutations within the bI4 intron, we observed (1) that the bI4 maturase is still translated in the presence of the upstream mutation, albeit in very low amounts, and (2) that the limited amounts of bI4 maturase made under these conditions is no longer able to promote the splicing process of the aI4 intron . These observations, which further strengthen the maturase model, strongly suggest that bI4 maturase acts sequentially on the bI4 intron and then on the aI4 intron. Mol Cell Biol, 1989 Oct, 9(10), 4447 - 58 Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae; Kunkel TA et al.; We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae . To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity . In assays that score polymerase errors within the lacZ alpha-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions . For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold . Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo . The specificity data also suggest models to explain -1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back synthesis. Genetics, 1989 Oct, 123(2), 269 - 79 Ty1 transposition in Saccharomyces cerevisiae is nonrandom; Natsoulis G et al.; A large collection of Ty1 insertions in the URA3 and LYS2 loci was generated using a GAL1-Ty1 fusion to augment the transposition frequency . The sites of insertion of most of these Ty elements were sequenced . There appears to be a gradient of frequency of insertion from the 5' end (highest frequency) to the 3' end (lowest frequency) of both loci . In addition we observed hotspots for transposition . Twelve of the 82 Ty1 insertions in the URA3 locus were inserted in exactly the same site . Hotspots were also observed in the LYS2 locus . All hotspots were in the transcribed part of the genes . Alignment of the sites of insertion and of the neighboring sequences only reveals very weak sequence similarities. J Virol, 1989 Oct, 63(10), 4422 - 5 Trans activation by the bovine papillomavirus E2 protein in Saccharomyces cerevisiae; Morrissey LC et al.; The papillomavirus E2 protein functions as an enhancer-binding factor to promote transcription in mammalian cells . We found that one copy of the E2 binding site acted as an E2 protein-dependent upstream activating sequence in Saccharomyces cerevisiae . Additional copies of the binding motif further augmented transcription . These results imply that the E2 protein functionally interacts with highly conserved transcriptional elements. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1989 Oct, 11(5), 331 - 7 {Human epidermal growth factor gene is expressed in saccharomyces cerevisiae}; Huang BR; Saccharomyces cerevisiae was transformed with plasmids containing the synthetic hEGF gene under control of a alpha-factor promoter . The expressed protein was efficiently secreted into the medium broth and was shown to be a biologically active hEGF. Can J Microbiol, 1989 Oct, 35(10), 945 - 50 Effects of iron binding agents on Saccharomyces cerevisiae growth and cytochrome P450 content; Wright GD et al.; The yeast Saccharomyces cerevisiae Y222 was studied in the presence of the following iron-binding agents: Desferal, dipyridyl, and human and bovine transferrins . We report that cell growth and lanosterol 14 alpha-demethylase cytochrome P450 are not affected by Desferal but that dipyridyl and serum transferrins decrease the cytochrome P450 content of the yeast . Paradoxically, while both human and bovine transferrins reduce cytochrome P450 content, only bovine transferrin appears to affect cell growth in this strain . No evidence for siderophore production by this strain was found under low iron conditions. Proc Natl Acad Sci U S A, 1989 Oct, 86(20), 7866 - 70 ATP-sensitive K+ channels in a plasma membrane H+-ATPase mutant of the yeast Saccharomyces cerevisiae; Ramirez JA et al.; A mutant in the plasma membrane H+-ATPase gene of the yeast Saccharomyces cerevisiae with a reduced H+-ATPase activity, when examined at the single-channel level with the patch-clamp technique, was found to exhibit K+ channels activated by intracellular application of ATP . In the parent strain, the same channel, identified by its conductance and selectivity, is not activated by ATP . This activity in the mutant is blocked by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide . ADP and the ATP analog adenosine 5'-{gamma-{35S}thio}triphosphate do not activate the channel . These findings suggest a tight physical coupling between the plasma membrane ATPase and the K+ channel. J Photochem Photobiol B, 1989 Oct, 4(1), 57 - 74 Molecular dosimetry of 8-MOP + UVA-induced DNA photoadducts in Saccharomyces cerevisiae: correlation of lesions number with genotoxic potential; Bankmann M et al.; Acid hydrolysis of purified DNA extracted from cells of a haploid repair-proficient (RAD) yeast strain that had been treated with 8-MOP + UVA revealed the existence of two major and one minor thymine photoproduct . At survival levels of the RAD strain between 100% and 1% furanside monoadducts constituted the major DNA lesion, followed by diadducts that, at the lowest survival level, nearly reached 50% of the thymine photoproducts; pyrone-side monoadducts were only detectable at the highest UVA exposure dose applied and clearly constitute a minority photoproduct . The number of induced diadducts was verified by determination of interstrand cross-links via denaturation and renaturation of 8-MOP + UVA-treated DNA from RAD and rad2 yeast strain . 8-MOP + UVA was shown to induce two types of locus-specific mutations: reversion of the lys1-1 ochre allele was between 20- to 50-fold higher than that of the his4-38 frameshift allele . Mutant yield for the lys 1-1 reversion was the same in RAD and excision repair-deficient rad2-20 strains whereas frameshift mutagenesis was about eightfold higher in the rad2-20 background. Eur J Biochem, 1989 Oct 1, 184(3), 651 - 6 Nuclearly inherited diuron-resistant mutations conferring a deficiency in the NADH--or succinate--ubiquinone oxidoreductase activity in Saccharomyces cerevisiae; Meunier B et al.; In Saccharomyces cerevisiae, diuron, antimycin and myxothiazol block the respiratory pathway at the bc1 complex level . Nuclearly inherited mutations located at the DIU3 and DIU4 loci confer in vitro resistance to diuron and cross-resistance to antimycin and myxothiazol at the NADH oxidase level . The mutant strains do not exhibit diuron resistance at the quinol-cytochrome-c oxidoreductase level . Thus, the apparent resistance does not seem to be the result of a modification of the inhibitory sites . Instead, the quinone reduction rate was found to be altered in the mutant . The diu3 mutations lead to a deficiency of the NADH--ubiquinone oxidoreductase activity, and the diu4 mutations to a deficiency of the succinate--ubiquinone oxidoreductase activity . On the basis of the model of Kroger and Klingenberg, a decrease of quinone reduction could explain the resistance to the bc1 complex inhibitors . Thus, the apparent resistance to the bc1 complex inhibitors was found to be due to a modification of the electron transfer kinetics. Nucleic Acids Res, 1989 Sep 25, 17(18), 7487 - 93 Similarity between the picornavirus VP3 capsid polypeptide and the Saccharomyces cerevisiae virus capsid polypeptide; Bruenn LA et al.; We have compared the sequence of the capsid polypeptide of the Saccharomyces cerevisiae double-stranded RNA virus, ScV, with those of the picornaviruses . A central region of 245 amino acids in the ScV capsid polypeptide of 680 amino acids has significant similarity to the picornavirus VP3 . This similarity is more extensive than that already noted for the alphavirus capsid polypeptide and the picornavirus VP3 (Fuller, S.D . and Argos, P, EMBO J . 6, 1099, 1987) . Together with the similarity between the ScV RNA polymerase and the picornavirus RNA polymerases, this result implies an evolutionary relationship between a simple double-stranded RNA virus of fungi and the small plus strand RNA animal viruses. Eur J Biochem, 1989 Sep 15, 184(2), 305 - 11 Purification and characterization of a nuclear factor which binds specifically to the upstream activation sequence of Saccharomyces cerevisiae enolase 1 gene; Machida M et al.; The nuclear factor which specifically binds to the upstream activation sequence (UAS) of the enolase 1 gene (ENO1) of yeast Saccharomyces cerevisiae was purified by sequence-specific affinity chromatography . The purified factor gave two closely migrated bands at 32 kDa on SDS/PAGE . The binding activities were eluted from a gel filtration column at molecular masses of 110 kDa and 60 kDa, suggesting a dimeric and a tetrameric assembly of the factor in the native form . The region protected by the purified factor against deoxyribonuclease I digestion contained the sequence ACCCAAACACC which is highly similar to the consensus sequence present in the 5'-flanking region of the ribosomal protein genes (RPG box) . We also identified the other factor specific to the ENO1 UAS which gave a single peak at a molecular mass of 120 kDa in gel filtration . We suggest the existence of multiple binding to the ENO1 UAS by at least two factors: one is the factor which we purified with a molecular mass of 32 kDa on SDS/PAGE and the other is the factor like RAP1 protein which generally recognizes the RPG-box-like sequence. Biochem Biophys Res Commun, 1989 Sep 15, 163(2), 908 - 15 Nucleotide sequence of AMS1, the structure gene of vacuolar alpha-mannosidase of Saccharomyces cerevisiae; Yoshihisa T et al.; AMS1, a structure gene of the vacuolar membrane alpha-mannosidase of Saccharomyces cerevisiae, has been characterized and found to encode both constituent polypeptides of the enzyme, a 107 kDa polypeptide and a 73 kDa polypeptide . The nucleotide sequence of AMS1 demonstrates that the gene encodes 1083 amino acids with a molecular weight 124,497 . Although the enzyme is considered to exist on the inner surface of the vacuolar membrane, the predicted primary amino acid sequence does not have a hydrophobic stretch suitable for a signal sequence in its N-terminal region. Eur J Biochem, 1989 Sep 15, 184(2), 433 - 43 Guanylate kinase from Saccharomyces cerevisiae . Isolation and characterization, crystallization and preliminary X-ray analysis, amino acid sequence and comparison with adenylate kinases; Berger A et al.; This paper describes a large-scale purification of guanylate kinase (ATP + GMP in equilibrium ADP + GDP) from Saccharomyces cerevisiae, the crystallization of the enzyme and preliminary X-ray investigations . Furthermore the complete amino acid sequence of the enzyme has been determined and was compared to adenylate kinase sequences . 1 . Guanylate kinase was purified in five steps to homogeneity: crude extract, ion-exchange chromatography, affinity chromatography and gel filtration twice . 2 . The enzyme was crystallized to single octahedral bipyramids with sizes up to 500 x 200 x 150 microns 3 . Preliminary X-ray results are given . 3 . The final sequence shows 186 amino acids (Mr = 20,548), containing one cysteine and one tryptophan . It was determined from peptides of five cleavages of the whole protein . Three cleavages were used for determination of the whole polypeptide chain . From the other two, only some peptides were used to secure overlaps and the cysteine position . The N-terminal blocking group was identified by 1H-NMR spectroscopy . 