S. cerevisiae

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 major ester components throughout the culture cycle . Like the wild type, however, the mutant showed an increase in the proportion of ergosta-5,7-dienyl esters in the stationary phase of the culture cycle . The data did not indicate a sterol/fatty acid specificity during the culture cycle.

Genetics, 1989 Jul, 122(3), 543 - 50
gcd12 mutations are gcn3-dependent alleles of GCD2, a negative regulator of GCN4 in the general amino acid control of Saccharomyces cerevisiae; Paddon CJ et al.; GCD12 encodes a translational repressor of the GCN4 protein, a transcriptional activator of amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae . gcd12 mutations override the requirement for the GCN2 and GCN3 gene products for derepression of GCN4 expression, suggesting that GCN2 and GCN3 function indirectly as positive regulators by negative regulation of GCD12 . In addition to their regulatory phenotype, gcd12 mutants are temperature-sensitive for growth (Tsm-) and, as shown here, deletion of the GCD12 gene is unconditionally lethal . Both the regulatory and the Tsm- phenotypes associated with gcd12 point mutations are completely overcome by wild-type GCN3, implying that GCN3 can promote or partially substitute for the functions of GCD12 in normal growth conditions even though it antagonizes GCD12 regulatory function in starvation conditions . The GCD12 gene has been cloned and mapped to the right arm of chromosome VII, very close to the map position reported for GCD2 . We demonstrate that GCD12 and GCD2 are the same genes; however, unlike gcd12 mutations, the growth defect and constitutive derepression phenotypes associated with the gcd2-1 mutation are expressed in the presence of the wild-type GCN3 gene . These findings can be explained by either of two alternative hypotheses: (1) gcd12 mutations affect a domain of the GCD2 protein that directly interacts with GCN3, and complex formation stabilizes mutant gcd12 (but not gcd2-1) gene products; (2) gcd12 mutations selectively impair one function of GCD2 that is replaceable by GCN3, whereas gcd2-1 inactivates a different GCD2 function for which GCN3 cannot substitute . Both models imply a close interaction between these two positive and negative regulators in general amino acid control.

Genetics, 1989 Jul, 122(3), 535 - 42
Disruption of the RAD52 gene alters the spectrum of spontaneous SUP4-o mutations in Saccharomyces cerevisiae; Kunz BA et al.; Defects in the RAD52 gene of the yeast Saccharomyces cerevisiae confer a mutator phenotype . To characterize this effect in detail, a collection of 238 spontaneous SUP4-o mutations arising in a strain having a disrupted RAD52 gene was analyzed by DNA sequencing . The resulting mutational spectrum was compared to that derived from an examination of 222 spontaneous mutations selected in a nearisogenic wild-type (RAD52) strain . This comparison revealed that the mutator phenotype was associated with an increase in the frequency of base-pair substitutions . All possible types of substitution were detected but there was a reduction in the relative fraction of A.T----G.C transitions and an increase in the proportion of G.C----C.G transversions . These changes were sufficient to cause a twofold greater preference for substitutions at G.C sites in the rad52 strain despite a decrease in the fraction of G.C----T.A transversions . There were also considerable differences between the distributions of substitutions within the SUP4-o gene . Base-pair changes occurred at fewer sites in the rad52 strain but the mutated sites included several that were not detected in the RAD52 background . Only two of the four sites that were mutated most frequently in the rad52 strain were also prominent in the wild-type strain and mutation frequencies at almost all sites common to both strains were greater for the rad52 derivative . Although single base-pair deletions occurred in the two strains with similar frequencies, several classes of mutation that were recovered in the wild-type background including multiple base-pair deletions, insertions of the yeast transposable element Ty, and more complex changes, were not detected in the rad52 strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetics, 1989 Jul, 122(3), 519 - 34
Genetic and physical analysis of double-strand break repair and recombination in Saccharomyces cerevisiae; Rudin N et al.; We have investigated HO endonuclease-induced double-strand break (DSB) recombination and repair in a LACZ duplication plasmid in yeast . A 117-bp MATa fragment, embedded in one copy of LACZ, served as a site for initiation of a DSB when HO endonuclease was expressed . The DSB could be repaired using wild-type sequences located on a second, promoterless, copy of LACZ on the same plasmid . In contrast to normal mating-type switching, crossing-over associated with gene conversion occurred at least 50% of the time . The proportion of conversion events accompanied by exchange was greater when the two copies of LACZ were in direct orientation (80%), than when inverted (50%) . In addition, the fraction of plasmids lost was significantly greater in the inverted orientation . The kinetics of appearance of intermediates and final products were also monitored . The repair of the DSB is slow, requiring at least an hour from the detection of the HO-cut fragments to completion of repair . Surprisingly, the appearance of the two reciprocal products of crossing over did not occur with the same kinetics . For example, when the two LACZ sequences were in the direct orientation, the HO-induced formation of a large circular deletion product was not accompanied by the appearance of a small circular reciprocal product . We suggest that these differences may reflect two kinetically separable processes, one involving only one cut end and the other resulting from the concerted participation of both ends of the DSB.

Genetics, 1989 Jul, 122(3), 503 - 17
Genetic and molecular analysis of recombination events in Saccharomyces cerevisiae occurring in the presence of the hyper-recombination mutation hpr1; Aguilera A et al.; The hyper-recombination mutation hpr1 specifically increases mitotic intrachromatid crossovers, with no effect on other mitotic recombination events such as unequal sister chromatid exchange and plasmid-chromosome recombination, and no effect on meiotic recombination and a lesser effect on intrachromosomal gene conversion . The excision repair RAD1 gene is partially required for the expression on the hpr1 phenotype . The simplest hypothesis to account for some of the hpr1 stimulated recombination events is that a heteroduplex DNA intermediate and localized gene conversion are involved . hpr1 stimulated crossover events are independent of intrachromosomal gene conversion events stimulated by the hyper-gene conversion mutation hpr5 . This result suggests that different intrachromosomal recombination processes are affected in each mutant strain . We propose that HPR1 may function to inhibit intrachromatid crossovers.

Arch Biochem Biophys, 1989 Jul, 272(1), 203 - 9
Synthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Man2GlcNAc2 and Man1GlcNAc2 are transferred from dolichol to protein in vivo; Jackson BJ et al.; Transfer of truncated oligosaccharides to protein in vivo and the structure of Man2GlcNAc2 synthesized by intact yeast (Saccharomyces cerevisiae) were investigated in the alg2 mutant . At the nonpermissive temperature the alg2 mutant accumulates lipid-linked oligosaccharides that migrate on Bio-Gel P4 in the range expected for Man2GlcNAc2 and Man1GlcNAc2 (T.C . Huffaker and P.W . Robbins (1983) Proc . Natl . Acad . Sci . USA 80, 7466-7470) . We characterized the oligosaccharides, derived from protein and lipid, by comigration with standards on HPLC and by Smith degradation followed by HPLC . Man2GlcNAc2 and Man1GlcNAc2 are found on protein in alg2, since their release from a protein-containing precipitate of alg2 cells is N-glycanase (peptide-N4{N-acetyl-beta-glucosaminyl}asparagine amidase) dependent . Transfer also occurred in alg2/pAC3 cells, which carry ALG2 on a multicopy plasmid that confers partial correction of the oligosaccharide phenotype . The alg2/pAC3 cells are viable at 36 degrees C . Two isomers of Man2GlcNAc2, Man1----3ManGlcNAc2 and Man1----6ManGlcNAc2, were present on lipid and protein . The transfer of Man2GlcNAc2 and Man1GlcNAc2 to protein by intact cells supports topological models that postulate access by early intermediates to the lumen of the endoplasmic reticulum.

J Virol, 1989 Jul, 63(7), 3176 - 9
N myristylation of the human immunodeficiency virus type 1 gag polyprotein precursor in Saccharomyces cerevisiae; Bathurst IC et al.; A semisynthetic gene precisely encoding the 502 amino acids of the human immunodeficiency virus type 1 gag precursor (Pr53gag) was expressed in the yeast Saccharomyces cerevisiae . Amino acid sequence analysis of the recombinant Pr53gag showed that the amino terminus was fully blocked . Labeling of Pr53gag with {3H}myristic acid demonstrated that, as with Pr53gag isolated from virus-infected cells, the yeast-derived protein was demethionylated and N myristylated on glycine, the second amino acid residue.

Proc Natl Acad Sci U S A, 1989 Jul, 86(14), 5419 - 23
Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae; Erdmann R et al.; Two mutants of Saccharomyces cerevisiae affected in peroxisomal assembly (pas mutants) have been isolated and characterized . Each strain contains a single mutation that results in (i) the inability to grow on oleic acid, (ii) accumulation of peroxisomal matrix enzymes in the cytosol, and (iii) absence of detectable peroxisomes at the ultrastructural level . These lesions (pas1-1 and pas2) are shown to be nonallelic and recessive . Crossing of pas1-1 and pas2 strains resulted in diploid cells that had regained the ability to grow on oleic acid as sole carbon source and to form peroxisomes . These pas mutants may provide useful tools for future studies on the molecular mechanisms involved in peroxisomal assembly.

Biofizika, 1989 Jul-Aug, 34(4), 630 - 4
{Regularity of changes in magnetic characteristics of Saccharomyces cerevisiae yeast cells at various stages of culture growth}; Tsapin AI et al.; Correlation between cell cycle of the synchronous yeast culture and ESR signal intensity at g 2.2 and 77 K was studied . It was shown that the maximal intensity of ESR signal was reached 10-15 min before the beginning of intensive cell division . The ESR signal with g 2.2 (77 K) is caused by the spin-glass like structure . The "freezing" temperature of these spin-glasses was measured.

Yeast, 1989 Jul-Aug, 5(4), 259 - 69
Functional exploration of the yeast (Saccharomyces cerevisiae) genome: use of a mini-Mu transposon to analyse randomly cloned DNA sequences; Daignan-Fornier B et al.; The development of mega-sequencing techniques requires new methods for global functional analysis of cloned DNA fragments . We have developed a mini-Mu transposon adapted to yeast cloned DNA fragment analysis . This transposon allows us to do the following in a single construction: (i) to probe yeast cells for the presence of expressed open reading frames (ORFs) in the cloned DNA fragment; (ii) to localize these ORFs in the fragment and determine their transcription orientation; (iii) to use beta-galactosidase protein fusions to study regulation of these ORFs; and (iv) to disrupt the corresponding chromosomal genes . On a 5-kb yeast DNA sequence, we have verified the reliability of this new tool by comparing the data obtained with the mini-Mu transposon to those obtained by classical methods . This transposon should be of immediate use in the yeast genome sequencing programme.

Eur J Biochem, 1989 Jul 1, 182(3), 613 - 20
Characterization of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of Saccharomyces cerevisiae; Vandercammen A et al.; The properties of yeast trehalose-6-phosphate synthase were reinvestigated in relation with the recent claim made by Panek et al . {Panek, A . C., de Araujo, P . S., Moura-Neto, V . and Panek, A . D . (1987) Curr . Genet . II, 459-465} that the enzyme would be stimulated by ATP and partially inactivated by cAMP-dependent protein kinase . Trehalose-6-phosphate synthase activity was measured by the sum of {14C}trehalose 6-phosphate and {14C}trehalose formed from UDP-{14C}glucose and glucose 6-phosphate . The activity measured in an extract of Saccharomyces cerevisiae was not affected by any treatment of the cells, such as incubation in the presence of glucose or of dinitrophenol, which are known to greatly increase the intracellular concentration of cAMP, nor by preincubation of the extract in the presence of ATP-Mg, cAMP and bovine heart cAMP-dependent protein kinase . The activity was also not significantly different in several mutants affected in the cAMP system . The kinetic properties of the partially purified enzyme were investigated; no effect of ATP could be detected but Pi acted as a potent noncompetitive inhibitor (Ki = 2 mM) . The activity of trehalose-6-phosphate phosphatase was measured by the amount of {14C}trehalose formed from {14C}trehalose 6-phosphate . The enzyme could be separated from other phosphatases and appeared to be highly specific for trehalose 6-phosphate . It was Mg dependent and its kinetics for trehalose 6-phosphate was hyperbolic . Studies performed with intact cells, crude extracts or the purified enzyme did not reveal any cAMP-dependent change in its activity . Remarkably, trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase copurified in the course of different chromatographic procedures, suggesting that they are part of a single bifunctional protein . A 50-fold purification of the two enzymes could be achieved with a yield of only 2% by chromatography on Mono S followed by gel filtration on Superose 6B.