4 . Since guanylate kinase shows the mononucleotide binding pattern GXXGXGK, it was compared to other proteins containing this pattern . But no further homology signal could be detected . A comparison with adenylate kinases revealed significant similarity in another chain segment . This led to the conclusion that guanylate kinase is at least partially homologous to the adenylate kinases. J Biol Chem, 1989 Sep 15, 264(26), 15593 - 9 The Saccharomyces cerevisiae SOC8-1 gene and its relationship to a nucleotide kinase; Choi WJ et al.; The yeast SOC8-1 gene was originally identified by partial complementation of cdc8 mutant strains . We have carried out Bal31 deletion analysis of the SOC8-1 gene to define the minimal size which is required for the complementation of the cdc8 mutation . When the SOC8-1 gene is cloned in a multicopy plasmid, it enables temperature-resistant growth in the cdc8 mutant strain, while the SOC8-1 gene in a single copy plasmid does not . Thus, its suppression of the cdc8 mutant is dosage dependent . The high copy number vector carrying the SOC8-1 gene can complement five different cdc8 alleles, indicating that the suppression is not allele specific . Since CDC8 encodes thymidylate kinase, cells bearing a high copy number plasmid containing SOC8-1 gene were tested for the ability to phosphorylate several nucleoside monophosphates, including UMP, GMP and dTMP . Significantly increased phosphorylation activity was observed, suggesting that SOC8-1 encodes a nucleotide kinase . Both restriction enzyme analysis of the SOC8-1 gene and partial purification of the overproduced kinase in SOC8-1 overproducing strains suggest that SOC8-1 may be allelic with URA6 . Consistent with these results, both SOC8-1 and URA6 are located on chromosome XI . Thus, one possible suppression mechanism is that SOC8-1 may provide a trans-acting dTMP kinase activity, bypassing the cdc8 gene defect. J Biol Chem, 1989 Sep 5, 264(25), 15022 - 7 Expression of the four subunits of the Torpedo californica nicotinic acetylcholine receptor in Saccharomyces cerevisiae; Jansen KU et al.; Yeast expression vectors were constructed containing complementary DNA encoding the alpha-, beta-, gamma-, and delta-subunits of the Torpedo californica nicotinic acetylcholine receptor under the control of the Saccharomyces cerevisiae alcohol dehydrogenase promoter . All four plasmids were integrated into the yeast genome of a single yeast cell . The resulting yeast strain synthesized polypeptides novel to yeast that had the molecular weights and antigenic properties similar to the authentic T . californica receptor alpha-, gamma, and delta-subunits . The beta-subunit polypeptide could not be detected in this yeast strain, even though the poly(A)+ RNA from this strain contained all the information necessary for the expression of functional acetylcholine receptors in Xenopus laevis oocytes . The replacement of the beta-subunit mRNA 5'-untranslated leader and its N-terminal signal sequence by the corresponding alpha-subunit sequences, however, resulted in the expression of the beta-subunit polypeptide in yeast grown at 5 degrees C. J Gen Microbiol, 1989 Sep, 135 ( Pt 9), 2429 - 37 Characterization of AAT1: a gene involved in the regulation of amino acid transport in Saccharomyces cerevisiae; Garrett JM; A new class of Saccharomyces cerevisiae mutants (aat1 - amino acid transport) has been identified . These mutants are unable to grow on rich medium or on minimal medium supplemented with certain amino acids (isoleucine, methionine, phenylalanine, tyrosine or valine) . This phenotype is directly linked to the presence of the leu2 allele in these strains: aat1 LEU2 organisms grow normally on all media tested . Leucine uptake through the leucine-specific permease is inhibited to less than 35% of wild-type levels in aat1 cells preincubated in nonpermissive media, and the activity of the general amino acid permease is also low in these conditions . aat1 cells are therefore unable to grow on rich media because they cannot take up enough leucine to supplement their auxotrophic requirement. J Gen Microbiol, 1989 Sep, 135 ( Pt 9), 2407 - 11 Controlling the growth rate of Saccharomyces cerevisiae cells using the glucose analogue D-glucosamine; McGoldrick EM et al.; By using competition between glucose and its analogue D-glucosamine, we have produced a system in which it is possible to vary the steady-state growth rate of populations of Saccharomyces cerevisiae cells without otherwise altering the composition of the medium or significantly affecting catabolite repression . We demonstrate that D-glucosamine inhibits the accumulation of glucose derived label and the phosphorylation of glucose by hexokinase (EC 2.7.1.1). Mol Gen Mikrobiol Virusol, 1989 Sep, (9), 14 - 20 {Repair of a double-stranded gap in plasmid DNA in radiosensitive mutants of Saccharomyces cerevisiae: effectiveness and precision}; Glazer VM et al.; The repair of a double strand gap in plasmid DNA in radiosensitive mutants of Saccharomyces cerevisiae has been studied . The proportion of repair events resulting in the complete doublestrand gap recovery of the plasmid DNA has been found to be close to 100% in Rad+ cells . The mutation rad55 did not interfere in the doublestrand gap repair efficiency and accuracy . The mutant rad57 is capable of the effective doublestrand gap repair without restoration of the DNA sequence deleted by the gap . The mutation rad53 substantially inhibited the efficiency of the doublestrand gap repair but did not influence the accuracy of the repair . Plasmid DNA doublestranded gap repair is completely blocked by mutations rad50 and rad54. Mol Endocrinol, 1989 Sep, 3(9), 1477 - 87 Expression of human placental aromatase in Saccharomyces cerevisiae; Pompon D et al.; A full-length human placental aromatase cDNA clone, Aro 2, was isolated upon screening a human placental cDNA library with an aromatase cDNA probe and an oligonucleotide probe whose sequence was derived from a human aromatase genomic clone . Nucleotide sequence microheterogeneity was found in the 3'-untranslated region among Aro 2 and in two previously described human aromatase cDNA clones . Both the minor sequence differences and the expression of a single protein species in placental tissue suggest the presence of different alleles for aromatase . Northern blot analyses using one cDNA and two oligonucleotide probes are consistent with the two mRNA messages of 2.9 and 2.5 kilobases arising in human placenta as a consequence of differential processing . Several yeast expression plasmids containing the aromatase cDNA we cloned were constructed . The enzyme was expressed in Saccharomyces cerevisiae . The expressed activity was inhibited by the known aromatase inhibitor, 4-hydroxyandrostenedione . A level of 2 micrograms aromatase/mg partially purified yeast microsomes was estimated by analyses of carbon monoxide difference spectra on microsomal fractions from yeast carrying plasmid pHARK/VGAL . Using {1 beta, 2 beta-3H}androst-4-ene-3,17-dione as the substrate, an apparent Michaels-Menken constant (Km) of 34 nM and a maximum velocity (Vmax) of 23 pmol {3H}water formed per min/mg protein were obtained for the yeast synthesized aromatase by transformation with plasmid pHARK/VGAL . The kinetic results are similar to those determined for human placental aromatase, and suggest that the yeast synthesized aromatase will be useful for further structure-function studies. Genetika, 1989 Sep, 25(9), 1703 - 4 {Genetic mapping of the NYS1 gene in Saccharomyces cerevisiae}; Kamilova TA; The NYS1 gene of Saccharomyces cerevisiae yeasts is linked to the centromere marker of chromosome IV - gene TRP1 and is located at 16.2 cM distance from it. Genetics, 1989 Sep, 123(1), 69 - 80 Length and distribution of meiotic gene conversion tracts and crossovers in Saccharomyces cerevisiae; Borts RH et al.; We have measured gene conversion tract length in strains of the yeast Saccharomyces cerevisiae containing three to six restriction site heterozygosities in a 9-kb interval . Tetrads containing a conversion were identified genetically by nonmendelian segregation of a marker in the middle of the interval . Gene conversions accompanied by a crossover have a tract length of 1.4 kb +/- 0.7 kb, which is indistinguishable from a tract length of 1.6 +/- 0.8 for conversions without an associated exchange . Among tetrads identified first as having a crossover in the interval, the average gene conversion tracts were apparently significantly shorter (0.71 +/- 1) . We provide evidence that this apparent difference is due to the method of measuring conversion tracts and does not reflect a real difference in tract length . We also provide evidence that the number and position of restriction site markers alters the apparent distribution of the conversion tracts . More than ninety percent of the conversion tracts spanning three or more sites were continuous. Genetics, 1989 Sep, 123(1), 55 - 68 A general screen for mutant of Saccharomyces cerevisiae deficient in tRNA biosynthesis; van Zyl WH et al.; We have devised a general screen for isolating conditional lethal mutants defective in synthesis of mature tRNA in Saccharomyces cerevisiae . Using this screen, we have identified several new genes in yeast that are required for production of mature tRNA . These genes most likely encode essential functions, since the mutations we isolated are recessive and cause temperature-sensitive growth . One of the mutants, tpd3, is defective in de novo transcription of 4S RNA at the nonpermissive temperature . A second mutant, tpd1, is specifically defective in excision of intervening sequence from a variety of tRNA species . Finally, two other mutants are defective in production of tRNA from a suppressor tRNA locus, as measured by an in vitro suppression assay . The specific lesion in these strains, though, is not known . These data confirm that the screen does, in fact, yield a broad spectrum of mutants defective in tRNA maturation. Genetics, 1989 Sep, 123(1), 29 - 43 Pachytene arrest and other meiotic effects of the start mutations in Saccharomyces cerevisiae; Shuster EO et al.; Mutations in the Start class of cell division cycle genes (CDC28, CDC36 and CDC39) define the point in the G1 phase of the vegetative cycle at which the cell becomes committed to completing another round of cell division . Genetic, cytological and biochemical data demonstrate that these mutations cause meiotic cells to become arrested at pachytene following completion of both chromosomal DNA replication and spindle pole body (SPB) duplication . In contrast these mutations have previously been found to cause arrest of the mitotic cell cycle prior to either of these landmark events, so the role of the Start genes in these events during vegetative growth must be indirect . Our observations are consistent with the hypothesis that CDC28, CDC36 and CDC39 are required for irreversible commitment to nuclear division in both the mitotic and meiotic pathways . CDC28 was additionally found to be required for the SPB separation that precedes spindle formation in preparation for the second meiotic division . Cytological and genetic analyses of this requirement revealed both that such separation may fail independently at either SPB and that ascospore formation can proceed independently of SPB separation. Biochem Cell Biol, 1989 Sep, 67(9), 612 - 31 Pyrimidine biosynthesis in Saccharomyces cerevisiae: the ura2 cluster gene, its multifunctional enzyme product, and other structural or regulatory genes involved in de novo UMP synthesis; Denis-Duphil M; There are six enzymatic steps in the de novo biosynthesis of uridine monophosphate (UMP) . In yeast, six structural genes (ura2, ura4, ura1, ura5, ura10, and ura3) and one regulatory gene (PPR1) are involved in this metabolic pathway . Gene ura2 codes for a multifunctional protein that carries the first two enzymatic activities of the pathway, i.e., carbamylphosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) . Gene ura2 has been cloned and sequenced, revealing the presence of three open reading frames, one of which codes for the multifunctional protein, a polypeptide of 2212 amino acids, with a mRNA of 7 +/- 0.3 kilobases . Expression of gene ura2 is regulated at the transcriptional level . As I indicate here, it could also be controlled at the posttranscriptional level since all the consensus sequences for a 1.2-kilobases intron are present in the coding sequence of the open reading frame . The deducted amino acid sequence has allowed the identification of four domains . Starting from the amino terminus of the protein, these are glutamine amido transferase, CPSase, a domain that resembles dihydroorotase (DHOase-like) but does not have DHOase activity, and ATCase . There are also two sites of interest that match known concensus phosphorylation sites; one is located in the distal part of the CPSase domain, the other in the connector region between DHOase-like and ATCase domains . The protein has been purified as a multienzyme aggregate and as a multifunctional protein . The latter form, when isolated from a protease B deficient strain of Saccharomyces cerevisiae, contained mostly polypeptide chains of 220 kilodaltons . Work is currently in progress to determine the site(s) of phosphorylation of this protein in vitro . ATCase activity of both wild-type and protease-deficient strains has been found to be localized in the nucleus . Channeling of carbamyl phosphate, the first intermediate in the pathway, has been demonstrated both in vitro and in permeabilized cells . The other genes of UMP biosynthesis, except for ura5, are regulated by induction of their transcription by the combined action of the product of the ppr1 gene and the inducer, dihydroorotate . Dihydroorotate dehydrogenase activity was found in the cytoplasm . Two isoenzymes of orotate phosphoribosyl transferase have been found, coded for by ura5 and ura10 . The products of genes ura10 and ura3 are proposed to participate in the channeling of orotidine monophosphate . The discussion considers the problem posed by the isolation of both multienzyme complexes and multifunctional proteins resulting from the expression of the same cluster genes.(ABSTRACT TRUNCATED AT 400 WORDS) Appl Environ Microbiol, 1989 Sep, 55(9), 2242 - 6 Transformation of Saccharomyces cerevisiae by electroporation; Delorme E; A method for introducing heterologous DNA into Saccharomyces cerevisiae rapidly and efficiently by electroporation was developed . Transformant colonies appeared somewhat sooner than by the LiCl or spheroplast transformation method, and the time spent in manipulation was much less than for these two methods . The pores in the cell membrane formed by the high voltage of electroporation were resealed within 6 to 7 min after electroporation . At a capacitance of 25 microF, the optimum voltage was 2.0 to 2.25 kV/cm . Log-phase cells concentrated to 10 to 20 units at an optical density of 600 nm in 200 microliters of fresh rich medium and electroporated at 2.25 kV/cm in the presence of 0.1 microgram of supercoiled plasmid DNA will yield 1,000 to 4,500 colonies per microgram of DNA. Mol Cell Biol, 1989 Sep, 9(9), 4064 - 8 Conservation of function and regulation within the Cdc28/cdc2 protein kinase family: characterization of the human Cdc2Hs protein kinase in Saccharomyces cerevisiae; Wittenberg C et al.; Whereas the Cdc28 protein kinase of the budding yeast Saccharomyces cerevisiae plays an essential role in cell cycle progression during the G1 interval, a function in the progression from the G2 interval into M phase has been inferred for its homologs, including the Cdc2Hs protein kinase of humans . To better understand these apparently disparate roles, we constructed a yeast strain in which the resident CDC28 gene was replaced by its human homolog, CDC2Hs . This transgenic yeast strain was able to perform the G1 functions attributed to the Cdc28 protein kinase, including the ability to grow and divide normally, to respond to environmental signals that induce G1 arrest, and to regulate the Cdc2Hs protein kinase appropriately in response to these signals. Mol Cell Biol, 1989 Sep, 9(9), 3869 - 77 A gene product needed for induction of allantoin system genes in Saccharomyces cerevisiae but not for their transcriptional activation; Bricmont PA et al.; The allantoin-degradative pathway of Saccharomyces cerevisiae consists of several genes whose expression is highly induced by the presence of allophanic acid . Induced expression requires a functional DAL81 gene product . Analysis of these genes has demonstrated the presence of three cis-acting elements in the upstream regions: (i) an upstream activation sequence (UAS) required for transcriptional activation in an inducer-independent fashion, (ii) an upstream repression sequence (URS) that mediates inhibition of this transcriptional activation, and (iii) an upstream induction sequence (UIS) needed for a response to inducer . The UIS element mediates inhibition of URS-mediated function when inducer is present . We cloned and characterized the DAL81 gene and identified the element with which it was associated . The gene was found to encode a rare 3.2-kilobase-pair mRNA . The amount of DAL81-specific RNA responded neither to induction nor to nitrogen catabolite repression . Deletion of the DAL81 gene resulted in loss of induction but did not significantly affect basal level expression of the DAL7 and DUR1,2 genes or the UAS and URS functions present in plasmid constructions . These data suggest that (i) transcriptional activation of the DAL genes and their responses to inducer are mediated by different factors and cis-acting sequences and (ii) the UIS functions only when a wild-type DAL81 gene product is available. Mol Cell Biol, 1989 Sep, 9(9), 3638 - 46 Isolation and characterization of STI1, a stress-inducible gene from Saccharomyces cerevisiae; Nicolet CM et al.; We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a 2.0-kilobase heat-inducible mRNA . This gene, which we have designated STI1, for stress inducible, was also induced by the amino acid analog canavanine and showed a slight increase in expression as cells moved into stationary phase . The STI1 gene encodes a 66-kilodalton protein, as determined from the sequence of the longest open reading frame . The putative STI1 protein, as identified by two-dimensional gel electrophoresis, migrated in the region of 73 to 75 kilodaltons as a series of four isoforms with different isoelectric points . STI1 is not homologous to the other conserved HSP70 family members in yeasts, despite similarities in size and regulation . Cells carrying a disruption mutation of the STI1 gene grew normally at 30 degrees C but showed impaired growth at higher and lower temperatures . Overexpression of the STI1 gene resulted in substantial trans-activation of SSA4 promoter-reporter gene fusions, indicating that STI1 may play a role in mediating the heat shock response of some HSP70 genes. Eur J Biochem, 1989 Sep 1, 184(1), 173 - 9 Functional complementation of catalase-defective peroxisomes in a methylotrophic yeast by import of the catalase A from Saccharomyces cerevisiae; Hansen H et al.; A mutant of the methanol-utilizing yeast Hansenula polymorpha defective in catalase was isolated . It lacks the ability to grow on methanol as the sole source of carbon and energy due to a loss of peroxisomal function that is required for the dissimilation and assimilation of this substrate . Growth of the mutant on glucose or glycerol was not impaired . Transformation of mutant cells with the gene coding for catalase A from Saccharomyces cerevisiae {Cohen, G., Fessl, F., Traczyk, J., Rytka, J . & Ruis, H . (1985) Mol . Gen . Genet . 200, 74-79} conferred constitutive expression of catalase activity . When the gene was placed under control of the regulatory methanol oxidase promoter from H . polymorpha, high levels of activity subject to glucose repression were obtained . In both cases efficient targeting of catalase A to the heterologous peroxisomes and assembly into an active form could be demonstrated . Concomitantly, growth on methanol was restored in the transformed mutant . The results are in line with a high conservation of transport signals on peroxisomal proteins . Expression of a cytosolic catalase in H . polymorpha did not confer the ability to grow on methanol . Therefore, proper localization of the catalase activity is a prerequisite for peroxisomal function. Mutat Res, 1989 Sep, 224(1), 31 - 78 Detection of induced mitotic chromosome loss in Saccharomyces cerevisiae--an interlaboratory study; Whittaker SG et al.; The diploid yeast strain D61.M was used to study induction of mitotic chromosome loss . The test relies upon the uncovering and expression of multiple recessive markers reflecting the presumptive loss of the chromosome VII homologue carrying the corresponding wild-type alleles . The underlying 'loss event' is probably complex since the predicted centromere-linked lethal tetrad segregations for chromosome VII are not recovered . Instead, the homologue bearing the multiple recessive markers is patently homozygous . An interlaboratory study was performed in which 16 chemicals were tested under code in 2 laboratories . The results generated by the Berkeley and Darmstadt laboratories were in close agreement . Acetonitrile, ethyl acetate, 4-acetylpyridine, propionitrile and nocodazole were identified as potent inducers of mitotic chromosome loss . Acetone, dimethyl sulfoxide and 2-methoxyethyl acetate either elicited weak responses or yielded ambiguous results . Water, carbon tetrachloride, 4-fluoro-D,L-phenylalanine, amphotericin B, griseofulvin, cadmium chloride, ethyl methanesulfonate and methylmercury(II) chloride failed to induce chromosome loss . These data suggest that the system described herein represents a reliable assay for chemically induced chromosome loss in yeast. Mutat Res, 1989 Sep, 224(1), 11 - 29 Quantitative approaches for assessing chromosome loss in Saccharomyces cerevisiae: general methods for analyzing downturns in dose response; Piegorsch WW et al.; Statistical methods are considered for analysis of data arising from a mitotic chromosome loss assay in Saccharomyces cerevisiae strain D61.M . The methods make use of reproducibility trial data from the assay (presented herein) and previous data, which suggest a unimodal, 'umbrella-patterned' dose response . Computer simulations are employed to illustrate the operating characteristics of the umbrella response methods . These methods are generally applicable to any toxicity assay that exhibits a downturn in dose response . Experimental design considerations are also discussed . These include applications of 2-stage sampling rules to first gauge the dose window of peak response, then test if the response deviates significantly from untreated levels. Mutat Res, 1989 Sep, 218(2), 111 - 24 PSO4: a novel gene involved in error-prone repair in Saccharomyces cerevisiae; Henriques JA et al.; The haploid xs9 mutant, originally selected for on the basis of a slight sensitivity to the lethal effect of X-rays, was found to be extremely sensitive to inactivation by 8-methoxypsoralen (8MOP) photoaddition, especially when cells are treated in the G2 phase of the cell cycle . As the xs9 mutation showed no allelism with any of the 3 known pso mutations, it was now given the name of pso4-1 . Regarding inactivation, the pso4-1 mutant is also sensitive to mono- (HN1) or bi-functional (HN2) nitrogen mustards, it is slightly sensitive to 254 nm UV radiation (UV), and shows nearly normal sensitivity to 3-carbethoxypsoralen (3-CPs) photoaddition or methyl methanesulfonate (MMS) . Regarding mutagenesis, the pso4-1 mutation completely blocks reverse and forward mutations induced by either 8MOP or 3CPs photoaddition, or by gamma-rays . In the cases of UV, HN1, HN2 or MMS treatments, while reversion induction is still completely abolished, forward mutagenesis is only partially inhibited for UV, HN1, or MMS, and it is unaffected for HN2 . Besides severely inhibiting induced mutagenesis, the pso4-1 mutation was found to be semi-dominant, to block sporulation, to abolish the diploid resistance effect, and to block induced mitotic recombination, which indicates that the PSO4 gene is involved in a recombinational pathway of error-prone repair, comparable to the E . coli SOS repair pathway. Eur J Biochem, 1989 Sep 1, 184(1), 21 - 8 Purification and characterization of an acetyl-CoA hydrolase from Saccharomyces cerevisiae; Lee FJ et al.; Acetyl-CoA hydrolase, which hydrolyzes acetyl-CoA to acetate and CoASH, was isolated from Saccharomyces cerevisiae and demonstrated by protein sequence analysis to be NH2-terminally blocked . The enzyme was purified 1080-fold to apparent homogeneity by successive purification steps using DEAE-Sepharose, gel filtration and hydroxylapatite . The molecular mass of the native yeast acetyl-CoA hydrolase was estimated to be 64 +/- 5 kDa by gel-filtration chromatography . SDS/PAGE analysis revealed that the denatured molecular mass was 65 +/- 2 kDa and together with that for the native enzyme indicates that yeast acetyl-CoA hydrolase was monomeric . The enzyme had a pH optimum near 8.0 and its pI was approximately 5.8 . Several acyl-CoA derivatives of varying chain length were tested as substrates for yeast acetyl-CoA hydrolase . Although acetyl-CoA hydrolase was relatively specific for acetyl-CoA, longer acyl-chain CoAs were also hydrolyzed and were capable of functioning as inhibitors during the hydrolysis of acetyl-CoA . Among a series of divalent cations, Zn2+ was demonstrated to be the most potent inhibitor . The enzyme was inactivated by chemical modification with diethyl pyrocarbonate, a histidine-modifying reagent. Genes Dev, 1989 Sep, 3(9), 1336 - 48 Loss of Ras activity in Saccharomyces cerevisiae is suppressed by disruptions of a new kinase gene, YAKI, whose product may act downstream of the cAMP-dependent protein kinase; Garrett S et al.; The yeast Saccharomyces cerevisiae contains two functionally redundant genes RAS1 and RAS2, which are homologous to the mammalian ras gene family and are required for vegetative growth . We isolated and characterized five temperature-sensitive alleles of RAS2 . In a ras1 strain, these alleles cause growth arrest at the G1 stage of the cell cycle . Revertants capable of growth at the nonpermissive temperature define four recessive, extragenic complementation groups . Suppressors in one complementation group (designated yak1) are particularly intriguing because they appear to alleviate only the growth defect of the temperature-sensitive ras mutants and do not show any of the phenotypes, such as heat shock sensitivity or starvation sensitivity, associated with increased production of cAMP . The YAK1 gene has been cloned, and disruptions generated in vitro reveal that it is not essential for growth and that its loss confers growth to a strain deleted for tpk1, tpk2, and tpk3, the structural genes for the catalytic subunit of the cAMP-dependent protein kinase . These results place Yak1 downstream from, or on a parallel pathway to, the kinase step in the Ras/cAMP pathway . Finally, the coding region predicts a protein with significant homology to the family of protein kinases, suggesting that loss of cAMP-dependent protein kinase function can be suppressed by the loss of a second protein kinase. Curr Genet, 1989 Sep, 16(3), 139 - 43 Cloning and characterization of the SKI3 gene of Saccharomyces cerevisiae demonstrates allelism to SKI5; Hougan L et al.; We have identified a mutant strain of the yeast Saccharomyces cerevisiae which overproduces killer toxin . This strain contains a single mutation which fails to complement defects in both the SKI3 and SKI5 genes, while a cloned copy of this gene complements both the ski3 and ski5 defects . The level of secreted toxin from a cDNA based plasmid is not increased in a ski3 strain, showing that the overproduction