Curr Genet, 1989 Jul, 16(1), 7 - 12
Cloning and physical characterization of linked lysine genes (lys4, lys15) of Saccharomyces cerevisiae; Wang L et al.; The plasmid pSC4 which carries a 7.8 kb yeast DNA insert at the BamHI site of the Vector YEp13, complemented simultaneously MO-59-13c lys4, LU75 lys15 and LU32 lys4lys15 (double) mutations of Saccharomyces cerevisiae . The 1.9 kb BamHI-XbaI DNA insert of the subclone pSO51 complemented the LU75 lys15 mutation . The 2.8 kb Xhol-XhoI DNA insert of the pSO52 subclone, like pSC4, complemented all three mutations . The 1.9 kb BamHI-XbaI DNA and the 2.8 kb Xhol-XhoI DNA were 100 bp apart in the pSC4 DNA insert and exhibited no homology with each other upon Southern hybridization . The 1.9 kb BamHI-XbaI DNA insert exhibited homology with the pSC4 and pSO51 DNA as well as the genomic DNA of MO-59-13c lys4, LU75 lys15, LU32 lys4lys15, and RC1 (LYS) when digested with appropriate restriction enzymes . The 2.8 kb XhoI-XhoI DNA insert exhibited homology with the pSC4 and pSO52 DNA as well as MO-59-13c lys4, LU75 lys15, LU32 lys4lys15, and RC1 (LYS) genomic DNA, when digested with XhoI enzyme . The 2.8 kb DNA probe also hybridized with ply(A)+ RNA from RC1 and lys4+ transformant but not that from MO-59-13c lys4 mutant.

Mol Cell Biol, 1989 Jul, 9(7), 3101 - 4
Two DNA repair and recombination genes in Saccharomyces cerevisiae, RAD52 and RAD54, are induced during meiosis; Cole GM et al.; The DNA repair and recombination genes of Saccharomyces cerevisiae, RAD52 and RAD54, were transcriptionally induced approximately 10- to 15-fold in sporulating MATa/alpha cells . Congenic MATa/a cells, which did not sporulate, did not show similar increases . Assays of beta-galactosidase activity in strains harboring either a RAD52- or RAD54-lacZ gene fusion indicated that this induction occurred at a time concomitant with a commitment to meiotic recombination, as measured by prototroph formation from his1 heteroalleles.

Mol Cell Biol, 1989 Jul, 9(7), 3009 - 17
Functional domains of a negative regulatory protein, GAL80, of Saccharomyces cerevisiae; Nogi Y et al.; To study the functional domains of a transcriptional repressor encoded by the GAL80 gene of Saccharomyces cerevisiae, we constructed various deletion and insertion mutations in the GAL80 coding region and determined the ability of these mutations to repress synthesis of galactose-metabolizing enzymes as well as the capacity of the mutant proteins to respond to the inducer . Two regions, from amino acids 1 to 321 and from amino acids 341 to 423, in the total sequence of 435 amino acids were required for repression . The internal region from amino acids 321 to 340 played a role in the response to the inducer . The 12 amino acids at the carboxy terminus were dispensable for normal functioning of the GAL80 protein . Using indirect immunofluorescence and subcellular fractionation techniques, we also found that two distinct regions (amino acids 1 to 109 and 342 to 405) within the putative repression domain were capable of directing cytoplasmically synthesized Escherichia coli beta-galactosidase to the yeast nucleus . In addition, three gal80 mutations were mapped at amino acid residues 183, 298, and 310 in the domain required for repression . On the basis of these results, we suggest that the GAL80 protein consists of a repression domain located in two separate regions (amino acid residues 1 to 321 and 341 to 423) that are interrupted by an inducer interaction domain (residues 322 to 340) and two nuclear localization domains (1 to 109 and 342 to 405) that overlap the repression domains.

Cytometry, 1989 Jul, 10(4), 475 - 83
Quantitative immunofluorescence in single Saccharomyces cerevisiae cells; Eitzman PD et al.; We have developed a staining procedure that allows the simultaneous determination of intracellular amounts of DNA and an antigen in Saccharomyces cerevisiae with a single laser flow cytometer . The antigen, beta-galactosidase from a cloned lacZ gene, is inducible and is detected with an indirect immunofluorescent stain . Cell preparation procedures, specifically cell fixation and cell wall removal, have significant effects on measured levels of immunofluorescence and have been optimized to prevent cell loss and maximize immunofluorescence . Average immunofluorescent levels of cell populations expressing different levels of beta-galactosidase show excellent correlation with measurements of average beta-galactosidase activity per cell based on cleavage of o-nitrophenyl-beta-D-galactopyranoside . Experiments with yeast populations containing various numbers of copies of the cloned gene indicate that the relationship between immunofluorescence and antigen content also holds at the single-cell level . Correlated measurements of DNA and beta-galactosidase content on a single-cell level permit the investigation of cellular enzyme content as a function of cell cycle position under various conditions . The procedure can be easily modified to detect other antigens by changing the primary antibody used.

Biochem Biophys Res Commun, 1989 Jun 30, 161(3), 1001 - 6
Identification of A1 protein as the fourth member of 13 kDa-type acidic ribosomal protein family in yeast Saccharomyces cerevisiae; Mitsui K et al.; The identity of protein A1 predicted by a cDNA clone from yeast Saccharomyces cerevisiae which has common carboxyl-terminus to 13 kDa-type acidic ribosomal proteins has been examined . The unique gene for A1 was isolated using the cDNA clone and found to possess two boxes similar to upstream activation sequences for ribosomal protein genes (UASrpg) in the 5'-flanking region . The in vitro-translation product directed by hybrid-selected mRNA with A1 cDNA comigrated with a minor component of split proteins from ribosome by electrofocusing . In addition, the mRNA level for A1 was found to be lower than other two major acidic ribosomal proteins suggesting that A1 is the fourth member of the protein family so far identified which is expressed at relatively low level.

Nucleic Acids Res, 1989 Jun 26, 17(12), 4433 - 9
Differential repair of UV damage in Saccharomyces cerevisiae; Terleth C et al.; Preferential repair of UV-induced damage is a phenomenon by which mammalian cells might enhance their survival . This paper presents the first evidence that preferential repair occurs in the lower eukaryote Saccharomyces cerevisiae . Moreover an unique approach is reported to compare identical sequences present on the same chromosome and only differing in expression . We determined the removal of pyrimidine dimers from two identical alpha-mating type loci and we were able to show that the active MAT alpha locus is repaired preferentially to the inactive HML alpha locus . In a sir-3 mutant, in which both loci are active this preference is not observed.

J Biol Chem, 1989 Jun 25, 264(18), 10542 - 6
Identification of neighboring protein pairs in the 60 S ribosomal subunits from Saccharomyces cerevisiae by chemical cross-linking; Xiang RH et al.; Protein-protein cross-linking was used to determine the spatial arrangement of proteins within the 60 S ribosomal subunits of Saccharomyces cerevisiae . Protein cross-links were generated by treatment of intact ribosomal subunits with dimethyl 3,3'-dithiobispropionimidate . Proteins were extracted from the treated subunits and fractionated by Cm-cellulose chromatography . Cross-linked proteins in these fractions were analyzed by electrophoresis on two-dimensional diagonal polyacrylamide gels containing sodium dodecyl sulfate . Component members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers . Seventeen pairs involving 16 of the 45 60 S subunit proteins were identified . Several proteins were detected in numerous cross-linked dimers and were used as foci for constructing a model depicting the arrangement of proteins within the 60 S ribosomal subunit . The model also incorporated previously published data on structure and function of proteins from the yeast 60 S subunit.

Science, 1989 Jun 16, 244(4910), 1346 - 8
Control of gene expression by artificial introns in Saccharomyces cerevisiae; Yoshimatsu T et al.; Artificial yeast introns that show cold-sensitive splicing have been constructed . These conditional introns can be inserted into a target gene as an "intron cassette" without disrupting the coding information, allowing expression of the gene to be cold sensitive . Insertion of these intron cassettes rendered the yeast URA3 gene cold sensitive in its expression . The advantage of this intron-mediated control system is that any gene can be converted to a controllable gene by simple insertion of an intron.

J Biol Chem, 1989 Jun 15, 264(17), 9923 - 31
Proton NMR comparison of the Saccharomyces cerevisiae ferricytochrome c isozyme-1 monomer and covalent disulfide dimer; Moench SJ et al.; Proton NMR studies of Saccharomyces cerevisiae (bakers yeast) isozyme-1 monomer and dimer ferricytochrome c have been carried out . The dimer is formed via a disulfide bridge between the Cys-102 residues of monomer proteins . Nuclear Overhauser effect (NOE) experiments have led to resonance assignments for many of the heme and axial ligand (Met-80; His-18) protons in both protein forms . Resonances of the following amino acids have also been assigned in both forms: Phe-10; Pro-30; Phe-82; Trp-59; Leu-68 . The proton NOE connectivity patterns of the monomer of yeast isozyme-1 ferricytochrome c are similar to those of horse, tuna, and yeast isozyme-2 ferricytochromes c, even though the observed hyperfine resonance spectra are significantly different for the various cytochromes . The pattern of dimer proton hyperfine resonances is distinct from the isozyme-1 monomer pattern, which indicates that the formation of a disulfide bridge via Cys-102 is detected at the heme site, approximately 10 A distant . It appears that a specific structural change is induced upon dimerization, which, in turn, causes specific perturbations in the vicinity of the heme . However, the general features of the NOE connectivity pattern in the dimer are the same as for the monomer indicating that dimerization does not result in drastic structural disruption . Furthermore, the 1H NMR spectrum of the dimer can be mimicked by the monomer form that results when the -SH group of Cys-102 is chemically modified with certain types of bulky, or hydrophilic reagents (i.e . 5,5'-dithiobis{2-nitrobenzoate}, indicating that perturbations of the yeast isozyme-1 ferricytochrome c proton resonance spectrum observed upon dimerization are essentially due to changes in intramolecular, rather than intermolecular, interactions . These results suggest that a possible regulatory site for yeast isozyme-1 cytochrome c exists at position 102, which could conceivably have a physiological role in altering the conformation of the molecule.

Chem Pharm Bull (Tokyo), 1989 Jun, 37(6), 1632 - 4
Photodynamic activities of food additive dyes on the yeast Saccharomyces cerevisiae; Iwamoto Y et al.; Photodynamic cell-inactivating activities of food additive dyes on the yeast Saccharomyces cerevisiae were investigated . Activities of dyes not permitted as food additives were also examined . Red No . 105 (rose bengal), Red No . 3 (erythrosine) and Red No . 104 (phloxine), which are permitted as food additives, markedly inactivated yeast cells by photodynamic action . Eosine, matius yellow and guinia green B, which are not permitted, also exhibited moderate cell-inactivating activity by photodynamic action . None of the dyes used in this experiment exhibited petite induction by photodynamic action.

Curr Genet, 1989 Jun, 15(6), 393 - 8
An alpha-specific gene, SAG1 is required for sexual agglutination in Saccharomyces cerevisiae; Doi S et al.; Seven alpha-specific mutants specifically defective in sexual agglutinability were isolated . The other alpha mating functions exhibited by these mutants, designated sag mutants, such as the production of alpha pheromone and response to a mating pheromone, were normal . While the MAT alpha sag1 cells did not agglutinate with wild-type a cells, the MATa sag1 cells did, indicating that the SAG1 gene is expressed only in alpha cells . The mutations were semi-dominant and fell into a single complementation group, SAG1, which was mapped near met3 on chromosome X . Complementation analysis showed that sag1 and ag alpha 1, the latter being a previously reported alpha-specific mutation, were mutations in the same gene.

Mol Gen Genet, 1989 Jun, 217(2-3), 419 - 26
The PSO4 gene is responsible for an error-prone recombinational DNA repair pathway in Saccharomyces cerevisiae; de Andrade HH et al.; The induction of mitotic gene conversion and crossing-over in Saccharomyces cerevisiae diploid cells homozygous for the pso4-1 mutation was examined in comparison to the corresponding wild-type strain . The pso4-1 mutant strain was found to be completely blocked in mitotic recombination induced by photoaddition of mono- and bifunctional psoralen derivatives as well as by mono- (HN1) and bifunctional (HN2) nitrogen mustards or 254 nm UV radiation in both stationary and exponential phases of growth . Concerning the lethal effect, diploids homozygous for the pso4-1 mutation are more sensitive to all agents tested in any growth phase . However, this effect is more pronounced in the G2 phase of the cell cycle . These results imply that the ploidy effect and the resistance of budding cells are under the control of the PSO4 gene . On the other hand, the pso4-1 mutant is mutationally defective for all agents used . Therefore, the pso4-1 mutant has a generalized block in both recombination and mutation ability . This indicates that the PSO4 gene is involved in an error-prone repair pathway which relies on a recombinational mechanism, strongly suggesting an analogy between the pso4-1 mutation and the RecA or LexA mutation of Escherichia coli.