phenotype is dependent upon an increased level of M1 dsRNA. Mol Cell Biol, 1989 Sep, 9(9), 4096 - 8 Integration of an aberrant retrotransposon in Saccharomyces cerevisiae; Wilke CM et al.; We describe an atypical composite Ty1 element that apparently resulted from the concurrent integration of two complete elements . A portion of the central region of one of these elements was inverted between two long terminal repeats . Inversions of this type have been detected among unintegrated retroviral circles . It now appears that such intermediates can be incorporated into the genome. Mol Cell Biol, 1989 Sep, 9(9), 3720 - 6 Regulation of postreceptor signaling in the pheromone response pathway of Saccharomyces cerevisiae; Blinder D et al.; alpha-Factor pheromone inhibits division of yeast a cells . After prolonged exposure to alpha-factor, the cells adapt to the stimulus and resume cell division . The sst2 mutation is known to inhibit adaptation . This report examines adaptation in scg1 (also designated gpa1) and STE4Hpl (Hpl indicates haploid lethal) mutants that exhibit constitutive activation of the pheromone response pathway . Recovery of the STE4Hpl mutant was blocked by the sst2-1 mutation, whereas recovery of the scg1-7 mutant was not completely blocked by sst2-1 . These results indicate that both SST2-dependent and -independent mechanisms regulate postreceptor events in the pheromone response pathway . Down regulation of receptors in response to alpha-factor was independent of the signal that was generated in the scg1 mutant. Biochem Int, 1989 Sep, 19(3), 571 - 81 Effect of alpha factor pheromone on the activity of enzymes which catalyze the synthesis and hydrolysis of cell wall structural polymers from Saccharomyces cerevisiae; Ruiz T et al.; Cells of Saccharomyces cerevisiae of the a mating type treated with alpha factor contain an increased amount of structural polymers (beta-glucans and chitin) in their cell walls and, consequently, exhibit higher glucan synthetase and chitin synthetase activities than untreated cells . However, alpha factor has no detectable effect on the activities of these enzymes when they are assayed, "in vitro", in the presence of the pheromone . On the other hand, the activity of beta-glucanases remains constant during the time that growth of a cells is kept arrested by alpha factor at the G1 phase of the cell division cycle and starts to increase when budding of the cells is reinitiated. Mol Cell Biol, 1989 Sep, 9(9), 3698 - 709 PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle; Bjorn SP et al.; The PRP4 (RNA4) gene product is involved in nuclear mRNA processing in yeast cells; we have previously cloned the gene by complementation of a temperature-sensitive mutation . Sequence and transcript analyses of the cloned gene predicted the gene product to be a 52-kilodalton protein, which was confirmed with antibodies raised against the PRP4 gene product . These antibodies inhibited precursor mRNA splicing in vitro, demonstrating a direct role of PRP4 in splicing . Immunoprecipitations with the antibodies indicated that the PRP4 protein is associated with the U4/U6 small nuclear ribonucleoprotein particle. J Biol Chem, 1989 Aug 25, 264(24), 14543 - 8 Molecular basis for resistance to myxothiazol, mucidin (strobilurin A), and stigmatellin . Cytochrome b inhibitors acting at the center o of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae; di Rago JP et al.; The respiratory bc1 complex transfers the electrons from ubiquinol to cytochrome c oxidase . Myxothiazol, strobilurin A (mucidin), and stigmatellin are center o inhibitors preventing electron transfer at the ubiquinone redox site Qo, which is located closer to the outer side of the inner mitochondrial membrane . The cytochrome b gene is carried by the organelle DNA . Yeast mutants resistant to myxothiazol and mucidin have been previously isolated and mapped to specific loci of the cytochrome b gene . In the present work, stigmatellin-resistant mutants were isolated and genetically analyzed . The mutated amino acid residues from seven myxothiazol-, four mucidin-, and six stigmatellin-resistant mutants have been identified by sequencing the relevant segments of the resistant cytochrome b gene . A third myxothiazol-resistant locus and the first stigmatellin-resistant locus were identified . The mutated codons were found to be clustered in two regions of the cytochrome b protein which appeared to be responsible for the resistance to Qo site inhibitors . The first region is within the end of the first, the second, and the beginning of the third exon whereas the second region is within exon five and the beginning of the sixth exon. J Biol Chem, 1989 Aug 15, 264(23), 13648 - 59 Structure of the phosphorylated N-linked oligosaccharides from the mnn9 and mnn10 mutants of Saccharomyces cerevisiae; Hernandez LM et al.; The N-linked oligosaccharides, from Saccharomyces cerevisiae mnn1 mnn9 mutant mannoprotein extracted from the cells in hot citrate buffer, were separated by ion exchange into a monophosphate diester, a monophosphate monoester, a diphosphate diester, and a diphosphate monoester diester . The structures of the major components with diesterified phosphate were assigned as follows (where M = mannose), according to a recently revised oligosaccharide structure for the mnn mutants (Hernandez, L . M., Ballou, L., Alvarado, E., Gillece-Castro, B . L., Burlingame, A . L., and Ballou, C . E . (1989) J . Biol . Chem . 264, 11849-11856) . formula; see text The monoester derivatives were mixtures of the possible isomers produced by removal of one or the other phosphoglycosyl-linked mannose units, and they were shown to arise by chemical degradation during isolation . The mnn1 mnn2 mnn10 acidic oligosaccharide fraction contained a mono- and a diphosphate ester . The monophosphate consisted predominantly of a single isomer with a mannosyl phosphate unit located at the end of the outer chain in an oligosaccharide with the following structure, where x may range from 2 to 12 . The diphosphate had a mannosyl phosphate in this formula; see text position as well as one on the terminal alpha 1----6-linked mannose in the core . The presence in the mnn1 mnn9 or mnn1 mnn2 mnn10 background of the mnn4 or mnn6 mutations, which are known to regulate phosphorylation in yeast, reduced phosphorylation by 90% but did not eliminate it . AI-12522 Gene, 1989 Aug 15, 80(2), 279 - 91 Simultaneous synthesis and assembly of various hepatitis B surface proteins in Saccharomyces cerevisiae; Jacobs E et al.; Yeast transposon of class-1-based vectors, allowing integration at a series of chromosomal loci by homologous recombination with resident transposons, were constructed . Using such vectors, we have introduced several copies of an expression cassette encoding the major hepatitis B surface protein as well as expression cassettes encoding the middle (M) or/and the large (L) surface protein into Saccharomyces cerevisiae . In extracts of such strains, coassembly of the different proteins into a single lipoprotein structure is observed . This was demonstrated by immunoprecipitation of the major protein using monoclonal antibodies directed specifically against epitopes that are present only on the M or the L protein . These results show that hepatitis B surface antigen envelope proteins synthesized in yeast are able to assemble into structures composed of different polypeptides . This opens the possibility of producing in yeast a variety of particles carrying well-defined amounts of preS epitopes on their surface . Also, one can envisage the production of mixed particles containing different foreign epitopes on their surface, in defined relative abundance, which could be useful for vaccine applications. J Biol Chem, 1989 Aug 5, 264(22), 13336 - 42 Presynapsis and synapsis of DNA promoted by the STP alpha and single-stranded DNA-binding proteins from Saccharomyces cerevisiae; Hamatake RK et al.; We previously purified an activity from meiotic cell extracts of Saccharomyces cerevisiae that promotes the transfer of a strand from a duplex linear DNA molecule to complementary circular single-stranded DNA, naming it Strand Transfer Protein alpha (STP alpha) (Sugino, A., Nitiss, J., and Resnick, M . A . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 3683-3687) . This activity requires no nucleotide cofactor but is stimulated more than 10-fold by the addition of yeast single-stranded DNA-binding proteins (ySSBs) . In this paper, we describe the aggregation and strand transfer of double-stranded and single-stranded DNA promoted by STP alpha and ySSB . There is a good correlation between the aggregation induced by various DNA-binding proteins (ySSBs, DBPs and histone proteins) and the stimulation of STP alpha-mediated DNA strand transfer . This implies that the stimulation by ySSBs and other binding proteins is probably due to the condensation of single-stranded and double-stranded DNA substrates into coaggregates . Within these coaggregates there is a higher probability of pairing between homologous double-stranded and single-stranded DNA, favoring the initiation of strand transfer . The aggregation reaction is rapid and precedes any reactions related to DNA strand transfer . We propose that condensation into coaggregates is a presynaptic step in DNA strand transfer promoted by STP alpha and that pairing between homologous double- and single-stranded DNA (synapsis) occurs in these coaggregates . Synapsis promoted by STP alpha and ySSBs also occurs between covalently closed double-stranded DNA and single-stranded linear DNA as well as linear double-stranded and linear single-stranded DNAs in the absence of any nucleotide cofactors. J Ultrastruct Mol Struct Res, 1989 Aug, 102(2), 95 - 108 Organization of the nuclear pore complex in Saccharomyces cerevisiae; Allen JL et al.; Fractions enriched for nuclear pore complexes (NPCs) have been isolated from Saccharomyces cerevisiae . The sequential extraction of nuclei with detergent, nucleases, and salt reveals an organization of the yeast NPC similar to other eukaryotes . Yeast NPCs contain a 30-nm "ring" structure not previously described in other organisms . This structure appears to organize 10-nm filaments into an assembly which exhibits an eight-fold rotational symmetry . Some proteins in the NPC fraction are capable of forming intermediate-sized filaments . These studies suggest that some component of the nuclear pore complex organizes an interaction between nuclear and cytoplasmic networks of intermediate filaments. Curr Genet, 1989 Aug, 16(2), 75 - 80 Reassessing the genotoxic potential of 8-MOP + UVA-induced DNA damage in the yeast Saccharomyces cerevisiae; Henriques JA et al.; Two different UVA irradiation systems were initially biologically calibrated with two haploid yeast strains proficient and deficient, respectively, in nucleotide excision repair . The number of DNA lesions introduced into the cell's genome by the photoactivated bifunctional furocoumarin 8-MOP was then calculated by means of the applied UVA exposure doses . At LD37 the repair-proficient wild type had about 14 ICL and 34 furan-side monoadducts in its DNA, while doubly blocked repair mutant rad3-12 pso1-1 had 2 ICL and 3 monoadducts . Locus-specific reversion of lys1-1 followed two-hit kinetics in the repair-proficient wild type and one-hit kinetics in an excision-deficient rad2-20 mutant, as would be expected if ICL was the main type of mutagenic lesion in the wild type and monoadducts the main mutagenic lesion type in the excision-deficient strain . Quantitative comparison of 8-MOP + UVA-induced ICL with those induced by bifunctional mustard revealed the former to have a much higher genotoxicity. Genetika, 1989 Aug, 25(8), 1356 - 63 {Restriction analysis of deletions and deletion mapping of point mutations in the ADE2 gene of Saccharomyces cerevisiae yeasts}; Gracheva LM et al.; The method of restriction analysis has been used to study the length of 10 deletion mutations in ADE2 locus of Saccharomyces cerevisiae . We showed that 7 deletions overlapped the whole transcribed region of the gene ADE2, while 3 deletions have one of the ends situated in this region . Four controlled sites were fixed on the genetic map of ADE2 locus, based on these results . Deletion mapping of great number of point mutations demonstrated non-random distribution of mutations of different types on the map of ADE2 locus. Biokhimiia, 1989 Aug, 54(8), 1344 - 7 {The level of cytochrome P-450 in Saccharomyces cerevisiae with disruptions of various stages of sterol synthesis}; Ekhvalova TV et al.; A spectral analysis of cytochromes P-450 in Saccharomyces cerevisiae cells and in mutant strains accumulating the ergosterol biosynthesis intermediates was carried out . Glucose repression and semianaerobiosis were found to induce cytochrome P-450 synthesis . No differences in the cytochrome P-450 content in mutant nys 3, nys 4 and parent strains were observed . Mutants nys 5 accumulated large amounts of cytochrome P-450 . Cytochrome P-420 was detected in wild type strains and in mutants nys 3 and nys 4 . The cultivation time and aeration conditions were shown to be unimportant for the generation of cytochrome P-420. Biokhimiia, 1989 Aug, 54(8), 1265 - 73 {Isolation and properties of phosphoribosyl-aminoimidazole-succinocarboxyamide-synthestase from Saccharomyces cerevisiae yeasts}; Ostanin KV et al.; An isolation procedure for phosphoribosyl succinocarboxamideaminoimidazole synthetase (SAICAR synthetase) (EC 6.3.2.6) has been developed . Pure SAICAR synthetase was found to be a monomeric protein with the apparent molecular weight of 36 kDa . The Michaelis constant for the three substrates of the reaction are 1.6 microM for CAIR, 14 microM for ATP and 960 microM for aspartic acid . The structural analogs of CAIR, 5-aminoimidazole ribotide and 5-aminoimidazole-4-carboxamide ribotide, act as competitive inhibitors of SAICAR synthetase . GTP and 2'-dATP can substitute for ATP in the reaction, while CTP and UTP inhibit the enzyme . No structural analogs of the aspartic acid were found to have affinity for SAICAR synthetase . The optimal reaction conditions for the enzyme were established to be at pH 8.0 and magnesium chloride concentration around 5 mM. J Biochem (Tokyo), 1989 Aug, 106(2), 223 - 7 The gene and the primary structure of acidic ribosomal protein A0 from yeast Saccharomyces cerevisiae which shows partial homology to bacterial ribosomal protein L10; Mitsui K et al.; Eukaryotic ribosomes contain an acidic ribosomal protein of about 38 kDa which shows immunological cross-reactivity with the 13 kDa-type acidic ribosomal proteins that are related to L7/L12 of bacterial ribosomes . By using a cDNA clone for 38 kDa-type acidic ribosomal protein A0 from the yeast Saccharomyces cerevisiae, we have cloned a genomic DNA encoding A0 and determined the sequence of 1,614 nucleotides including about 500 nucleotides in the 5'-flanking region . The gene lacks introns and possesses two boxes homologous to upstream activation sequences (UASrpg) in the 5'-flanking region . The amino acid sequence of A0 deduced from the nucleotide sequence shows that A0 shares a highly similar carboxyl-terminal region of about 40 amino acids in length with 13 kDa-type acidic ribosomal proteins, including an identical carboxyl-terminal, DDDMGFGLFD . In the amino-terminal region A0 contains an arginine-rich segment which shows a low but distinct similarity to that of bacterial ribosomal protein L10 through which L10 is thought to bind to 23S rRNA . On the other hand, the carboxyl-terminal half of A0 is enriched with hydrophobic amino acid residues including four pairs of phenylalanine residues which are all conserved in a human homologue. Mol Cell Biol, 1989 Aug, 9(8), 3464 - 72 Transcription by RNA polymerase I stimulates mitotic recombination in Saccharomyces cerevisiae; Stewart SE et al.; The recombination-stimulating sequence HOT1 is derived from the ribosomal DNA array of Saccharomyces cerevisiae and corresponds to sequences that promote transcription by RNA polymerase I . When inserted at a chromosomal location outside the ribosomal DNA array, HOT1 stimulates mitotic recombination in the adjacent sequences . To investigate the relationship between transcription and recombination, transcription promoted by HOT1 was directly examined . These studies demonstrated that transcription starts at the RNA polymerase I initiation site in HOT1 and proceeds through the chromosomal sequences in which recombination is enhanced . Linker insertion mutations in HOT1 were generated and assayed for recombination stimulation and for promoter function; this analysis demonstrated that the same sequences are required for both activities . These results indicate that the ability of HOT1 to enhance recombination is related to, and most likely dependent on, its ability to promote transcription. Mol Cell Biol, 1989 Aug, 9(8), 3155 - 65 AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating; Lipke PN et al.; We have cloned the alpha-agglutinin structural gene, AG alpha 1, by the isolation of alpha-specific agglutination-defective mutants, followed by isolation of a complementing plasmid . Independently isolated alpha-specific agglutination-defective mutations were in a single complementation group, consistent with biochemical results indicating that the alpha-agglutinin is composed of a single polypeptide . Mapping results suggested that the complementation group identified by these mutants is allelic to the ag alpha 1 mutation identified previously . Expression of AG alpha 1 RNA was alpha specific and inducible by a-factor . Sequences similar to the consensus sequences for positive control by MAT alpha 1 and pheromone induction were found upstream of the AG alpha 1 initiation codon . The AG alpha 1 gene could encode a 650-amino-acid protein with a putative signal sequence, 12 possible N-glycosylation sites, and a high proportion of serine and threonine residues, all of which are features expected for the alpha-agglutinin sequence . Disruption of the AG alpha 1 gene resulted in failure to express alpha-agglutinin and loss of cellular agglutinability in alpha cells . An Escherichia coli fusion protein containing 229 amino acids of the AG alpha 1 sequence was recognized by an anti-alpha-agglutinin antibody . In addition, the ability of this antibody to inhibit agglutination was prevented by this fusion protein . These results indicate that AG alpha 1 encodes alpha-agglutinin . Features of the AG alpha 1 gene product suggest that the amino-terminal half of the protein contains the a-agglutinin binding domain and that the carboxy-terminal half contains a cell surface localization domain, possibly including a glycosyl phosphatidylinositol anchor. Genes Dev, 1989 Aug, 3(8), 1206 - 16 Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae; Vijayraghavan U et al.; In this study we report the isolation of temperature-sensitive mutants that affect pre-mRNA splicing . A bank of approximately 1000 temperature-sensitive Saccharomyces cerevisiae strains was generated and screened on RNA gel blots by hybridization with an actin intron probe . We isolated 16 mutants defining 11 new complementation groups prp(rna)17-prp(rna)27 with four phenotypic classes of mutants and 21 mutants in the prp2-prp11 complementation groups (formerly rna2-rna11) . The majority of the complementation groups share a phenotype of pre-mRNA accumulation, seen in all of the prp(rna)2-prp(rna)11 mutants . Three novel classes of mutants were isolated in this study . One class, consisting of two complementation groups, exhibits an accumulation of the lariat intermediate of splicing, with no change in the levels of pre-mRNA . The second class, also represented by two complementation groups, shows an accumulation of the intron released after splicing . The third novel class, comprising one complementation group, accumulates both pre-mRNA and the released intron . All mutants isolated were recessive for the splicing phenotype . Only 2 of the 11 complementation groups, although recessive, were not temperature sensitive . This study, together with previous isolation of the prp(rna)2-prp(rna)11 groups and the spliceosomal snRNAs, puts at least 26 gene products involved directly or indirectly in pre-mRNA splicing. Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6240 - 4 Cloning human telomeric DNA fragments into Saccharomyces cerevisiae using a yeast-artificial-chromosome vector; Riethman HC et al.; Telomeric fragments of human DNA ranging in size from 50 to 250 kilobases were cloned into Saccharomyces cerevisiae using a yeast-artificial-chromosome (YAC) vector . Six human-telomeric YAC (HTY) strains were selected by virtue of the specific hybridization of their DNA with the human telomeric terminal-repeat sequence (TTAGGG)n, and the telomeric localization of this sequence within each YAC was demonstrated by its sensitivity to nuclease BAL-31 . In situ hybridization of DNA from three of these HTY strains with human metaphase chromosomes yielded discrete patterns of hybridization signals at the telomeres of a limited number of human chromosomes, different for each clone . DNA from selected cosmid subclones of one of the HTY strains was used to localize the origin of the cloned telomeric DNA by in situ hybridization to the tip of the long arm of chromosome 7. Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6225 - 9 A DNA double chain break stimulates triparental recombination in Saccharomyces cerevisiae; Ray A et al.; Mitotic recombination between his3 heteroalleles on heterologous chromosomes is stimulated by a DNA double chain break delivered in vivo at a site 8.6 kilobase pairs distant from one his3 allele and unlinked to the other . The induced recombination at his3 is accompanied by gap repair at the break site using the uncut homolog as a template . The DNA between the break site and his3 is not deleted in most of the His+ recombinants. Proc Natl Acad Sci U S A, 1989 Aug, 86(16), 6043 - 6 Translation in Saccharomyces cerevisiae: initiation factor 4A-dependent cell-free system; Blum S et al.; Yeast Saccharomyces cerevisiae genes TIF1 and TIF2 (translation initiation factor) encode a protein tentatively called translation initiation factor (Tif) due to the similarity of its amino acid sequence and its molecular weight to mammalian eukaryotic initiation factor 4A . To clarify whether Tif is involved in translation, we produced an affinity-purified anti-Tif antibody by using Tif isolated from a Tif-overproducing yeast strain as immunogen and an Escherichia coli strain expressing Tif from an expression vector to provide the extract for affinity purification of the antibody . By using chromatographic procedures and the affinity-purified anti-Tif antibody as probe to identify Tif-containing fractions, we purified Tif from wild-type yeast cells . When yeast cells containing the only TIF1 gene on a plasmid under the control of the galactose-inducible CYC1-GAL10 promoter were grown in medium containing glucose as the carbon source, the production of Tif was shut off and growth was arrested . Lysates made from these cells were inactive in in vitro translation . Addition of Tif to these lysates restored in vitro protein synthesis . These results show that Tif is a translation factor, the yeast homologue of mammalian translation initiation factor 4A. Mutat Res, 1989 Aug, 213(2), 105 - 115 Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion; Ivanov EL et al.; We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele . Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail . The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation . It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used . The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes) . The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele . Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected . Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination . Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes. Genetics, 1989 Aug, 122(4), 749 - 57 Extrachromosomal elements cause a reduced division potential in nib 1 strains of Saccharomyces cerevisiae; Sweeney R et al.; The nib 1 allele of yeast confers a sensitivity to an endogenous plasmid, 2 mu DNA, in that nib 1 strains bearing 2 mu DNA (cir+) exhibit a reduction in division potential . In the present study, the reduction in division potential characteristic of nib 1 cir+ strains is shown to be dependent on the simultaneous presence of both the A and the D open reading frames of 2 mu DNA as well as on the presence of an unidentified extrachromosomal element other than 2 mu DNA . Furthermore, in nib 1 strains, an uncharacterized extrachromosomal element can cause a less severe reduction of division potential in the absence of intact 2 mu DNA . Thus, the nib 1 allele may confer a generalized sensitivity to extrachromosomal elements. Curr Genet, 1989 Aug, 16(2), 65 - 74 Molecular characterization of the two genes SNQ and SFA that confer hyperresistance to 4-nitroquinoline-N-oxide and formaldehyde in Saccharomyces cerevisiae; Gompel-Klein P et al.; The genes SNQ and SFA confer hyperresistance to 4-NQO and FA when present on a multi-copy plasmid in yeast . Both are non-essential genes since transplacement of SNQ by a disrupted snq-0::LEU2 yielded stable and viable haploid integrants . Southern analysis revealed that SNQ and SFA are single-loci genes, and OFAGE analysis showed that they are located on chromosome XIII and IV, respectively . Northern blot analysis of SNQ and SFA revealed poly(A)+ RNA transcripts of 2 kb and 1.7 kb, respectively . Nuclease S1 mapping showed SNQ to have a coding region of 1.6 kb and SFA, one of 1.3 kb . The 5' coding regions were determined for both genes, while the 3' end could only be determined for gene SNQ . Both genes do not appear to contain introns . The SFA locus was also mapped by transposon mutagenesis . Tn10-LUK integrants disrupted the SFA gene function at sites that were determined by subcloning to lie within the SFA transcription unit. Mol Cell Biol, 1989 Aug, 9(8), 3517 - 23 Reconstitution of the vitamin D-responsive osteocalcin transcription unit in Saccharomyces cerevisiae; McDonnell DP et al.; The human