Genetics, 1989 Jun, 122(2), 317 - 30
Mutations in the Saccharomyces cerevisiae opi3 gene: effects on phospholipid methylation, growth and cross-pathway regulation of inositol synthesis; McGraw P et al.; We report the isolation of two new opi3 mutants by EMS mutagenesis, and construction of an insertion allele in vitro using the cloned gene . We have demonstrated that the opi3 mutations cause a deficiency in the two terminal phospholipid N-methyltransferase (PLMT) activities required for the de novo synthesis of PC (phosphatidylcholine) . The opi3 mutants, under certain growth conditions, produce membrane virtually devoid of PC although, surprisingly, none of the mutants displays a strict auxotrophic requirement for choline . Although the opi3 mutants grow without supplements, we have shown that the atypical membrane affects the ability of the mutant strains to initiate log phase growth and to sustain viability at stationary phase . The commencement of log phase growth is enhanced by addition of choline or to a lesser extent DME (dimethylethanolamine), and retarded by addition of MME (monomethylethanolamine) . The mutant cells lose viability at the stationary phase of the cell cycle in the absence of DME or choline, and are also temperature sensitive for growth at 37 degrees especially in media containing MME . These growth defects have been correlated to the presence of specific phospholipids in the membrane . The opi3 growth defects are suppressed by an unusual mutation in the phospholipid methylation pathway that perturbs the N-methyltransferase (PEMT) activity immediately preceding the reactions affected by the opi3 lesion . We believe this mutation, cho2-S, alters the substrate specificity of the PEMT . A secondary effect of opi3 mutations is disruption of the cross pathway regulation of the synthesis of the PI (phosphatidylinositol) precursor inositol . Synthesis of inositol is controlled through regulation of the INO1 gene which encodes inositol-1-phosphate synthase . This highly regulated gene is expressed constitutively in opi3 mutants . We have used the opi3 strains to demonstrate that synthesis of either PC or PD (phosphatidyldimethylethanolamine) will restore normal regulation of the INO1 gene.

EMBO J, 1989 Jun, 8(6), 1849 - 54
Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III; Boulet A et al.; Saccharomyces cerevisiae cdc2 mutants arrest in the S-phase of the cell cycle when grown at the non-permissive temperature, implicating this gene product as essential for DNA synthesis . The CDC2 gene has been cloned from a yeast genomic library in vector YEp13 by complementation of a cdc2 mutation . An open reading frame coding for a 1093 amino acid long protein with a calculated mol . wt of 124,518 was determined from the sequence . This putative protein shows significant homology with a class of eukaryotic DNA polymerases exemplified by human DNA polymerase alpha and herpes simplex virus DNA polymerase . Fractionation of extracts from cdc2 strains showed that these mutants lacked both the polymerase and proofreading 3'-5' exonuclease activity of DNA polymerase III, the yeast analog of mammalian DNA polymerase delta . These studies indicate that DNA polymerase III is an essential component of the DNA replication machinery.

Biotechnol Appl Biochem, 1989 Jun, 11(3), 273 - 87
Production of recombinant human serum albumin from Saccharomyces cerevisiae; Quirk AV et al.; Human serum albumin has been constitutively expressed in a Saccharomyces cerevisiae brewing yeast . After cell growth and disruption the product was associated with the insoluble fraction and represented approximately 1% of total cell protein . After the cell debris was extensively washed, the albumin was solubilized with 8 M urea and 28 mM 2-mercaptoethanol in 50 mM sodium carbonate buffer, pH 10 . The denatured albumin was refolded by dialysis and further purified by anion exchange and gel filtration chromatography . Losses of renatured material could be reduced, or higher protein concentrations used during refolding, if the denatured product was purified by cation-exchange chromatography in urea prior to refolding . Apart from an additional N-terminal N-acetyl methionine, the refolded product proved identical to human serum albumin derived from plasma when compared by a variety of physical, chemical, and biological analytical methods.

Microbiol Rev, 1989 Jun, 53(2), 256 - 71
Synthesis of ribosomes in Saccharomyces cerevisiae; Warner JR; The assembly of a eucaryotic ribosome requires the synthesis of four ribosomal ribonucleic acid (RNA) molecules and more than 75 ribosomal proteins . It utilizes all three RNA polymerases; it requires the cooperation of the nucleus and the cytoplasm, the processing of RNA, and the specific interaction of RNA and protein molecules . It is carried out efficiently and is exquisitely sensitive to the needs of the cell . Our current understanding of this process in the genetically tractable yeast Saccharomyces cerevisiae is reviewed . The ribosomal RNA genes are arranged in a tandem array of 100 to 200 copies . This tandem array has led to unique ways of carrying out a number of functions . Replication is asymmetric and does not initiate from every autonomously replicating sequence . Recombination is suppressed . Transcription of the major ribosomal RNA appears to involve coupling between adjacent transcription units, which are separated by the 5S RNA transcription unit . Genes for many ribosomal proteins have been cloned and sequenced . Few are linked; most are duplicated; most have an intron . There is extensive homology between yeast ribosomal proteins and those of other species . Most, but not all, of the ribosomal protein genes have one or two sites that are essential for their transcription and that bind a common transcription factor . This factor binds also to many other places in the genome, including the telomeres . There is coordinated transcription of the ribosomal protein genes under a variety of conditions . However, the cell seems to possess no mechanism for regulating the transcription of individual ribosomal protein genes in response either to a deficiency or an excess of a particular ribosomal protein . A deficiency causes slow growth . Any excess ribosomal protein is degraded very rapidly, with a half-life of 1 to 5 min . Unlike most types of cells, yeast cells appear not to regulate the translation of ribosomal proteins . However, in the case of ribosomal protein L32, the protein itself causes a feedback inhibition of the splicing of the transcript of its own gene . The synthesis of ribosomes involves a massive transfer of material across the nuclear envelope in both directions . Nuclear localization signals have been identified for at least three ribosomal proteins; they are similar but not identical to those identified for the simian virus 40 T antigen . There is no information about how ribosomal subunits are transported from the nucleus to the cytoplasm.(ABSTRACT TRUNCATED AT 400 WORDS)

Mol Microbiol, 1989 Jun, 3(6), 705 - 14
The proline transport protein of Aspergillus nidulans is very similar to amino acid transporters of Saccharomyces cerevisiae; Sophianopoulou V et al.; In Aspergillus nidulans, the gene prnB encoding the major proline transport system is one of a cluster of four genes necessary and sufficient for the utilization of proline as sole nitrogen and/or carbon source . The prn cluster has been cloned and the sequence and transcript map of the prnB gene are presented in this paper . The predicted translated sequence consists of 570 amino acids, resulting in a molecular weight of 63,028 Daltons . Its hydropathy profile shows 10 hydrophobic segments typical of integral membrane proteins . No N-terminal hydrophobic signal peptide is present, the N-terminal and C-terminal ends of the protein being hydrophilic . Similar results were previously found for the arginine and histidine transporters of Saccharomyces cerevisiae, with which the prnB transporter shares regions of highly conserved amino acid sequences . Using S1 mapping and Northern blot analyses, we confirm the presence of a unique inducible prnB transcript of 1.9 kb.

J Bacteriol, 1989 Jun, 171(6), 3539 - 44
Characteristics of galactose transport in Saccharomyces cerevisiae cells and reconstituted lipid vesicles; Ramos J et al.; Growth on galactose induces two transport processes, a high-affinity and a low-affinity process . The most important results of a comparison of the two processes were that (i) both depended on GAL2 expression, (ii) only the high-affinity process required galactokinase, (iii) both were down-regulated by catabolite inactivation, (iv) neither was significantly inhibited by carbonyl cyanide-p-trifluoromethoxy-phenyl-hydrazone, (v) neither was differentially inhibited by silver nitrate or mercuric chloride, and (vi) transport activity with a Km closer to that of the low-affinity process of whole cells was reconstituted in fused phospholipid membrane vesicles.

Mol Gen Genet, 1989 Jun, 217(2-3), 370 - 7
Three regulatory systems control expression of glutamine synthetase in Saccharomyces cerevisiae at the level of transcription; Benjamin PM et al.; The GLN1 gene of Saccharomyces cerevisiae was cloned by complementation of a gln1 auxotroph . A GLN1-lacZ fusion was constructed to assay GLN1 promoter activity . beta-Galactosidase and glutamine synthetase expression in chromosomally integrated GLN1-lacZ fusion strains were co-regulated in response to a shift from glutamine to glutamate as the nitrogen source, purine limitation, and 3-aminotriazole-induced histidine starvation . Regulation of GLN1 expression by each of the three pathways occurred at the transcriptional level . Increased accumulation of GLN1 mRNA was observed within 5 min after a shift from glutamine to glutamate as the nitrogen source . After 5 min, GLN1 mRNA levels were constant . The level of GLN1 transcript was reduced by approximately 75% within 5 min following glutamine addition to the cells growing with glutamate as nitrogen source . This indicates that the GLN1 message is unstable and has a half-life of approximately 3 min . Deletion analysis indicated that the sequences required for GLN1 expression are located within approximately 350 bp upstream from the transcriptional initiation site.

J Gen Microbiol, 1989 Jun, 135 ( Pt 6), 1453 - 60
cAMP- and RAS-independent nutritional regulation of plasma-membrane H+-ATPase activity in Saccharomyces cerevisiae; Mazon MJ et al.; The plasma-membrane ATPase of Saccharomyces cerevisiae is a proton pump whose activity, essential fro proliferation, is subject to regulation by nutritional signals . The previous finding that the CDC25 gene product is required for the glucose-induced H+-ATPase activation suggested that H+-ATPase activity is regulated by cAMP . Analysis of starvation-induced inactivation and glucose-induced activation of the H+-ATPase in mutants affected in activity of the RAS proteins, adenylyl cyclase or cAMP-dependent protein kinase showed that nutritional regulation of H+-ATPase activity does not depend directly on any of these factors . We conclude that adenlyl cyclase does not mediate all nutritional responses . This also indicates that the specific CDC25 requirement for the glucose-induced activation of the H+-ATPase identifies a new function for the CDC25 gene product, a function that appears to be independent of CDC25-mediated modulation of the RAS/adenylyl cyclase/cAMP pathway.

Genetics, 1989 Jun, 122(2), 297 - 305
The PET54 gene of Saccharomyces cerevisiae: characterization of a nuclear gene encoding a mitochondrial translational activator and subcellular localization of its product; Costanzo MC et al.; The product of the nuclear Saccharomyces cerevisiae gene PET54 is specifically required, along with at least two other nuclear gene products, for translation of the mitochondrial mRNA encoding subunit III of cytochrome c oxidase (coxIII) . We have genetically mapped PET54 (to the right arm of chromosome VII, 4.8 cM centromere-distal to SUF15), and have biochemically characterized the gene and its product . We determined the nucleotide sequence of a 1.6-kb DNA fragment carrying PET54 and identified the PET54 reading frame by determining the sequence of an ochre mutant allele as well as frameshift and frameshift-revertant alleles of the gene . The wild-type PET54 gene encodes a slightly basic 293-amino acid protein . PET54 is expressed from two mRNAs, both with unusual features: a major transcript with an extremely short 5'-untranslated leader, and a minor transcript with a relatively long 5'-leader carrying three short open reading frames . Antiserum raised against a trpE-PET54 fusion protein was used to probe subcellular fractions . These experiments showed that the PET54 protein is specifically associated with mitochondria, suggesting that it is likely to act directly in coxIII translation.

Mol Cell Biol, 1989 Jun, 9(6), 2715 - 23
Cell size modulation by CDC25 and RAS2 genes in Saccharomyces cerevisiae; Baroni MD et al.; A detailed kinetic analysis of the cell cycle of cdc25-1, RAS2Val-19, or cdc25-1/RAS2Val-19 mutants during exponential growth is presented . At the permissive temperature (24 degrees C), cdc25-1 cells show a longer G1/unbudded phase of the cell cycle and have a smaller critical cell size required for budding without changing the growth rate in comparison to an isogenic wild type . The RAS2Val-19 mutation efficiently suppresses the ts growth defect of the cdc25-1 mutant at 36 degrees C and the increase of G1 phase at 24 degrees C . Moreover, it causes a marked increase of the critical cell mass required to enter into a new cell division cycle compared with that of the wild type . Since the critical cell mass is physiologically modulated by nutritional conditions, we have also studied the behavior of these mutants in different media . The increase in cell size caused by the RAS2Val-19 mutation is evident in all tested growth conditions, while the effect of cdc25-1 is apparently more pronounced in rich culture media . CDC25 and RAS2 gene products have been showed to control cell growth by regulating the cyclic AMP metabolic pathway . Experimental evidence reported herein suggests that the modulation of the critical cell size by CDC25 and RAS2 may involve adenylate cyclase.

Mol Cell Biol, 1989 Jun, 9(6), 2431 - 44
Some of the signals for 3'-end formation in transcription of the Saccharomyces cerevisiae Ty-D15 element are immediately downstream of the initiation site; Yu K et al.; Fragments from the Ty-D15 element of Saccharomyces cerevisiae were assayed for the ability to direct 3'-end formation for RNA initiated by the GAL1 promoter . The delta, the direct repeat at each end of the element, was capable of forming 3' ends at two sites, an inefficient upstream site and an efficient downstream site near the end of the delta . Different sequences were required for 3'-end formation at these sites . For the efficient site, all transcripts had 3' ends in the delta and no downstream transcription was detected, which suggested that these sequences terminate transcription . Surprisingly, the delta region downstream of the initiation site for Ty RNA comprised part of this major site and terminated more than 50% of the transcripts that read into it . Sequences necessary for the efficient site were localized to two small regions . Both regions were upstream of the 3' end and contained similarities to a tripartite consensus sequence that has been proposed as a terminator element . Sequences near the position of the 3' end could also affect termination; a short G + C-rich sequence inserted just downstream changed an efficient terminator to an inefficient one . Initiation in the delta had no effect on the efficiency or positions or termination in that delta . A new initiation site was seen when the same delta terminated transcription, but transcriptional interference did not occur, since the amount of initiation was not decreased.