osteocalcin gene is regulated in mammalian osteoblasts by 1,25(OH)2D3-dependent and -independent mechanisms . The sequences responsible for this activity have been mapped to within the -1339 region of the gene . We show here that this enhancer region functions analogously in Saccharomyces cerevisiae cells engineered to produce active 1,25(OH)2D3 receptor . When fused to the proximal promoter elements of the yeast iso-1-cytochrome c gene, the enhancer demonstrated substantial promoter activity . This activity was elevated further by 1,25(OH)2D3 when the reporter constructs were assayed in cells containing the 1,25(OH)2D3 receptor . This system affords a model for 1,25(OH)2D3 action and represents a simple assay system that will enable definition of the important cis-acting regulatory sequences within the osteocalcin gene and identification of their cognate transcription factors. Mol Cell Biol, 1989 Aug, 9(8), 3342 - 9 A 125-base-pair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae; Cottarel G et al.; Saccharomyces cerevisiae centromeres contain a conserved region ranging from 111 to 119 base pairs (bp) in length, which is characterized by the three conserved DNA elements CDEI, CDEII, and CDEIII . We isolated a 125-bp CEN6 DNA fragment (named ML CEN6) containing only these conserved elements and assayed it completely separated from its chromosomal context on circular minichromosomes and on a large linear chromosome fragment . The results show that this 125-bp CEN6 DNA fragment is by itself sufficient for complete mitotic and meiotic centromere functions. Genetics, 1989 Aug, 122(4), 759 - 72 Mitotic and meiotic gene conversion of Ty elements and other insertions in Saccharomyces cerevisiae; Vincent A et al.; We examined meiotic and mitotic gene conversion events involved in deletion of Ty elements and other insertions from the genome of the yeast Saccharomyces cerevisiae . We found that Ty elements and one other insertion were deleted by mitotic gene conversion less frequently than point mutations at the same loci . One non-Ty insertion similar in size to Ty, however, did not show this bias . Mitotic conversion events deleting insertions were more frequently associated with crossing over than those deleting point mutations . In meiosis, conversion events duplicating the element were more common than those that deleted the element for one of the loci (HIS4) examined. Mol Gen Genet, 1989 Aug, 218(2), 240 - 8 Alpha-factor leader sequence-directed transport of Escherichia coli beta-galactosidase in the secretory pathway of Saccharomyces cerevisiae; Das RC et al.; The construction of two fused genes is described . One involves the in-frame fusion of the yeast prepro-alpha-factor coding sequence, and the Escherichia coli lac Z gene . The second gene fusion utilizes a 103 bp yeast invertase NH2-terminal coding sequence at the fusion junction of the hybrid gene described above . The gene fusions, under the control of the alpha-factor promoter, expressed active beta-galactosidase in alpha haploid yeast cells . The activity could be regulated in a temperature-sensitive sir3 mutant . The incorporation of the invertase coding sequence at the MF alpha 1-lacZ fusion junction provided significantly higher levels of beta-galactosidase activity . A substantial quantity of the hybrid proteins generated from the gene fusions was primarily localized in the intracellular membranes of yeast cells, while a processed form could be secreted into the periplasm. Mol Cell Biol, 1989 Aug, 9(8), 3260 - 8 The most abundant small cytoplasmic RNA of Saccharomyces cerevisiae has an important function required for normal cell growth; Felici F et al.; The most abundant RNA visible between 5.8S and 18S rRNA on an ethidium bromide-stained gel of total Saccharomyces cerevisiae RNA has an apparent size of about 600 nucleotides . By purifying the band and using it as a probe to screen a genomic library, we isolated and sequenced the unique gene for this RNA . The transcribed sequence, determined to be 519 nucleotides long, contains elements typical of RNA polymerase III transcription . The RNA is predominantly cytoplasmic, so we called it small cytoplasmic RNA 1 (scR1) . ScR1 is neither 3'-polyadenylated nor 5'-trimethylguanosine capped . We constructed a null mutation of the gene by deleting 252 base pairs from the transcribed region . Haploid strains carrying the scr1-delta lesion grew very slowly, segregated cytoplasmic petites {( rho-}) at high frequency, and showed signs of aberrant cell division . A secondary structure model for scR1 shows some of the conserved features of the signal recognition particle 7SL RNAs. Nucleic Acids Res, 1989 Jul 25, 17(14), 5781 - 92 Transcriptional analysis of the CDC7 protein kinase gene of Saccharomyces cerevisiae; Ham J et al.; The 5' flanking region of the CDC7 gene of Saccharomyces cerevisiae has been sequenced to a point 797 nucleotides upstream of the putative translational initiation codon (designated +1) . The sequence reveals a number of symmetry elements between -100 to -380 and two blocks of high local AT content between -29 to -75 and -112 to -144 . Transcription initiates heterogeneously at about 10 discrete sites up to 110 nucleotides upstream of the putative translational initiation codon . Some minor transcriptional start sites were also observed between the ATG at +1 and a second in-frame ATG at +55, suggesting that CDC7 may also be translationally heterogeneous . Deletion analysis of the CDC7 upstream region has shown that the gene lacks a functional TATA box, and has identified a 30bp element that is necessary for normal CDC7 promoter function during mitosis . This motif has significant homology with a component of the c-fos promoter which acts to regulate c-fos expression by binding a transcription activating factor . The results suggest that the 30bp motif may play a similar role in regulating CDC7 expression and that there may be similarities between factors that regulate CDC7 expression in yeast and c-fos expression in vertebrate cells. J Biol Chem, 1989 Jul 25, 264(21), 12339 - 43 Molecular cloning and sequencing of a cDNA encoding N alpha-acetyltransferase from Saccharomyces cerevisiae; Lee FJ et al.; Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eukaryotic proteins and is catalyzed by an N alpha-acetyltransferase . Recently, a eukaryotic N alpha-acetyltransferase was purified to homogeneity from Saccharomyces cerevisiae, and its substrate specificity was partially characterized (Lee, F.-J . S., Lin L.-W., and Smith, J . A . (1988) J . Biol . Chem . 263, 14948-14955) . This article describes the cloning from a yeast lambda gt11 cDNA library and sequencing of a full length cDNA encoding yeast N alpha-acetyltransferase . DNA blot hybridizations of genomic and chromosomal DNA reveal that the gene (so-called AAA1, amino-terminal, alpha-amino, acetyltransferase) is present as a single copy located on chromosome IV . The use of this cDNA will allow the molecular details of the role of N alpha-acetylation in the sorting and degradation of eukaryotic proteins to be determined. J Biol Chem, 1989 Jul 25, 264(21), 12172 - 8 Intracellular Mn (II)-associated superoxide scavenging activity protects Cu,Zn superoxide dismutase-deficient Saccharomyces cerevisiae against dioxygen stress; Chang EC et al.; Three Cu,Zn superoxide dismutase (SOD-1)-deficient Saccharomyces cerevisiae mutants do not grow in 100% O2 in rich medium and require Met and Lys when grown in air (Bilinski, T., Krawiec, Z., Liczmanski, A., and Litwinska, J . (1985) Biochem . Biophys . Res . Commun . 130, 533-539) . We show herein that medium manganese (II) accumulated by the mutants rescues these O2-sensitive phenotypes; 2 mM medium Mn2+ represented the threshold required for cell growth . The accumulation of Mn2+ was not oxygen-inducible since mutants grown aerobically and anaerobically accumulated the same amount of Mn2+ . Mn2+ accumulation is not unique to these mutants since wild type accumulated almost twice as much Mn2+ as did mutant . ESR spectra of the cell extracts and whole cells loaded with Mn2+ were typical of free Mn(II) ion . These spectra could not account quantitatively for the total cellular Mn2+, however . A screen for soluble antioxidant activities in the Mn2+-supplemented cells detected O2- (superoxide) scavenging activity, with no change in catalase or peroxidase activities . This O2- scavenging activity was CN- and heat-resistant . No achromatic bands were revealed in nondenaturing gels of Mn2+- containing cell extracts stained for O2- scavenging activity . The Mn2+-dependent O2- scavenging activity in the cell extracts was quenched by EDTA and dialyzable . More than 60% of both the intracellular Mn2+ and the O2- scavenging activity was removed by 2-h dialysis . Dialyzed cells were not viable in air unless resupplemented with either Met or Mn2+ . Although Mn2+ supported the aerobic growth of these mutants, excess Mn2+, which correlated with an elevated O2- scavenging activity, was toxic to both mutant and wild type . The results indicate that free or loosely bound Mn2+ ion protects the mutants against oxygen stress by providing an intracellular, presumably cytosolic, O2- scavenging activity which replaces the absent SOD-1. J Mol Biol, 1989 Jul 20, 208(2), 257 - 67 Function of DNA topoisomerases as replication swivels in Saccharomyces cerevisiae; Kim RA et al.; We have examined the roles of eukaryotic DNA topoisomerases I and II in DNA replication by the use of a set of four isogenic strains of Saccharomyces cerevisiae that are TOP1+ TOP2+, TOP1+ top2 ts, delta top1 TOP2+, and delta top1 top2 ts . Cells synchronized by treatment with the alpha-mating factor, or by cycles of feeding and starvation, were released from cell-cycle arrest, and the size distribution of DNA chains that were synthesized after the cells reentered the S-phase was determined as a function of time . The results indicate that synthesis of short DNA chains several thousand nucleotides in length can initiate in the absence of both topoisomerases, but their further elongation requires at least one of the two topoisomerases . Inactivation of DNA topoisomerase II does not alter significantly the time dependence of the patterns of nascent DNA chain synthesis, which is consistent with the notion that the requirement of this enzyme for viability is due to its essential role during mitosis, when pairs of intertwined newly replicated chromosomes are being segregated . The absence of DNA topoisomerase I leads to a temporary delay in the extension of the short DNA chains; this delay in chain elongation is also reflected in the rate of total DNA synthesis in the delta top1 mutant during the early S-phase . Thus, in wild-type cells, DNA topoisomerase I is probably the major replication swivel . The patterns of DNA synthesis in asynchronously grown delta top1 top2 ts cells at permissive and non-permissive temperatures are also consistent with the above conclusions. Gene, 1989 Jul 15, 79(2), 199 - 206 High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression; Lopes TS et al.; Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with a high mitotic stability under non-selective conditions . Since high stability can best be assured by integration of the vector into chromosomal DNA we have set out to design a vector that is able to integrate into the yeast genome in a large number of copies . The rDNA locus appeared to be an attractive target for such multiple integration since it encompasses 100-200 tandemly repeated units . Plasmids containing several kb of rDNA for targeted homologous recombination, as well as the deficient LEU2-d selection marker were constructed and, after transformation into yeast, tested for both copy number and stability . One of these plasmids, designated pMIRY2 (for multiple integration into ribosomal DNA in yeast), was found to be present in 100-200 copies per cell by restriction analysis . The pMIRY2 transformants retained 80-100% of the plasmid copies over a period of 70 generations of growth in batch culture under non-selective conditions . To explore the potential of pMIRY2 as an expression vector we have inserted the homologous genes for phosphoglycerate kinase (PGK) and Mn2+-dependent superoxide dismutase (SOD) as well as the heterologous genes for thaumatin from Thaumatococcus danielli (under the GAPDH promoter), into this plasmid and analyzed the yield of the various proteins . Under optimized conditions the level of PGK in cells transformed with pMIRY2-PGK was about 50% of total soluble protein . The yield of thaumatin in the pMIRY2-thaumatin transformants exceeded by about a factor of 100 the level of thaumatin observed in transformants carrying only a single thaumatin gene integrated at the TRP1 locus in chromosome IV. Eur J Biochem, 1989 Jul 15, 183(1), 155 - 60 Molecular cloning of the nuclear gene for mitochondrial ribosomal protein YmL31 from Saccharomyces cerevisiae; Grohmann L et al.; The nuclear gene for mitochondrial ribosomal protein YmL31 (MRP-L31) of Saccharomyces cerevisiae was cloned using synthetic oligonucleotide mixtures which correspond to the