Mol Cell Biol, 1989 Jun, 9(6), 2289 - 97
GPA1Val-50 mutation in the mating-factor signaling pathway in Saccharomyces cerevisiae; Miyajima I et al.; The GPA1 gene of Saccharomyces cerevisiae encodes a protein that is highly homologous to the alpha subunit of mammalian hetrotrimeric G proteins and is essential for haploid cell growth . A mutation of the GPA1 protein, GPA1Val-50, in which Gly-50 was replaced by valine, could complement the growth defect of a GPA1 disruption, gpal::HIS3 . However, cells with gpa1::HIS3 expressing the GPA1Val-50 protein were supersensitive to alpha-factor in a short-term incubation but resumed growth after long-term incubation even after exposure to high concentrations of alpha-factor . The former phenotype associated with GPA1Val-50 is recessive, and the latter phenotype is dominant to GPA1+ . The supersensitivity of GPA1Val-50 to alpha-factor was dependent on STE2 and STE4, which demonstrates that this GPA1Val-50-produced phenotype requires the mating-factor receptor and the beta subunit of the G protein . The double mutant of sst2-1 GPA1Val-50 recovered from division arrest, which suggested that SST2 is not required for recovery of the GPA1Val-50 mutant.

J Bacteriol, 1989 Jun, 171(6), 3545 - 8
Role of cyclic-AMP-dependent protein kinase in catabolite inactivation of the glucose and galactose transporters in Saccharomyces cerevisiae; Ramos J et al.; The derepressed high-affinity glucose transport system and the induced galactose transport system are catabolite inactivated when cells with these transport systems are incubated with glucose . The role of the cyclic AMP cascade in the catabolite inactivation of these transport systems was shown by using mutants affected in the activity of cyclic-AMP-dependent protein kinase (cAPK) . In tpk1(w) mutants with reduced cAPK activity, the sugar transport systems were expressed but were not catabolite inactivated . In bcy1 mutants with unbridled cAPK activity resulting from a defective regulatory subunit, the transport systems were absent or present at low levels.

Biochem Biophys Res Commun, 1989 May 30, 161(1), 46 - 51
Alpha-agglutinin expression in Saccharomyces cerevisiae; Wojciechowicz D et al.; A polyclonal antiserum raised against purified alpha-agglutinin was made specific for alpha-agglutinin after adsorption with a cells . The adsorbed antiserum identified alpha-agglutinin peptides on Western blots and bound to cell surface alpha-agglutinin, inhibiting the binding of alpha cells to a cells . Using the antibody, we have determined that 1) the surface distribution of alpha-agglutinin on alpha cells is polar, 2) about 5 x 10(4) molecules/cell are constitutively expressed on strain X2180-1B (alpha) cells, and 3) treatment of alpha cells with the sex pheromone a-factor causes an increase in cell surface alpha-agglutinin, consistent with the a-factor induced increase in cell agglutinability.

Biochem Biophys Res Commun, 1989 May 30, 161(1), 182 - 6
Properties of revertants of lys2 and lys5 mutants as well as alpha-aminoadipate-semialdehyde dehydrogenase from Saccharomyces cerevisiae; Storts DR et al.; alpha-Aminoadipate-semialdehyde dehydrogenase catalyzes the conversion of alpha-aminoadipate to alpha-aminoadipate-semialdehyde in the biosynthetic pathway of lysine in yeasts and molds . Mutants belonging to lys2 and lys5 loci of Saccharomyces cerevisiae lacked the alpha-aminoadipate-semialdehyde dehydrogenase activity . Complementation in vitro was demonstrated by combining the extracts from different lys2 and lys5 mutants . Some of the revertants of lys2 and lys5 mutants exhibited lower specific activity and higher thermolability of alpha-aminoadipate-semialdehyde dehydrogenase than the enzyme from wild-type cells . The enzyme was partially purified from wild-type cells and the molecular weight of the enzyme was estimated on a Sephacryl S-300 column at 180,000 . Results from the revertant analysis and in vitro complementation indicated LYS2 and LYS5 as structural genes, each encoding a subunit of this large enzyme.

J Biol Chem, 1989 May 25, 264(15), 8670 - 5
Identification of an upstream repressor site controlling the expression of an anaerobic gene (ANB1) in Saccharomyces cerevisiae; Mehta KD et al.; The Saccharomyces cerevisiae anaerobic gene (ANB1) is negatively regulated both by oxygen and heme . A 299-base pair-long fragment from the 5'-flanking region of the ANB1 gene was found to confer oxygen-mediated negative regulation to an heterologous CYC1-LacZ hybrid gene . Studies with deletions of predefined length in this fragment demonstrated the presence of separate elements that comprise an upstream promoter that is active in the absence or presence of oxygen, and an upstream repressor site (URS) which confers strong repression upon the promoter element when oxygen is present . The promoter element is located 5' to the URS in the ANB1 gene . Mixed oligonucleotide-directed mutagenesis was used to obtain nucleotide substitutions in the URS which partially or completely inactivated this sequence without affecting the promoter activity . The URS region has three short direct repeats which seem to be important for function, as nucleotide substitutions within the repeats and not outside them, inactivated URS function . A model to explain the negative regulation of the ANB1 gene by oxygen and heme is proposed.

J Biol Chem, 1989 May 25, 264(15), 8641 - 5
Purification and characterization of phosphatidate phosphatase from Saccharomyces cerevisiae; Lin YP et al.; Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae . The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, Mono Q, and Superose 12 . The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Phosphatidate phosphatase activity was associated with the purified 91,000 subunit . The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12 . Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7 . The Km value for phosphatidate was 50 microM, and the Vmax was 30 mumol/min/mg . The turnover number (molecular activity) for the enzyme was 2.7 x 10(3) min-1 at pH 7 and 30 degrees C . The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 degrees C . Phosphatidate phosphatase activity was sensitive to thioreactive agents . Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.

Biochemistry, 1989 May 16, 28(10), 4233 - 40
Interaction of the Saccharomyces cerevisiae alpha-factor with phospholipid vesicles as revealed by proton and phosphorus NMR; Jelicks LA et al.; Proton and phosphorus-31 nuclear magnetic resonance (1H and 31P NMR) studies of the interaction between a tridecapeptide pheromone, the alpha-factor of Saccharomyces cerevisiae, and sonicated lipid vesicles are reported . 31P NMR studies demonstrate that there is interaction of the peptide with the phosphorus headgroups, and quasielastic light scattering (QLS) studies indicate that lipid vesicles increase in size upon addition of peptide . Previous solution (aqueous and DMSO) studies from this laboratory indicate that alpha-factor is highly flexible with only one long-lived identifiable structural feature, a type II beta-turn spanning the central portion of the peptide . Two-dimensional (2D) 1H nuclear Overhauser effect spectroscopy (NOESY) studies demonstrate a marked ordering of the peptide upon interaction with lipid, suggesting a compact N-terminus, in addition to a stabilized beta-turn . In contrast to our results in both solution and lipid environment, Wakamatsu et al . {Wakamatsu, K., Okada, A., Suzuki, M., Higashijima, T., Masui, Y., Sakakibara, S., & Miyazawa, T . (1986) Eur . J . Biochem . 154, 607-615} proposed a lipid environment conformation, on the basis of one-dimensional transferred NOE studies in D2O, which does not include the beta-turn.

Eur J Biochem, 1989 May 15, 181(3), 663 - 8
Purification of GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase from Saccharomyces cerevisiae; Haselbeck A; The enzyme GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase (GDP-Man:DolP mannosyltransferase) catalyzing the reaction: GDP-man + DolP in equilibrium DolP-Man + GDP has been purified from Saccharomyces cerevisiae to homogeneity . The purification was achieved using a combination of column chromatographic methods with preparative gel electrophoresis . The enzyme has an apparent molecular mass of 30 kDa on SDS/polyacrylamide gels . Enzymatic activity could be correlated directly with this band . Antibodies against the transferase were raised in rabbits . The immune serum obtained removed enzymatic activity from a detergent extract of yeast membranes and reacted specifically with the 30-kDa band on immunoblots . Experiments addressing the orientation of this enzyme in the endoplasmic reticulum membrane are presented by using selective trypsin and N-ethylmaleimide treatment.

J Biol Chem, 1989 May 15, 264(14), 8366 - 74
The primary structure of the aspartate transcarbamylase region of the URA2 gene product in Saccharomyces cerevisiae . Features involved in activity and nuclear localization; Nagy M et al.; The yeast URA2 locus encodes a multifunctional protein which possesses the carbamylphosphate synthetase and aspartate transcarbamylase activities and which catalyzes the first two reactions of the pyrimidine pathway . We report here the nucleotide sequence of the central and the 3' region of this locus . The latter encodes that part of the multifunctional protein which has the aspartate transcarbamylase activity . The deduced amino acid sequence shows a high degree of homology with the known aspartate transcarbamylases of various organisms from Escherichia coli to mammals . The amino acid residues that have been shown to be involved in the catalytic site of the E . coli enzyme are all conserved suggesting that, in the more complex structure of the yeast protein, the catalytic sites are also located at subunit interfaces . There is also an important conservation of the amino acid pairs that, in E . coli, are implicated in intra- and interchain interactions . As well as the oligomeric structure suggested by these two features, the three-dimensional structure of the yeast enzyme must also be organized to account for the channeling of carbamylphosphate, from the carbamylphosphate synthetase catalytic site to that of aspartate transcarbamylase, and for the concomitant feedback inhibition of the two activities by the end product UTP . The URA2 gene product was shown to be localized in the nucleus . With the aim of identifying the regions that may be involved in this transport, we have determined by electron microscopy the subcellular distribution of aspartate transcarbamylase in three strains expressing different fragments of the URA2 locus . In the first strain the protein lacks 190 residues at the N terminus, but accumulates normally in the nucleus . In the second strain the protein lacks 382 residues in the central part and seems impaired in the nuclear transport process . In the third strain the 476-residue protein encoded by the 3' region of URA2 locus and catalyzing the aspartate transcarbamylase reaction is able by itself to migrate to and accumulate in the nucleus . This suggests that two regions are involved in the nuclear accumulation . On the basis of their conservation in analogous proteins of other eukaryotes and their similarity to sequences already identified as nuclear location signals, a sequence in the central region of the protein and two short sequences in the C-terminal region are good candidates for the nuclear location signal involved in the targeting of the URA2 product.

J Gen Microbiol, 1989 May, 135 ( Pt 5), 1217 - 27
Parallel changes in catabolite repression of haem biosynthesis and cytochromes in repression-resistant mutants of Saccharomyces cerevisiae; Borralho LM et al.; Effects of three mutant genes, CAT1-2d, cat2-1 and hex2-3, on catabolite repression of mitochondrial cytochromes and the first two enzymes of haem biosynthesis were compared . The CAT1-2d mutation gave no resistance to glucose, whereas cat2-1 endowed both cytochromes and 5-aminolaevulinate dehydratase with resistance, but did not alter the effect of glucose on 5-aminolaevulinate synthase . The hex2-3 mutation caused repression resistance of cytochromes and of the two haem biosynthetic enzymes . hex2-3 strains also accumulated intracellular 5-aminolaevulinate . Co-inheritance of the latter traits, sensitivity to maltose inhibition and ability to grow on raffinose in the presence of 2-deoxyglucose, demonstrated that the pleiotropic phenotype is a function of the single gene hex2-3 . Revertants which grew on maltose regained sensitivity to deoxyglucose and exhibited normal sensitivity of cytochromes and haem biosynthesis enzymes to repression . Addition of the hex1-18 mutation, which renders cytochromes resistant to repression, to a cat2-1 strain did not produce the same effect on 5-aminolaevulinate synthase as hex2-3 . It is concluded that repression patterns of haem and cytochrome biosynthesis are substantially affected by hex2-3 and cat2-1 but not by CAT1-2d.

Mikrobiologiia, 1989 May-Jun, 58(3), 471 - 4
{Stability to the extreme influences and significance of internal pH of Saccharomyces cerevisiae depending on the density of cellular suspensions}; Zikmanis PB et al.; The resistance of a Saccharomyces cerevisiae population against gamma-irradiation (400 krd), heating (60 degrees C), repeated freezing--thawing and rehydration of dehydrated cells was shown to depend on the density of a cell suspension . The percentage of damaged cells (stained with primulin) was found to increase in a suspension with decreasing cell concentrations . The effect was also exerted in an oxygen-free medium . The percentage of undamaged cells increased in populations with a lower cell concentration when a more viscous medium (0.2% agar) was used . The intracellular pH dropped down in a denser intact yeast suspension and rose with an increase in the concentration of dehydrated-rehydrated cells.

Mol Gen Genet, 1989 May, 217(1), 40 - 6
Function of the PHO regulatory genes for repressible acid phosphatase synthesis in Saccharomyces cerevisiae; Yoshida K et al.; Expression of the repressible acid phosphatase (rAPase) gene, PHO5, of Saccharomyces cerevisiae is repressed by a certain level of inorganic phosphate (Pi) in the medium and is derepressed when the Pi concentration is lowered . The Pi signals are conveyed to PHO5 by a regulatory system consisting of proteins coded for by the PHO2, PHO4, PHO80 and PHO81 genes . We have found that the transcription of PHO81 is regulated by Pi through the PHO regulatory system . Increasing the dosage of PHO4 and PHO81 by ligating each gene to YEp13 gives rise to, respectively, considerable and weak synthesis of rAPase by cultivation of the transformants in high-Pi medium; but in low-Pi medium, increased dosage of PHO4 stimulates the rAPase synthesis significantly, whereas PHO81 has no effect . Increased dosage of PHO2 stimulates rAPase synthesis considerably in low-Pi but not in high-Pi . A coordinate increase of PHO80 cancels the dosage effect of PHO4, but not that of PHO81 . Coordinate increases of PHO80 and PHO2 give rise to the same phenotype as an increased dosage of PHO80 alone . The level of the PHO4 protein was found to be the limiting factor of the rAPase synthesis and the copy number of the PHO5 gene not to be . These facts accord with the idea that the PHO80 protein transmits the Pi signals to the PHO5 gene via the PHO4 protein, whereas the PHO2 protein does not have a direct function in the signal transmission.

Prikl Biokhim Mikrobiol, 1989 May-Jun, 25(3), 397 - 400
{The effect of a mutagenic factor on the morphologo-biochemical properties of the yeast Saccharomyces cerevisiae}; Abramov ShA et al.; A yeast mutant valuable for the baking industry was obtained by using laser radiation . Its morphological and biochemical characteristics were studied.

Mol Gen Mikrobiol Virusol, 1989 May, (5), 15 - 9
{Synthesis of heat-shock proteins in Saccharomyces cerevisiae protoplasts}; Bekker ML et al.; The effect of cellular capsule elimination in Saccharomyces cerevisiae yeasts (protoplast formation) on the heat-shock protein synthesis and the synthesis of the proteins in protoplasts were studied . The methods of mono- and dimeric electrophoresis have demonstrated that (1) about 18 heat-shock proteins with the molecular masses 26-98 Kd are synthesized in cells at 41 degrees C; (2) protoplast formation per se does not induce the synthesis of heat-shock proteins, but the induction of these proteins in protoplasts at 41 degrees C is similar to the one in intact cells . The protoplast formation induces the synthesis of specific proteins different from heat-shock proteins and the synthesis is inhibited by the heat-shock . The heat-shock induces modification of 88 and 86 Kd heat-shock proteins . It inhibits the synthesis of a number of peptides (15-50 Kd) in cells and protoplasts.

Mol Cell Biol, 1989 May, 9(5), 2142 - 52
A transcriptional cascade governs entry into meiosis in Saccharomyces cerevisiae; Smith HE et al.; Two signals activate meiosis in yeast: starvation and expression of the a1 and alpha 2 products of the mating-type locus . Prior studies suggest that these signals stimulate expression of an activator of meiosis, the IME1 (inducer of meiosis) product . We have cloned a gene, IME2, with properties similar to those of IME1: both genes are required for meiosis, and both RNAs are induced in meiotic cells . Elevated dosage of IME1 or IME2 stimulates the meiotic recombination pathway without starvation; thus, the IME products may be part of the switch that activates meiosis . IME1 was found to be required for IME2 expression, and a multicopy IME2 plasmid permitted meiosis in an ime1 deletion mutant . Accordingly, we propose that the IME1 product stimulates meiosis mainly through activation of IME2 expression.

Mol Cell Biol, 1989 May, 9(5), 1882 - 96
Cloning and sequence analysis of the Saccharomyces cerevisiae RAD9 gene and further evidence that its product is required for cell cycle arrest induced by DNA damage; Schiestl RH et al.; Procaryotic and eucaryotic cells possess mechanisms for arresting cell division in response to DNA damage . Eucaryotic cells arrest division in the G2 stage of the cell cycle, and various observations suggest that this arrest is necessary to ensure the completion of repair of damaged DNA before the entry of cells into mitosis . Here, we provide evidence that the Saccharomyces cerevisiae RAD9 gene, mutations of which confer sensitivity to DNA-damaging agents, is necessary for the cell cycle arrest phenomenon . Our studies with the rad9 delta mutation show that RAD9 plays a role in the cell cycle arrest of methyl methanesulfonate-treated cells and is absolutely required for the cell cycle arrest in the temperature-sensitive cdc9 mutant, which is defective in DNA ligase . At the restrictive temperature, cell cycle progression of cdc9 cells is blocked sometime after the DNA chain elongation step, whereas cdc9 rad9 delta cells do not arrest at this point and undergo one or two additional divisions . Upon transfer from the restrictive to the permissive temperature, a larger proportion of the cdc9 cells than of the cdc9 rad9 delta cells forms viable colonies, indicating that RAD9-mediated cell cycle arrest allows for proper ligation of DNA breaks before the entry of cells into mitosis . The rad9 delta mutation does not affect the frequency of spontaneous or UV-induced mutation and recombination, suggesting that RAD9 is not directly involved in mutagenic or recombinational repair processes . The RAD9 gene encodes a transcript of approximately 4.2 kilobases and a protein of 1,309 amino acids of Mr 148,412 . We suggest that RAD9 may be involved in regulating the expression of genes required for the transition from G2 to mitosis.

Yeast, 1989 May-Jun, 5(3), 149 - 58
Structure and nuclear localization signal of the SKI3 antiviral protein of Saccharomyces cerevisiae; Rhee SK et al.; The yeast chromosomal genes SKI2, SKI3, SKI4, SKI6, SKI7 and SKI8 repress the replication of double-stranded RNA viruses, protecting the host from the otherwise lethal effects of the virus . We cloned and sequenced the SKI3 gene and found that it encodes a 163 kDa protein including a typical nuclear localization signal . Cell fractionation experiments show that the SKI3 gene product is indeed tightly associated with nuclei and that the putative nuclear localization sequence directs beta-galactosidase into the nucleus . However, fusion of a part of the SKI3 protein lacking this signal with beta-galactosidase also directs beta-galactosidase into the nucleus, suggesting the presence of a second nuclear localization signal . The SKI3 gene is only essential in the presence of an M double-stranded RNA virus.

Yeast, 1989 May-Jun, 5(3), 141 - 8
Induction of cytochrome P-450 and catalase activity in Saccharomyces cerevisiae by UV and X-ray irradiation . Possible role for cytochrome P-450 in cell protection against oxidative damage; Morichetti E et al.; Cytochrome P-450 was induced both in the diploid wild-type D7 strain and in two isogenic DNA-repair-deficient strains (rad3 and rad56) of Saccharomyces cerevisiae following UV- and X-irradiation . The induction occurred only in logarithmic growth phase cells and it was transient showing a peak 3 h after irradiation . The maximal amount of cytochrome P-450 was directly proportional to the radiation dose applied . Under the same experimental conditions an increase of the catalase activity was also observed, suggesting that activated oxygen species produced by irradiation might be implicated in the induction of both enzymes . The sensitivity to H2O2 of cells containing high cytochrome P-450 levels was enhanced when this enzyme was specifically inhibited by tetrahydrofuran and metyrapone . This supports the hypothesis that cytochrome P-450, as well as catalase, might be involved in cell protection against oxidative damage.

Genetics, 1989 May, 122(1), 19 - 27
A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae; Sikorski RS et al.; A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae . Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations . A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT . These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3) . They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well . Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.

Eur J Biochem, 1989 May 1, 181(2), 357 - 61
Positive and negative regulatory elements control the expression of the UGA4 gene coding for the inducible 4-aminobutyric-acid-specific permease in Saccharomyces cerevisiae; Vissers S et al.; In Saccharomyces cerevisiae, the pathway of 4-aminobutyric acid catabolism, for use as a nitrogen source, involves a specific permease (encoded by the UGA4 gene) and two enzymes (encoded by the UGA1 and UGA2 genes, respectively) . The synthesis of these proteins is induced by 4-aminobutyrate . It also requires the product of the UGA3 gene . Here, we describe four additional regulatory mutations which provide evidence for the existence of both positive and negative regulatory elements which control the final expression of the UGA4 gene . Some of them simultaneously control the expression of the UGA1 and UGA2 genes . Three classes of mutant with a constitutive 4-aminobutyrate-specific permease have been isolated . (a) Recessive mutations in the UGA43 gene suggest that the product of the UGA43 gene behaves like a trans-acting negative regulator of UGA4 gene expression . (b) The semi-dominant mutation (uga11), closely linked to the UGA4 gene, might affect the receptor of the UGA43 gene product . In these two classes of mutant, only the permease is constitutive . (3) The uga81 mutation, closely linked to the UGA3 gene, makes the whole UGA regulon constitutive . On the other hand, recessive mutations at the UGA35 gene locus lead to non-inducibility of the UGA regulon . Hence the UGA35 gene product behaves like a second trans-acting positive regulator in addition to UGA3.

Eur J Biochem, 1989 May 1, 181(2), 323 - 9
In vitro studies on the translocation of acid phosphatase into the endoplasmic reticulum of the yeast Saccharomyces cerevisiae; Krebs HO et al.; We demonstrate here the in vitro translocation of yeast acid phosphatase into rough endoplasmic reticulum . The precursor of the repressible acid phosphatase from Saccharomyces cerevisiae encoded by the PHO5 gene, was synthesized in a yeast lysate programmed with in vitro transcribed PHO5 mRNA . In the presence of yeast rough microsomes up to 16% of the acid phosphatase synthesized was found to be translocated into the microsomes, as judged by proteinase resistance, and fully core-glycosylated . The translocation efficiency however, decreased to 3% if yeast rough microsomes were added after synthesis of acid phosphatase had been terminated . When a wheat-germ extract was used for in vitro synthesis, the precursor of acid phosphatase was translocated into canine pancreatic rough microsomes and thereby core-glycosylated in a signal-recognition-particle-dependent manner . Replacing canine with yeast rough microsomes in the wheat-germ translation system, however, resulted in a significant decrease in the ability to translocate and glycosylate the precursor . Translocation and glycosylation were partially restored by a high-salt extract prepared from yeast ribosomes . The results presented here suggest that yeast-specific factors are needed to translocate and glycosylate acid phosphatase efficiently in vitro.

Mol Gen Genet, 1989 May, 217(1), 149 - 54
Structure of the HOM2 gene of Saccharomyces cerevisiae and regulation of its expression; Thomas D et al.; In Saccharomyces cerevisiae the HOM2 gene encodes aspartic semi-aldehyde dehydrogenase (ASA DH) . The synthesis of this enzyme had been shown to be derepressed by growth in the presence of high concentrations of methionine . In the present work we have cloned and sequenced the HOM2 gene and found that the promoter region of this gene bears one copy of the consensus sequence for general control of amino acid synthesis . This prompted us to study the regulation of the expression of the HOM2 gene . We have found that ASA DH is the first reported enzyme of the related threonine and methionine pathway to be regulated by the general control of amino acid synthesis.

J Gen Microbiol, 1989 May, 135 ( Pt 5), 1209 - 16
The hexokinase isoenzyme PII of Saccharomyces cerevisiae ia a protein kinase; Herrero P et al.; The HXK2 gene product has an important role in controlling carbon catabolite repression in Saccharomyces cerevisiae . We have raised specific antibodies against the hexokinase PII protein and have demonstrated that it is a 58 kDa phosphoprotein with protein kinase activity . The predicted amino acid sequence of the HXK2 gene product has significant homology to the conserved catalytic domain of mammalian and yeast protein kinases . Protein kinase activity was located in a different domain of the protein from the hexose-phosphorylating activity . The hexokinase PII protein level remained unchanged in P2T22D mutant cells (hxk1 HXK2 glk1) growing in a complex medium with glucose . The protein kinase activity of hexokinase PII is regulated by the glucose concentration of the culture medium . Exit from the carbon catabolite repression phase and entry into derepression phase may be controlled, in part, by modulation of the 58 kDa protein kinase activity by changes in cyclic AMP concentration.

J Cell Biol, 1989 May, 108(5), 1665 - 72
Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae; Cabib E et al.; Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme . However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed . Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar . Lysis occurs at a low pH and is prevented by buffering the medium above pH 5 . A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation . The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH . Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells . Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1 . The latter emerges as an auxiliary or emergency enzyme . Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.