N-terminal amino acid sequence of the mature YmL31 . The gene MRP-L31 codes for a basic protein with a calculated molecular mass of 15.5 kDa and resides on chromosome XI . A comparison of the amino acid sequence deduced from the nucleotide sequence of the MRP-L31 gene and the N-terminal sequence of the isolated protein revealed the existence of a leader peptide sequence of 12 amino acid residues . No significant similarity to known ribosomal protein sequences of other organisms was found. J Biol Chem, 1989 Jul 15, 264(20), 12106 - 12 Purification and characterization of human immunodeficiency virus (HIV) core precursor (p55) expressed in Saccharomyces cerevisiae; Vlasuk GP et al.; The core structure of retroviruses, including the human immunodeficiency virus (HIV), consists of proteins that are initially synthesized as polyprotein precursors and then processed by a virally encoded protease yielding the mature core polypeptides . To obtain sufficient quantities of the purified HIV core precursor p55 for detailed studies, a segment of HIV DNA encoding the full length core precursor polyprotein p55 was expressed in Saccharomyces cerevisiae using a plasmid containing a constitutive galactose promoter . The expression of this DNA produced a protein with an estimated molecular size of 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); this protein was immunoreactive to anti-HIV p24 antisera . Following cell lysis, freezing, and thawing, the expressed protein was an insoluble aggregate that served as the starting material for the purification process . Solubilization of the insoluble p55 with guanidine HCl followed by phenyl-Sepharose column chromatography and high performance liquid chromatography resulted in a preparation of p55 that was greater than 95% pure by SDS-PAGE, immunoreactive to anti-HIV core protein antibodies, and completely soluble in aqueous solution . The expressed p55 appeared to be myristoylated as evidenced by the incorporation of radiolabel following incubation of recombinant yeast cells with {3H}myristic acid; in addition the amino terminus of the final purified protein was blocked . Proteolytic digestion of purified p55 with synthetic HIV protease yielded the predicted amino- and carboxyl-terminal products; these were confirmed by amino acid sequence analysis . In contrast, digestion of purified p55 by the protease derived from the avian myeloblastosis virus resulted in fragments that were different in size from those produced by the HIV protease . The availability of the purified, full length water-soluble HIV core precursor will be useful in identifying agents that inhibit its processing by the HIV protease. J Biol Chem, 1989 Jul 15, 264(20), 12091 - 6 Dispensable presequence for cellular localization and function of mitochondrial malate dehydrogenase from Saccharomyces cerevisiae; Thompson LM et al.; The nucleotide sequence corresponding to codons for the 17-amino acid residues in the presumed targeting presequence for yeast mitochondrial malate dehydrogenase was removed by oligonucleotide-directed mutagenesis of the isolated gene (MDH1) . Integrative transformation was used to insert the "leaderless" gene (mdhl-) into the MDH1 chromosomal locus of a strain containing a disrupted MDH1 gene . Expression of the mature form of malate dehydrogenase as a primary translation product was verified by demonstrating that the mature form is synthesized in mdhl- cells at the same rate as the precursor form in MDH1 cells in the presence of carbonyl cyanide m-chlorophenylhydrazone and by comparison of in vitro translation products of RNAs from mdhl- and MDH1 cells . Expression of mdhl- restores total cellular malate dehydrogenase activity to levels comparable to those in wild type cells and reverses the phenotype associated with strains containing MDH1 disruptions by restoring wild type rates of growth in media containing acetate as a carbon source . Immunochemical analyses and enzyme assays show comparable levels of malate dehydrogenase in the matrix fractions from mitochondria isolated from mdhl- and MDH1 cells and give no evidence for accumulation of the mature enzyme in the cytosol of mdhl- cells . These results indicate that the presequence for malate dehydrogenase is not essential for efficient mitochondrial localization or function in yeast. J Biol Chem, 1989 Jul 15, 264(20), 11865 - 73 RAS2 protein of Saccharomyces cerevisiae is methyl-esterified at its carboxyl terminus; Deschenes RJ et al.; Yeast and mammalian RAS gene products are GTP-binding proteins that are posttranslationally localized to the inner surface of the plasma membrane . This localization is accomplished by the addition of a lipid moiety to a conserved cysteine residue close to the carboxyl terminus . In a previous report we showed that the mammalia Ha-ras protein is also modified posttranslationally by methyl esterification . Here we show that the yeast RAS2 protein is posttranslationally modified by methyl esterification at or near the carboxyl terminus . We also present evidence indicating that the methyl ester is linked to the conserved cysteine residue, implying that RAS2 protein is cleaved to expose this cysteine as the carboxyl-terminal residue . This maturation pathway may be shared by a family of proteins that are initially synthesized as soluble proteins and must become membrane-localized to function. J Biol Chem, 1989 Jul 15, 264(20), 11857 - 64 Protein glycosylation defects in the Saccharomyces cerevisiae mnn7 mutant class . Support for the stop signal proposed for regulation of outer chain elongation; Ballou L et al.; Total cell mannoprotein was isolated from Saccharomyces cerevisiae X2180 mutants that have defects in elongation of the outer chain attached to the N-linked core oligosaccharides (mnn7, mnn8, mnn9, and mnn10) (Ballou, L., Cohen, R . E., and Ballou, C . E . (1980) J . Biol . Chem . 255, 5986-5991) . Comparison of the oligosaccharides released by endoglucosaminidase H digestion confirmed that the mnn9 mutation eliminates all but two mannoses of the outer chain, whereas the mnn8 and mnn10 strains produce outer chains of variable but similar lengths . The isolate designated mnn7 was found to be allelic with mnn8 . Haploid mutants of the type mnn8 mnn9 or mnn9 mnn10 had the mnn9 phenotype, which established that the mnn9 defect is dominant and presumably acts at a processing step prior to the steps affected by mnn8 and mnn10 . Analysis of the mnn1 mnn2 mnn10 oligosaccharides revealed that the heterogeneous outer chain contained 6-16 alpha 1----6-linked mannose units and each was terminated by a single alpha 1----2-linked mannose unit, whereas the core lacked one such unit that was present in the mnn9 oligosaccharide . The results are consistent with and support the hypothesis (Gopal, P . K., and Ballou, C . E . (1988) Proc . Natl . Acad . Sci . U.S.A . 84, 8824-8828) that addition of such a side-chain mannose unit is associated with termination of outer chain elongation in these mutants and may serve as a stop signal that regulates outer chain synthesis in the parent wild-type strain. J Biol Chem, 1989 Jul 15, 264(20), 11849 - 56 A new Saccharomyces cerevisiae mnn mutant N-linked oligosaccharide structure; Hernandez LM et al.; We find that the N-linked Man8GlcNAc2- core oligosaccharide of Saccharomyces cerevisiae mnn mutant mannoproteins is enlarged by the addition of the outer chain to the alpha 1----3-linked mannose in the side chain that is attached to the beta 1----4-linked mannose rather than by addition to the terminal alpha 1----6-linked mannose . This conclusion is derived from structural studies on a phosphorylated oligosaccharide fraction and from mass spectral fragment analysis of neutral core oligosaccharides. Gene, 1989 Jul 15, 79(2), 227 - 37 Biochemical and immunological characterization of the bovine leukemia virus (BLV) envelope glycoprotein (gp51) produced in Saccharomyces cerevisiae; Legrain M et al.; The nucleotide sequence coding for bovine leukemia virus (BLV) envelope glycoprotein gp51 was inserted into a yeast-Escherichia coli shuttle vector carrying the promoter and secretion signal sequence of PHO5 (the yeast gene coding for repressible acid phosphatase) and the CYC1 transcriptional terminator . Yeast cells transformed by this construction synthesized gp51 after PHO5 induction by inorganic phosphate deprivation . The yeast-expressed gp51 was partially glycosylated into heterodisperse protein molecules ranging from 40 to 48 kDa . No gp51 was excreted in the culture medium . The amount of protein accumulated in yeast cells was estimated to reach 0.06% of soluble proteins . This modest level of expression seemed to be due to the toxicity of gp51 to the yeast cell . The yeast-expressed gp51 products were used in enzyme-linked immunosorbent assays for the detection of antibodies in sera from BLV-infected animals; they were also screened for the presence of well-defined biological epitopes . In both studies poor reactivity was observed . Rabbits immunized with the recombinant gp51 showed high antibody titers to native BLV gp51 . However, these antibodies did not neutralize BLV in vitro. Gene, 1989 Jul 15, 79(2), 219 - 26 High yield synthesis of the bovine leukemia virus (BLV) p24 major internal protein in Saccharomyces cerevisiae; Dumont J et al.; Bovine leukemia virus (BLV) p24 gene was expressed in Saccharomyces cerevisiae under the control of the PHO5 (encoding repressible acid phosphatase, rAPase) promoter . Yeast cells were transformed by a yeast-E . coli shuttle vector carrying the PHO5 promoter, the p24 gene and the CYC1 transcription terminator . After low inorganic phosphate (Pi) induction of the PHO5 promoter, p24 accumulated in the producing cells up to a concentration representing 10% of total soluble proteins . The expression level of p24 gene was not increased by insertion of the positive regulatory gene PHO4 on the p24 expression vector . The p24 produced in this system and incubated in crude yeast extract showed a remarkably high resistance to proteolytic degradation, a feature that presumably correlates with the compact globular conformation of the protein combined to the stabilizing effect of the N-terminal residue. Biochem Biophys Res Commun, 1989 Jul 14, 162(1), 253 - 7 Total synthesis of the lipopeptide a-mating factor of Saccharomyces cerevisiae; Xue CB et al.; The a-mating factor of Saccharomyces cerevisiae was synthesized using both solution phase and solid phase strategies . Structure of the final peptide was confirmed using amino acid analysis, fast atom bombardment mass spectroscopy and 400 MHz proton NMR . The synthetic farnesylated dodecapeptide, YIIKGVFWDPAC (S-farnesyl) OCH3, exhibited chromatographic and spectroscopic properties identical to the natural pheromone and had significant biological activity at nanomolar concentrations. Biochem Biophys Res Commun, 1989 Jul 14, 162(1), 357 - 63 Different conformation of purified human recombinant interleukin 1 beta from Escherichia coli and Saccharomyces cerevisiae is related to different level of biological activity; Casagli MC et al.; Human recombinant interleukin 1 beta produced in Escherichia coli and in Saccharomyces cerevisiae was purified to homogeneity by a combination of ion exchange, gel filtration and hydroxylapatite column chromatography . The two proteins, both expressed in the mature form, differ in that the protein secreted from yeast is glycosylated and lacks the first four amino acids . The biological activity of IL-1 obtained from E . coli is comparable to that of the natural protein, while the protein produced from yeast showed very low specific activity . The analysis of the state of oxidation of the two cysteine residues present in the IL-1 molecule and the evaluation of the immunoreactivity of the two proteins have proved that a different conformation is at the basis of the different biological activity of the two proteins. Biochim Biophys Acta, 1989 Jul 13, 975(2), 222 - 30 Mitochondrial H+-ATPase in mutants of Saccharomyces cerevisiae with defective subunit 8 of the enzyme complex; Marzuki S et al.; Mutants of Saccharomyces cerevisiae carrying defined lesions in the mitochondrial aap1 gene, coding for membrane subunit 8 of the H+-ATPase, have been investigated to examine the consequence of the mutations on the function and assembly of the enzyme complex . These include three mit- mutants, which cannot grow by oxidative metabolism due to their inability to synthesize full-length subunit 8, and three partial revertants of one of the mutants . The mutations in these strains have been previously characterized by DNA sequencing . The use of a monoclonal antibody to the beta subunit of the H+-ATPase as a probe of assembly defect revealed that the presence of subunit 8 is essential for the assembly of subunit 6 to the enzyme complex . Mitochondria isolated from the mit- mutants have negligible {32Pi}ATP exchange activity and they exhibited ATPase activity which is not sensitive to inhibition by oligomycin, indicating a defective membrane F0 sector . Normal assembly of subunit 8 (and subunit 6) was observed in the revertant strains, despite 8-9 amino-acid substitutions in the membrane-spanning region of the H+-ATPase subunit 8 in two of the strains . The assembled complex, however, exhibited reduced {32Pi}ATP exchange activity and low sensitivity to oligomycin, indicating that the product of the aap1 gene is a functional subunit of the mitochondrial H+-ATPase. J Biol Chem, 1989 Jul 5, 264(19), 11444 - 9 Isolation and characterization of a glycosylated form of human insulin-like growth factor I produced in Saccharomyces cerevisiae; Gellerfors P et al.; Expression and secretion of human insulin-like growth factor-I (IGF-I) in Saccharomyces cerevisiae was achieved by linking an actin (ACT) promoter to an MF alpha 1 prepro leader peptide/IGF-I gene fusion . Purified human IGF-I from yeast culture media was found to contain, in addition to the native form, also a glycosylated variant . Structural studies showed that both IGF-I forms were processed identically, resulting in 70-amino-acid long polypeptides, with intact N-terminal and C-terminal residues of glycine and alanine, respectively . The glycosylation site was determined to threonine-29 (Thr29), by 1H NMR spectroscopy and protein sequence analysis of an isolated tryptic peptide(22-36) . No other glycosylation sites were found . Only mannose was detected in the sugar analysis, with an estimated content of 4.5% w/w corresponding to 2 mannose residues per molecule of IGF-I . The carbohydrate structure, determined by 1H and 13C NMR spectroscopy, was found to be alpha-D-Manp(1----2)alpha-D-Manp(1----3)Thr corresponding to an O-linked glycoprotein structure . No other post-translational modifications could be identified in the glycosylated IGF-I form . Furthermore, this form was highly active, comparable to native IGF-I, exhibiting a specific activity of 20,500 units/mg, as determined by a radio-receptor assay. Genetika, 1989 Jul, 25(7), 1179 - 87 {Regularities in formation of gamma-induced mutants of Saccharomyces cerevisiae}; Chepurnoi AI et al.; As shown by an example of the formation of reversions in the leu2 gene of Saccharomyces cerevisiae, the process of mutant formation after gamma-irradiation is associated with the post-irradiation replication phases . When no DNA replication takes place, the mutants are not formed . The periods of realizing premutation lesions into mutations depend on what cell cycle phase the cells were in when irradiated. FEMS Microbiol Lett, 1989 Jul 1, 51(1), 61 - 5 Electrofusion of fragile mutants of Saccharomyces cerevisiae; Tsoneva I et al.; This communication presents experimental evidence that intact fragile (osmotic sensitive) yeasts can be electrofused and give viable hybrids . The yield increases with one order of magnitude for electrofusion of intact fragile yeasts with protoplasts of non-fragile ones . The yield of viable hybrids, obtained by electrofusion of protoplasts of fragile and non-fragile yeasts, is one order of magnitude higher than the yield from protoplasts of non-fragile yeasts . The destabilized cell wall and plasma membrane of the mutant yeasts could be a possible explanation for this phenomenon. EMBO J, 1989 Jul, 8(7), 2057 - 65 Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae; Rothman JH et al.; To further our studies of protein sorting and biogenesis of the lysosome-like vacuole in yeast, we have isolated spontaneous mutations in 11 new VPL complementation groups, as well as additional alleles of the eight previously described VPL genes . These mutants were identified by selecting for cells that mislocalize vacuolar proteins to the cell surface . Morphological examination of the vpl mutants indicated that most contain vacuoles of normal appearance; however, some of the mutants generally lack a large vacuole, and instead accumulate smaller organelles . Of the 19 VPL complementation groups, 12 were found to be identical to 12 of 33 VPT complementation groups identified in a separate study . Moreover, the end1 mutant and all of the previously reported pep mutants, with the exception of pep4, were found to exhibit a profound vacuolar protein sorting defect, and complementation tests between the PEP, VPL VPT and END1 groups demonstrated that there are extensive overlaps between these groups . Collectively, mutants in these four collections define 49 complementation groups required to deliver or retain soluble vacuolar enzymes, including carboxypeptidase Y (CPY) and proteinase A . We have also isolated 462 new mutants that lack normal levels of vacuolar CPY activity . Among these latter mutants, only pep4 mutants were found to be specifically defective in vacuolar zymogen activation . We conclude that there is a large number of gene products required for sorting or retention of vacuolar proteins in yeast, and only a single gene, PEP4, that is essential for activation of CPY and other vacuolar zymogens. Biochem Cell Biol, 1989 Jul, 67(7), 352 - 7 Relationships between sensitivity to hydroxyurea and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIO) and ribonucleotide reductase RNR2 mRNA levels in strains of Saccharomyces cerevisiae; Rittberg DA et al.; Ribonucleotide reductase is responsible for providing the deoxyribonucleotide precursors for DNA synthesis . In most species the enzyme consists of a large and a small subunit, both of which are required for activity . In mammalian cells, the small subunit is the site of action of several antitumor agents, including hydroxyurea and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (MAIQ) . The mRNA levels for the small subunit of ribonucleotide reductase (RNR2) and sensitivity to hydroxyurea and MAIQ were determined in four strains of the yeast, Saccharomyces cerevisiae . Two strains exhibited significantly different sensitivities to both hydroxyurea and MAIQ, which closely correlated with differences in the levels of RNR2 mRNA . These results are consistent with recent observations with mammalian cells in culture, and indicate that a common mechanism of resistance to hydroxyurea and related drugs occurs through the elevation in ribonucleotide reductase message levels . A transplason mutagenized strain with marked structural modifications in RNR2 DNA and mRNA showed an extreme hypersensitivity to hydroxyurea but not to MAIQ, providing evidence that the two drugs do not inhibit the RNR2 subunit by the same mechanism . In addition, a yeast strain isolated for low but reproducible resistance to MAIQ exhibited a sensitivity to hydroxyurea similar to the parental wild-type strain, supporting the idea that the two drugs inhibit the activity of RNR2 by unique mechanisms . These yeast strains provide a useful approach for further studies into the regulation of eucaryotic ribonucleotide reduction and drug resistance mechanism involving a key rate-limiting step in DNA synthesis. Yeast, 1989 Jul-Aug, 5(4), 271 - 84 Saccharomyces cerevisiae mutants defective in chromosome segregation; McGrew JT et al.; We have devised a genetic screen to identify trans-acting factors involved in chromosome transmission in yeast . This approach was designed to potentially identify a subset of genes encoding proteins that interact with centromere DNA . It has been shown that mutations in yeast centromere DNA cause aberrant chromosome segregation during mitosis and meiosis . We reasoned that the function of an altered centromere should be particularly sensitive to changes in factors with which it interacts . We constructed a disomic strain containing one copy of chromosome III with a wild-type centromere and one copy of chromosome III bearing the SUP11 gene and a mutant CEN3 . This strain forms white colonies with red sectors due to nondisjunction of the chromosome bearing the mutant centromere . After mutagenesis we picked colonies that exhibited increased nondisjunction of the mutant chromosome as evidenced by increased red-white sectoring . Using this approach, we have isolated three trans-acting chromosome nondisjunction (cnd) mutants that are defective in maintaining chromosomes during mitosis in yeast. Mol Cell Biol, 1989 Jul, 9(7), 2989 - 99 Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes; Traglia HM et al.; The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion . This lesion interferes with the processing and production of all major classes of RNA . Each class of RNA is affected at a distinct and presumably unrelated step . Furthermore, RNA does not appear to exit the nucleus . To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes . The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein . No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products . Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type . We found that neither single-amino-acid change alone resulted in temperature sensitivity . The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400 . We generated genomic deletions that removed C-terminal regions of this protein . Deletion of amino acids 397 to 407 did not appear to affect cell growth . Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth . This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus . We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing . Removal of amino acids 330 to 407 resulted in loss of viability. Mol Cell Biol, 1989 Jul, 9(7), 2914 - 21 The OBF1 protein and its DNA-binding site are important for the function of an autonomously replicating sequence in Saccharomyces cerevisiae; Walker SS et al.; The autonomously replicating sequence ARS121 was cloned as a 480-base-pair (bp) long DNA fragment that confers on plasmids autonomous replication in Saccharomyces cerevisiae . This fragment contains two OBF1-binding sites (sites I and II) of different affinities, as identified by a gel mobility shift assay and footprint analysis . Nucleotide substitutions (16 to 18 bp) within either of the two sites obliterated detectable in vitro OBF1 binding to the mutagenized site . Linker substitution (6 bp) mutations within the high-affinity site I showed effects similar to those of the complete substitution, whereas DNA mutagenized outside the binding site bound OBF1 normally . We also tested the mitotic stability of centromeric plasmids bearing wild-type and mutagenized copies of ARS121 . Both deletion of the sites and the extensive base alterations within either of the two OBF1-binding sites reduced the percentage of plasmid-containing cells in the population from about 88% to 50 to 63% under selective growth and from about 46% to 15 to 20% after 10 to 12 generations of nonselective growth . Furthermore, linker (6 bp) substitutions within site I, the high-affinity binding site, showed similar deficiencies in plasmid stability . In contrast, plasmids containing linker substitutions in sequences contiguous to site I displayed wild-type stability . In addition, plasmid copy number analysis indicated that the instability probably resulted not from nondisjunction during mitosis but rather from inefficient plasmid replication . The results strongly support the notion that the OBF1-binding sites and the OBF1 protein are important for normal ARS function as an origin of replication. Mol Cell Biol, 1989 Jul, 9(7), 2906 - 13 Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences; Francesconi SC et al.; We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S . Eisenberg C . Civalier, and B . K . Tye, Proc . Natl . Acad . Sci . USA 85:743-746, 1988) . OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography . Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides . The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons . Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients . Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes . The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively . These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers . Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related . In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively . Furthermore, in the accompanying paper (S . S . Walker, S . C . Francesconi, B . K . Tye, and S . Eisenberg, Mol . Cell . Biol . 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication . These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS. Lipids, 1989 Jul, 24(7), 625 - 9 Gas chromatographic analysis of intact steryl esters in wild type Saccharomyces cerevisiae and in an ester accumulating mutant; Fenner GP et al.; The steryl ester faction from wild type and mutant strains of the yeast Saccharomyces cerevisiae was analyzed without saponification by a non-polar capillary gas chromatographic column . When expressed as microgram ester/mg dry wt, the total ester fraction remained constant or declined slightly from log to stationary phase in the wild type . In the mutant the decrease was more dramatic . No individual ergosteryl ester species was dominant throughout the culture cycle in the wild type . A compound tentatively identified as zymosteryl palmitate was the most prevalent ester in wild type log phase cells, ergosta-5,7-dienyl palmitate and ergosta-5,7-dienyl palmitoleate were the major esters in stationary cells . In the mutant strain, ergosteryl esters of palmitate, palmitoleate, oleate, and stearate were the