Mol Gen Genet, 1989 May, 217(1), 31 - 9
Mode of expression of the positive regulatory genes PHO2 and PHO4 of the phosphatase regulon in Saccharomyces cerevisiae; Yoshida K et al.; The mode of expression was investigated for two positive regulatory genes, PHO2 and PHO4, whose products are indispensable for the transcriptional control of the structural genes of repressible acid phosphatase and the inorganic phosphate (Pi) transport system in Saccharomyces cerevisiae . Northern analysis of poly(A)+ RNA of the wildtype and the pho regulatory mutants with PHO4 DNA as hybridization probe and expressional analysis of a pho4'-'lacZ fused gene on a YEp plasmid revealed that PHO4 is expressed at a low level, constitutively, and independently of the PHO regulatory system and Pi in the medium . Similar analyses with PHO2 DNA indicated that PHO2 is expressed at an even lower level than PHO4, and is repressed by Pi and by the active PHO2 product, possibly at the translational level, while retaining a substantial level of basal activity.

Proc Natl Acad Sci U S A, 1989 May, 86(10), 3713 - 7
Specificity of mismatch repair following transformation of Saccharomyces cerevisiae with heteroduplex plasmid DNA; Bishop DK et al.; A method is described for genetic detection of mismatch repair products following transformation of Saccharomyces cerevisiae . The method is based on the detection of beta-galactosidase activity in clonal derivatives of cells transformed with heteroduplex plasmid DNA . Heteroduplex plasmid substrates were constructed by insertion of an oligonucleotide heteroduplex into the coding sequence of the Escherichia coli lacZ gene . The plasmid and oligonucleotides were designed so that one strand of the construct would code for a functional beta-galactosidase and the other strand would contain an in-frame nonsense codon . The frequencies of transformed clones containing only Lac+ cells, only Lac- cells, or a mixture of the two Lac phenotypes provided information on the efficiency of the repair reaction . With this method, plasmids carrying single-base substitution mismatches, a single-base frameshift mismatch (T/delta), or a 3-base-pair substitution mismatch (TGA/GAA) were tested . A/C, G/T, G/A, G/G, and T/delta mismatches were repaired with significantly greater efficiencies than C/C, A/A, T/T, and TGA/GAA . T/C was repaired with an intermediate efficiency . The frequencies of products obtained with G/G, G/A, and T/delta mismatches suggested modest inequality of repair in the two possible directions . Strains carrying the repair-deficient pms1-1 mutation were also tested . The efficiencies of repair of A/C, G/T, G/G, and A/A mismatches were reduced in pms1-1 cells compared with wild-type cells . In addition, a change in repair inequality was detected when transformation of the two strains with an A/C mismatch was compared.

Genetics, 1989 May, 122(1), 29 - 46
Genetic and molecular characterization of suppressors of SIR4 mutations in Saccharomyces cerevisiae; Schnell R et al.; In order to learn more about other proteins that may be involved in repression of HML and HMR in Saccharomyces cerevisiae, extragenic suppressor mutations were identified that could restore repression in cells defective in SIR4, a gene required for function of the silencer elements flanking HML and HMR . These suppressor mutations, which define at least three new genes, SAN1, SAN2 and SAN3, arose at the frequency expected for loss-of-function mutations following mutagenesis . All san mutations were recessive . Suppression by san1 was allele-nonspecific, since san1 could suppress two very different alleles of SIR4, and was locus-specific since san1 was unable to suppress a SIR3 mutation or a variety of mutations conferring auxotrophies . The SAN1 gene was cloned, sequenced, and used to construct a null allele . The null allele had the same phenotype as the EMS-induced mutations and exhibited no pleiotropies of its own . Thus, the SAN1 gene was not essential . SAN1-mediated suppression was neither due to compensatory mutations in interacting proteins, nor to translational missense suppression . SAN1 may act posttranslationally to control the stability or activity of the SIR4 protein.

Biochem Biophys Res Commun, 1989 Apr 28, 160(2), 525 - 34
Evidence for the presence of DNA primase in mitochondria of Saccharomyces cerevisiae; Desai SD et al.; Chromatography of a DNA polymerase preparation from mitochondria of Saccharomyces cerevisiae on DNA-cellulose column, using Tris-HCl (pH 7.5) buffer containing 0.6 M NaCl as eluent, was found to yield a fraction exhibiting DNA primase-like activity free of DNA polymerase . This fraction could support the synthesis of 12-15 residue-long oligoribonucleotides on single-stranded natural or synthetic DNA templates . The oligoribonucleotides could be further elongated by incorporation of deoxyribonucleotides in the presence of Klenow fragment.

J Biol Chem, 1989 Apr 25, 264(12), 6979 - 83
Molecular cloning and sequencing of genomic DNA encoding aminopeptidase I from Saccharomyces cerevisiae; Chang YH et al.; Yeast aminopeptidase I is a vacuolar enzyme, which catalyzes the removal of amino acids from the NH2 terminus of peptides and proteins (Frey, J., and Rohm, K-H . (1978) Biochim . Biophys . Acta 527, 31-41) . A yeast genomic DNA encoding aminopeptidase I was cloned from a yeast EMBL3A library and sequenced . The DNA sequence encodes a precursor protein containing 514 amino acid residues . The "mature" protein, whose NH2-terminal sequence was confirmed by automated Edman degradation, consists, based only on the DNA sequence, of 469 amino acids . A 45-residue presequence contains positively and negatively charged as well as hydrophobic residues, and its NH2-terminal residues could be arrayed in an amphiphilic alpha-helix . This presequence differs from the signal sequences which direct proteins across bacterial plasma membranes and endoplasmic reticulum or into mitochondria . It remains to be established how this unique presequence targets aminopeptidase I to yeast vacuoles and how this sorting utilizes classical protein secretory pathways . Further, the aminopeptidase I gene, localized previously by genetic mapping to yeast chromosome XI and called the LAP4 gene (Trumbly, R . J., and Bradley, G . (1983) J . Bacteriol . 156, 36-48), was determined by DNA blot analyses to be a single copy gene located on chromosome XI.

FEBS Lett, 1989 Apr 24, 247(2), 235 - 8
Leucine transport in plasma membrane vesicles of Saccharomyces cerevisiae; Calahorra M et al.; Yeast plasma membrane vesicles were obtained by the fusion of liposomes with purified yeast membranes by means of the freeze thaw-sonication technique . Beef heart mitochondria cytochrome-c oxidase was incorporated into the vesicles . Addition of substrate (ascorbate/TMPD/cytochrome c) generated a membrane potential negative inside, and an alkaline pH gradient inside the vesicle, that served as the driving force for leucine transport . Both delta pH and delta psi could drive leucine transport . When delta pH was increased in the presence of valinomycin and potassium, at the expense of delta psi, leucine uptake increased by 10%.

J Biol Chem, 1989 Apr 15, 264(11), 6557 - 65
Purification of DNA polymerase II, a distinct DNA polymerase, from Saccharomyces cerevisiae; Budd ME et al.; Yeast DNA polymerases I and III have been well characterized physically, biochemically, genetically and immunologically . DNA polymerase II is present in very small amounts, and only partially purified preparations have been available for characterization, making comparison with DNA polymerases I and III difficult . Recently, we have shown that DNA polymerases II and III are genetically distinct (Sitney et al., 1989) . In this work, we show that polymerase II is also genetically distinct from polymerase I, since polymerase II can be purified in equal amounts from wild-type and mutant strains completely lacking DNA polymerase I activity . Thus, yeast contains three major nuclear DNA polymerases . The core catalytic subunit of DNA polymerase II was purified to near homogeneity using a reconstitution assay . Two factors that stimulate the core polymerase were identified and used to monitor activity during purification and analysis . The predominant species of the most highly purified preparation of polymerase II is 132,000 Da . However, polymerase activity gels suggest that the 132,000-Da form of DNA polymerase II is probably an active proteolytic fragment derived from a 170,000-Da protein . The highly purified polymerase fractions contain a 3'----5'-exonuclease activity that purifies at a constant ratio with polymerase during the final two purification steps . However, DNA polymerase II does not copurify with a DNA primase activity.

Eur J Biochem, 1989 Apr 15, 181(1), 243 - 7
Hormone-induced expression of the CHS1 gene from Saccharomyces cerevisiae; Appeltauer U et al.; When MATa cells of Saccharomyces cerevisiae have been treated with the mating hormone alpha-factor an increase in chitin synthase zymogen, as well as chitin content in the cell-wall fraction, have been reported . With a DNA probe derived from the cloned CHS1 gene that codes for chitin synthase I {Bulawa, C . E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W . L . and Robbins, P . (1986) Cell 46, 213-225} a Northern analysis was conducted of CHS1-specific transcripts . alpha-Factor-treated MATa cells revealed more than sixfold elevated steady-state levels of CHS1 mRNA as compared to control cells . MAT alpha cells responded the same way when treated with a-factor although induction rate was somewhat smaller . After hormone application a rapid increase in CHS1 mRNA levels could be observed that occurred also in the absence of ongoing protein synthesis . In order to minimize possible side effects of CHS1-coding sequences on expression and mRNA stability a CHS1::SUC2 chimaeric gene was constructed where 730 bp of the CHS1 promoter region (+20 bp of the coding region) were fused in frame to a fragment of the SUC2 coding region . The fusion protein exhibits invertase activity that has been used to monitor CHS1 promoter activity . By analysis of shortened versions of the CHS1 promoter a 94-bp DNA fragment has been identified that confers hormone inducibility to the CHS1 promoter . According to the published sequence of the CHS1 gene, this fragment contains four repeats of a TGAAACA consensus sequence previously identified in the alpha-factor-inducible BAR1 promoter {Kronstad, J . W., Holly, J . A . and MacKay, V . L . (1987) Cell 50, 369-377} . This heptamer may represent the cis-acting element involved in mating-hormone-mediated gene expression in yeast.

Biochemistry, 1989 Apr 4, 28(7), 2856 - 62
Purification and characterization of a protein from Saccharomyces cerevisiae that binds tightly to single-stranded DNA and stimulates a cognate strand exchange protein; Heyer WD et al.; A single-stranded DNA binding protein (yeast SSB protein) was purified to near-homogeneity from mitotic Saccharomyces cerevisiae cells . The Mr 34,000 protein specifically eluted at high salt (approximately 1200 mM NaCl) during chromatography on a single-stranded DNA-cellulose column . The protein formed stable complexes with single-stranded DNA in an apparent cooperative fashion . As judged from titration and competition experiments, the affinity of the protein was much higher for single-stranded DNA than for double-stranded DNA or single-stranded RNA . The SSB protein also was found to stimulate the strand exchange reaction between linear M13mp19 RF DNA and circular M13mp19 viral DNA as catalyzed by a yeast strand exchange protein previously purified in this laboratory {Kolodner, R., Evans, D . H., & Morrison, P . T . (1987) Proc . Natl . Acad . Sci . U.S.A . 84, 5660-5664} . Titration experiments showed maximum stimulation of joint molecule formation at a stoichiometry of about 1 Mr 34,000 monomer yeast SSB per 18 nucleotides of single-stranded DNA . Kinetic experiments demonstrated at least an 18-fold increase in the rate of strand exchange due to the presence of the SSB in reactions where the amount of strand exchange protein was limiting . The yeast SSB protein stimulated the Escherichia coli RecA protein in the strand exchange reaction involving linear M13mp19 RF DNA and circular M13mp19 viral DNA as efficiently as E . coli SSB . However, the E . coli SSB protein did not substitute for the yeast SSB protein in reactions with the yeast strand exchange protein . This suggests that the stimulation of the yeast strand exchange protein by the yeast SSB may involve specific protein/protein interactions.

J Gen Microbiol, 1989 Apr, 135 ( Pt 4), 1037 - 45
Host-plasmid interactions in Saccharomyces cerevisiae: effect of host ploidy on plasmid stability and copy number; Spalding A et al.; The segregational stability of two chimaeric plasmids has been examined in an isogenic series of haploid, diploid and tetraploid strains of Saccharomyces cerevisiae, constructed by transformation-associated spheroplast fusion . For the highly unstable, ARS-based plasmid YRp7M, a significant increase in its segregational stability was observed with increasing ploidy, while the relatively stable, 2 microns-based plasmid pMA3a showed only a small increase in stability in strains of higher ploidy . The copy number of both pMA3a and the endogenous 2 microns plasmid increased in proportion with the host cell ploidy, while the copy number of TRp7M was increased in the higher ploidy strains but did not correlate with ploidy . These results suggest that the copy numbers of both the 2 microns plasmid and a plasmid derived from it are controlled by a nuclear gene and that, in addition, there are 2 microns sequences, other than those required for the FLP-mediated recombination system, that play a role in maintaining copy number.

Yeast, 1989 Apr, 5 Spec No, S441 - 5
Evidence for two mechanisms of flocculation in Saccharomyces cerevisiae; Stratford M; Inhibition of flocculation by sugars was studied in 41 strains of Saccharomyces cerevisiae . Two distinct groupings were found . The first type, including all strains containing FLO 1, FLO 4, FLO 5, FLO 8 and TUP 1, were partially inhibited by mannose only . The second type (NewFLO) were completely inhibited by mannose, maltose, glucose and sucrose . Sugar inhibitions were reversible and were exacerbated by increased agitation . NewFLO strains were more sensitive to inhibition by inorganic salts than the FLO 1 type strains . This inhibition is probably chaotropic in nature . Most FLO 1 type strains are constitutive, expressing flocculation throughout growth, whereas, NewFLO strains are completely repressed by the presence of excess ammonium ions . It is proposed that this indicates two entirely distinct mechanisms of flocculation, probably involving a mannose-specific lectin in the FLO 1 type and a broad specificity lectin in the NewFLO type.

Yeast, 1989 Apr, 5 Spec No, S287 - 91
Isolation and characterization of Saccharomyces cerevisiae mutants resistant to sulphite; Casalone E et al.; Several spontaneous and UV-induced sulphite resistant mutants of Saccharomyces cerevisiae have been isolated and characterized . Some of the UV-induced mutants appeared to be much more resistant than the spontaneous ones, as judged by their plating efficiency and cellular growth in the presence of increasing concentrations of sulphite . All the resistant mutants seemed to have an intracellular glutathione content and glutathione reductase activity higher than and an extracellular glutathione concentration lower than the parental strain.

Mol Cell Biol, 1989 Apr, 9(4), 1659 - 66
Molecular analysis of GPH1, the gene encoding glycogen phosphorylase in Saccharomyces cerevisiae; Hwang PK et al.; In yeast cells, the activity of glycogen phosphorylase is regulated by cyclic AMP-mediated phosphorylation of the enzyme . We have previously cloned the gene for glycogen phosphorylase (GPH1) in Saccharomyces cerevisiae . To assess the role of glycogen and phosphorylase-catalyzed glycogenolysis in the yeast life cycle, yeast strains lacking a functional GPH1 gene or containing multiple copies of the gene were constructed . GPH1 was found not to be an essential gene in yeast cells . Haploid cells disrupted in GPH1 lacked phosphorylase activity and attained higher levels of intracellular glycogen but otherwise were similar to wild-type cells . Diploid cells homozygous for the disruption were able to sporulate and give rise to viable ascospores . Absence of functional GPH1 did not impair cells from synthesizing and storing trehalose . Increases in phosphorylase activity of 10- to 40-fold were detected in cells carrying multiple copies of GPH1-containing 2 microns plasmid . Northern (RNA) analysis indicated that GPH1 transcription was induced at the late exponential growth phase, almost simultaneous with the onset of intracellular glycogen accumulation . Thus, the low level of glycogen in exponential cells was not primarily maintained through regulating the phosphorylation state of a constitutive amount of phosphorylase . GPH1 did not appear to be under formal glucose repression, since transcriptional induction occurred well in advance of glucose depletion from the medium.

Proc Natl Acad Sci U S A, 1989 Apr, 86(8), 2530 - 4
Multiple states of protein-DNA interaction in the assembly of transcription complexes on Saccharomyces cerevisiae 5S ribosomal RNA genes; Braun BR et al.; Multiple stages of protein-DNA interaction in the assembly of RNA polymerase III transcription complexes on a Saccharomyces cerevisiae 5S rRNA gene have been distinguished by DNase I "footprinting" and gel retardation . Transcription factor IIIA interacts with approximately 35 base pairs of the internal promoter region . Transcription factors IIIC and IIIB incrementally extend the interaction along the 5S gene, if, and only if, transcription factor IIIA is also bound . Complexes assembled from the complete set of purified transcription factors or from a complete transcription system extend over the entire transcription unit together with almost 50 base pairs of 5' flanking sequence.

J Biochem (Tokyo), 1989 Apr, 105(4), 588 - 93
Characterization of NADH kinase from Saccharomyces cerevisiae; Iwahashi Y et al.; At least two enzymes that phosphorylate diphosphopyridine nucleotides were detected in Saccharomyces cerevisiae: NADH-specific kinase was localized exclusively in the mitochondria, and NAD+-specific kinase was distributed in the microsomal and cytosol fractions but not in the mitochondria . The identity of NAD+ kinase detected in the two fractions remains equivocal . NADH kinase was highly purified 1,041-fold from the mitochondrial fraction . The Km values for NADH and ATP were 105 microM and 2.1 mM, respectively . The relative molecular mass was estimated to be 160,000 by means of molecular sieve chromatography . From inactivation studies with SH inhibitors and protection by NADH, it was demonstrated that a cysteine residue is involved in the binding site of NADH.

Biochemistry, 1989 Mar 21, 28(6), 2728 - 32
Important role of the proline residue in the signal sequence that directs the secretion of human lysozyme in Saccharomyces cerevisiae; Yamamoto Y et al.; To elucidate the role of the proline residue in the engineered signal sequence that directs the secretion of human lysozyme in Saccharomyces cerevisiae, we have remodeled an idealized signal sequence L8 = Met-Arg-(Leu)8-Pro-Leu-Ala-Ala-Leu-Gly {Yamamoto, Y., Taniyama, Y., Kikuchi, M., & Ikehara, M . (1987) Biochem . Biophys . Res . Commun . 149, 431-436} in the vicinity of the proline residue . By analyzing the secretory capability of 10 engineered signal sequences, we have shown the following . (1) The proline residue is important for the secretion of human lysozyme and is allowed at position -4, -5, or -6 . (2) The secretory capability of the engineered signal sequences is correlated with their predicted conformations . (3) The functional signal sequences that we have investigated can be generalized as follows: Met-Arg-(Leu)n-Pro-(Xaa)-Ala-Leu-Gly where n equals 6-12 and Xaa is Leu, Ala, or Leu-Ala or can be omitted.

Gene, 1989 Mar 15, 76(1), 89 - 97
Distribution of introns in frameshift-suppressor proline-tRNA genes of Saccharomyces cerevisiae; Winey M et al.; Mutations in the suf9, suf10, and suf11 genes of yeast suppress + 1 nucleotide (nt) insertions in proline codons . Nucleotide sequence analysis indicates that the suf9 and suf11 genes are members of the proline tRNA(UGG) gene family, which also includes three other previously identified genes, suf7, suf8, and trn1 . All five members of this gene family contain introns . The suf9 and suf11 introns are 31 and 30 nt in length, respectively, and are similar but not identical in sequence to other introns within the family . The suf10 gene is identical in sequence to suf2, which was shown previously to encode proline tRNA(IGG) . Both members of this gene family lack introns . Alleles of suf9, suf10, and suf11 that confer frameshift suppression were also analyzed . The SUF9-1 allele results in a G----U substitution at nt position 39 in the anticodon stem . The recessive suf11-1 allele is a double mutant containing the same nt position 39 alteration as in SUF9-1 plus a second U----A substitution at nt position 38 in the anticodon loop . The SUF10-1 suppressor mutation corresponds to a +1G insertion in the anticodon loop . Since the nt substitutions in suf11-1 alter the sequence of the 3' exon/intron boundary, the double mutant pre-tRNA was tested for its ability to be cleaved in vitro by tRNA-splicing endonuclease . It was found that suf11-1 pre-tRNA is cleaved with reduced efficiency at the 3' splice junction.

J Biol Chem, 1989 Mar 15, 264(8), 4412 - 6
Inducible overexpression, purification, and active site mapping of DNA topoisomerase II from the yeast Saccharomyces cerevisiae; Worland ST et al.; Overexpression of yeast DNA topoisomerase II was achieved by placing the coding sequences of the gene TOP2 downstream of an inducible promoter PGAL1 on a multicopy plasmid . By using a simple purification procedure, milligram amounts of the enzyme of a high specific activity can be obtained from a few liters of culture . In the presence of a drug VM-26 (teniposide), more than 90% of the enzyme molecules become covalently bound to DNA upon addition of the protein denaturant sodium dodecyl sulfate . The formation of the covalent complex was used to map the tyrosine residue that becomes covalently linked to DNA when the enzyme transiently breaks DNA . After exhaustive digestion of the DNA-protein complex with trypsin, a DNA-linked peptide was purified and sequenced directly to identify Tyr-783 as the active site residue.

FEBS Lett, 1989 Mar 13, 245(1-2), 261 - 6
Circular dichroism and fluorescence studies on five mutant forms of protein synthesis initiation factor eIF-4E, from the yeast Saccharomyces cerevisiae; McCubbin WD et al.; CD studies have shown that five tryptophan to phenylalanine (W----F) mutants of eukaryotic initiation factor-4E (eIF-4E) contain low amounts of alpha-helix, the main elements of secondary structure being beta-sheets/turns and aperiodic regions . Interactions with the cap analog m7GpppG are accompanied by changes in overall secondary structure which include reductions, and in one case an increase in alpha-helix content, as well as increases in total beta-structure (3 mutant forms) and decreases in total beta-structure (2 mutant forms) . These changes may also involve more significant perturbations of localized regions containing phenylalanine residues either involved in nucleotide binding, or close to the nucleotide-binding site . Measurements of intrinsic Trp fluorescence have shown different quantum yields and reduced m7GpppG-induced quenching (with one exception) . Acrylamide quenching studies yielded similar parameters for 4 of the mutants but 1 form displayed significantly reduced values . Melting experiments showed that the Trp fluorescence of 4 of the mutants decreased as the temperature was increased, this effect being reduced in 3 cases in the presence of m7GpppG . W 58 F showed an increase in fluorescence as the temperature was raised and this effect was accentuated in the presence of nucleotide . A preliminary attempt has been made to correlate the spectroscopic data with the known biological importance of the individual Trp residues.

Biochim Biophys Acta, 1989 Mar 13, 979(3), 375 - 7
Effect of the chronic ethanol action on the activity of the general amino-acid permease from Saccharomyces cerevisiae var . ellipsoideus; Ferreras JM et al.; The presence of ethanol and cycloheximide during growth were found to inhibit the function of the general amino-acid permease of Saccharomyces cerevisiae var . ellipsoideus . Contrary to cycloheximide, the effect of ethanol upon growth in alcohol-free medium was reversible . The effect of both inhibitors could be explained in terms of reduction of the number of active carrier molecules located in the plasma membrane.

FEBS Lett, 1989 Mar 13, 245(1-2), 131 - 6
Cyclic AMP controls the plasma membrane H+-ATPase activity from Saccharomyces cerevisiae; Ulaszewski S et al.; The thermosensitive G1-arrested cdc35-10 mutant from Saccharomyces cerevisiae, defective in adenylate cyclase activity, was shifted to restrictive temperature . After 1 h incubation at this temperature, the plasma membrane H+-ATPase activity of cdc35-10 was reduced to 50%, whereas that in mitochondria doubled . Similar data were obtained with cdc25, another thermosensitive G1-arrested mutant modified in the cAMP pathway . In contrast, the ATPase activities of the G1-arrested mutant cdc19, defective in pyruvate kinase, were not affected after 2 h incubation at restrictive temperature . In the double mutants cdc35-10 cas1 and cdc25 cas1, addition of extracellular cAMP prevented the modifications of ATPase activities observed in the single mutants cdc35-10 and cdc25 . These data indicate that cAMP acts as a positive effector on the H+-ATPase activity of plasma membranes and as a negative effector on that of mitochondria.

J Biol Chem, 1989 Mar 5, 264(7), 3723 - 31
Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae . II . Biochemical characterization of temperature-sensitive RIP1- mutations; Ljungdahl PO et al.; Although the function of the Rieske iron-sulfur protein is generally understood, little is known of how the structure of this protein supports its mechanistic role in electron transfer in the cytochrome bc1 complex . To better understand the structural basis of iron-sulfur protein function, we have undertaken a mutational analysis of the gene encoding this protein and initially isolated five temperature-sensitive iron-sulfur protein mutants (Beckmann, J . D., Ljungdahl, P . O., and Trumpower, B . L . (1989) J . Biol . Chem . 264, 3713-3722) . Each of the five ts-rip1- mutants exhibited pleiotropic effects . Although the mutant iron-sulfur proteins manifest several in vitro phenotypes in common, each exhibited unique characteristics . All of the ts-rip1- mutations resulted in membranes with decreased ubiquinol-cytochrome c oxidoreductase activities and decreased thermostability compared to membranes containing wild type iron-sulfur protein . All of the mutations conferred slight but significant resistance to the respiratory inhibitor myxothiazol, and one mutant was hypersensitive to inhibition by UHDBT, a structural analog of ubiquinone . In addition, one of the mutations completely blocks post-translational processing of the iron-sulfur protein, leading to accumulation of pre-iron-sulfur protein in mitochondrial membranes at nonpermissive temperatures . Finally, a mutation 12-amino acid residues away from the carboxyl terminus (203S) results in an extremely unstable protein . This region of the protein may be essential in blocking degradation of pre-iron-sulfur protein by cytoplasmic proteases as the protein is imported into the mitochondria, or may be a "degradation signal," which tags the iron-sulfur protein for turnover.

Nature, 1989 Mar 2, 338(6210), 35 - 9
An initiation site for meiotic gene conversion in the yeast Saccharomyces cerevisiae; Nicolas A et al.; An initiation site for meiotic gene conversion has been identified in the promoter region of the ARG4 gene of Saccharomyces cerevisiae . The chromosome on which initiation occurs is the recipient of genetic information during gene conversion.

J Biochem (Tokyo), 1989 Mar, 105(3), 417 - 22
Expression of bovine myoglobin cDNA as a functionally active holoprotein in Saccharomyces cerevisiae; Shimada H et al.; We isolated a cDNA clone for myoglobin mRNA from fetal bovine skeletal muscle using a DNA fragment of human myoglobin exon 2 as a probe . The complete coding sequence of myoglobin as well as the 3'- and part of the 5'-nontranslatable sequences (546 and 66 basepairs, respectively) were determined . The amino acid sequence predicted from the nucleotide sequence was in agreement with that determined in the purified protein from adult bovine cardiac muscle (Han, K . K., Dautrevaux, M., Chaila, X., & Biserte, G . {1970} Eur . J . Biochem . 16, 465-471), except for eight amino acid residues: Val-99----Ile,Ile-101----Val, Asn-122----Asp, Ala-124----Gly, Gly-129----Ala, Ala-142----Met, Glu-144----Ala, and Lys-145----Gln . When the myoglobin cDNA was expressed in Saccharomyces cerevisiae under the control of the GAL7 promoter, myoglobin was synthesized as a functionally active holoprotein which bound molecular oxygen reversibly . The amount of myoglobin reached nearly 1% of the total extractable protein in the yeast . N-terminal sequence analysis of the produced myoglobin revealed a glycine residue at the terminus, indicating that as in native muscle the N-terminal Met was removed in yeast by processing.

Mol Cell Biol, 1989 Mar, 9(3), 1368 - 70
Acquisition and processing of a conditional dicentric chromosome in Saccharomyces cerevisiae; Hill A et al.; The introduction of a conditional centromere into chromosome III of Saccharomyces cerevisiae provided an opportunity to evaluate phenotypic and karyotypic consequences in cells harboring dicentric chromosomes upon entry into mitosis . A mitotic pause ensued, and monocentric derivatives of chromosome III were generated at a high frequency.

Mol Cell Biol, 1989 Mar, 9(3), 1191 - 9
Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae; Bernstein M et al.; When incubated at a restrictive temperature, Saccharomyces cerevisiae sec59 mutant cells accumulate inactive and incompletely glycosylated forms of secretory proteins . Three different secretory polypeptides (invertase, pro-alpha-factor, and pro-carboxypeptidase Y) accumulated within a membrane-bounded organelle, presumably the endoplasmic reticulum, and resisted proteolytic degradation unless the membrane was permeabilized with detergent . Molecular cloning and DNA sequence analysis of the SEC59 gene predicted an extremely hydrophobic protein product of 59 kilodaltons . This prediction was confirmed by reconstitution of the sec59 defect in vitro . The alpha-factor precursor, which was translated in a soluble fraction from wild-type cells, was translocated into, but inefficiently glycosylated within, membranes from sec59 mutant cells . Residual glycosylation activity of membranes of sec59 cells was thermolabile compared with the activity of wild-type membranes . Partial restoration of glycosylation was obtained in reactions that were supplemented with mannose or GDP-mannose, but not those supplemented with other sugar nucleotides . These results were consistent with a role for the Sec59 protein in the transfer of mannose to dolichol-linked oligosaccharide.

Mol Cell Biol, 1989 Mar, 9(3), 1049 - 59
Dominant effects of tubulin overexpression in Saccharomyces cerevisiae; Burke D et al.; The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters . Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure . Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation . The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate . Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin . Transient overexpression of both tubulins resulted in a high frequency of chromosome loss . These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin . Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency . These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.

Genetics, 1989 Mar, 121(3), 477 - 89
Mutations in CEN3 cause aberrant chromosome segregation during meiosis in Saccharomyces cerevisiae; Gaudet A et al.; We investigated the structural requirements of the centromere from chromosome III (CEN3) of Saccharomyces cerevisiae by analyzing the ability of chromosomes with CEN3 mutations to segregate properly during meiosis . We analyzed diploid cells in which one or both copies of chromosome III carry a mutant centromere in place of the wild-type centromere and found that some alterations in the length, base composition and primary sequence characteristics of the central A+T-rich region (CDE II) of the centromere had a significant effect on the ability of the chromosome to segregate properly through meiosis . Chromosomes containing mutations which delete a portion of CDE II showed a high rate of premature disjunction at meiosis I . Chromosomes containing point mutations in CDE I or lacking CDE I appeared to segregate properly through meiosis; however, plasmids carrying centromeres with CDE I completely deleted showed an increased frequency of segregation to nonsister spores.

Genetics, 1989 Mar, 121(3), 463 - 76
Pheromones and pheromone receptors are the primary determinants of mating specificity in the yeast Saccharomyces cerevisiae; Bender A et al.; Saccharomyces cerevisiae has two haploid cell types, a and alpha, each of which produces a unique set of proteins that participate in the mating process . We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity . We show that the capacity to synthesize alpha-factor pheromone and a-factor receptor is sufficient to allow mating by mat alpha 1 mutants, mutants that normally do not express any alpha- or a-specific products . Likewise, the capacity to synthesize a-factor receptor and alpha-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells . Thus, the a-factor receptor and alpha-factor pheromone constitute the minimum set of alpha-specific proteins that must be produced to allow mating as an alpha cell . Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat alpha 2 mutants, cells that simultaneously express both pheromones and both receptors . We created a series of strains that express different combinations of pheromones and receptors in a mat alpha 2 background . These constructions reveal that mat alpha 2 mutants can be made to mate as either a cells or as alpha cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type . Moreover, these studies show that the inability of mat alpha 2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to alpha factor by the a-factor receptor.

Yeast, 1989 Mar-Apr, 5(2), 117 - 29
Mapping and characterizing a new DNA replication mutant in Saccharomyces cerevisiae; Eberly SL et al.; A detailed characterization of the mak1-3 mutation of Saccharomyces cerevisiae has been made possible by modifying its genetic background . The mak1-3 mutation, which confers temperature sensitivity for growth, was originally identified as one of four mak1 mutations (Wickner and Leibowitz, 1976) . Mak1-1, 1-2 and 1-4 mutants are deficient in DNA topoisomerase I activity and thus have been renamed 'top1' (Thrash et al., 1984) . Studies presented here show that the map position of MAK1-3 on chromosome XVI distinguishes it from TOP1 which maps on chromosome XV (Wickner and Leibowitz, 1976) . An investigation of in vivo macromolecular synthesis in the mak1-3 mutant shows that it is deficient in DNA replication at the restrictive temperature . Experiments in which DNA synthesis was measured in synchronized cell populations indicate that the mak1-3 mutant is deficient in the initiation step of DNA synthesis . Furthermore, crude extracts from the mak1-3 mutant cells support temperature-sensitive in vitro DNA synthesis on yeast chromosomal DNA replication origin containing plasmid pARS1, suggesting that the MAK1 gene product is directly required for in vitro DNA replication . The conclusion that mak1-3 is a newly identified DNA replication mutation is based on the observations that it (1) complements all DNA synthesis mutants examined, (2) maps to a previously undetected chromosomal location and (3) has a distinct terminal morphology . In light of these distinctions and of the role mak1-3 plays in DNA replication, it has been renamed 'dna1'.

J Bacteriol, 1989 Mar, 171(3), 1245 - 53
A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae; Schmidheini T et al.; The Saccharomyces cerevisiae ARO7 gene product chorismate mutase, a single-branch-point enzyme in the aromatic amino acid biosynthetic pathway, is activated by tryptophan and subject to feedback inhibition by tyrosine . The ARO7 gene was cloned on a 2.05-kilobase EcoRI fragment . Northern (RNA) analysis revealed a 0.95-kilobase poly(A)+ RNA, and DNA sequencing determined a 771-base-pair open reading frame capable of encoding a protein 256 amino acids . In addition, three mutant alleles of ARO7 were cloned and sequenced . These encoded chorismate mutases which were unresponsive to tyrosine and tryptophan and were locked in the on state, exhibiting a 10-fold-increased basal enzyme activity . A single base pair exchange resulting in a threonine-to-isoleucine amino acid substitution in the C-terminal part of the chorismate mutase was found in all mutant strains . In contrast to other enzymes in this pathway, no significant homology between the monofunctional yeast chorismate mutase and the corresponding domains of the two bifunctional Escherichia coli enzymes was found.

Biochimie, 1989 Mar, 71(3), 313 - 8
Comparative studies between the glucose-induced phosphorylation signal and the heat shock response in mutants of Saccharomyces cerevisiae; Panek AD et al.; Addition of glucose to derepressed yeast cells, as well as a heat shock treatment, trigger the phosphorylation of trehalase and of trehalose-6-phosphate synthase . In the present paper, evidence is provided for the requirement of the RAS protein in the transduction of the glucose signal . On the other hand, a heat shock at 52 degrees C for 2 min was able to produce a significant phosphorylating effect even in mutant strains deficient in the GTP binding protein . Moreover, it was shown that this treatment does not affect exclusively the cAMP-dependent protein kinase . The use of a series of mutant strains confirmed that low levels of cAMP favor thermotolerance; the role of trehalose in yeast viability is also discussed.

Mol Gen Genet, 1989 Mar, 216(1), 70 - 4
The bI4 RNA mitochondrial maturase of Saccharomyces cerevisiae can stimulate intra-chromosomal recombination in Escherichia coli; Goguel V et al.; When the bI4 RNA maturase, encoded by the fourth intron of the mitochondrial cytochrome b gene of Saccharomyces cerevisiae, was expressed in Escherichia coli, formation of intra-chromosomal Lac+ recombinants was stimulated threefold . This "hyper-rec" phenotype was recA as well as recBCD dependent . The most active form of the bI4 maturase stimulated homologous recombination whereas splicing deficient mutants of bI4 maturase were either deficient in or unable to stimulate homologous recombination.

Yeast, 1989 Mar-Apr, 5(2), 79 - 90
Cloning of CDC33: a gene essential for growth and sporulation which does not interfere with cAMP production in Saccharomyces cerevisiae; Verdier JM et al.; The CDC33 gene of Saccharomyces cerevisiae belongs to the class II 'START' genes . Its product is required for the initiation of a new cell division cycle (Hartwell, 1974) . Many results suggest that the cAMP signalling pathway is one of the major controlling elements of 'START' . Components of this pathway are encoded by class II 'START' genes . The aim of the present study is to determine whether or not the CDC33 gene interferes with the cAMP signalling pathway . We report here the molecular cloning of the CDC33 gene by complementation of the cdc33-1 thermosensitive mutant . The identity of the cloned gene is confirmed by site-specific reintegration and segregation analysis . This gene is transcribed into a 900-nucleotides mRNA and appears to be relatively abundant in the cell . We also show that the CDC33 gene product is essential for sporulation . cdc33-1 mutant cells are able to enter into the resting state . The cAMP intracellular pool is not modified when the cdc33-1 mutant is shifted to the restrictive temperature . The cdc33-1 mutation is not suppressed by other known elements of the cAMP cascade . All these results suggest that the CDC33 'START' gene does not interfere with the cAMP signalling pathway which controls cell division.

J Bacteriol, 1989 Mar, 171(3), 1417 - 22
Saccharomyces cerevisiae protein kinase dependent on Ca2+ and calmodulin; Miyakawa T et al.; A Ca2+- and calmodulin (CaM)-dependent protein kinase of Saccharomyces cerevisiae was partially purified by CaM affinity chromatography of the soluble fraction, and the properties of the enzyme were investigated . The protein kinase activity of the affinity-purified preparation was stimulated at least eightfold by the simultaneous presence of Ca2+ and CaM . The enzyme stimulation was strongly inhibited by trifluoperazine (TFP), a CaM antagonist . When the kinase was incubated in the presence of ATP, Ca2+, and CaM before the assay, the enzyme showed activity even in the presence of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and TFP . The conversion to this Ca2+- and CaM-independent form occurred very rapidly under the incubation conditions required for protein phosphorylation by the kinase . At the highest level of conversion, Ca2+- and CaM-independent kinase activity, which was measured in the presence of EGTA and TFP, was nearly equal to the total kinase activity, which was measured in the presence of Ca2+ and CaM . A protein with a molecular weight of 58,000 was the major species that was phosphorylated in a Ca2+- and CaM-dependent manner by incubation of the CaM affinity-purified proteins with {gamma-32P}ATP . The protein kinase activity of the protein with the same molecular weight was demonstrated by in situ protein phosphorylation in sodium dodecyl sulfate-polyacrylamide gels by using casein as the substrate, after removal of the detergent from electrophoresed CaM-binding proteins . These data indicate that phosphorylation of the kinase is responsible for the conversion of enzyme activity . Enzyme regulation by this mode may play an important role in integrating cellular functions during the cell cycle . A possible role for the Ca2+-and CaM-dependent protein kinase in the signal transduction of the mating pheromone alpha factor is also discussed.

Biochem Biophys Res Commun, 1989 Feb 28, 159(1), 305 - 11
Characterization of KEX2-encoded endopeptidase from yeast Saccharomyces cerevisiae; Mizuno K et al.; Yeast Saccharomyces cerevisiae KEX2 gene previously isolated was characterized as the gene encoding an endopeptidase required for proteolytic processing of precursors of alpha-factor and killer toxin . In this study, the cloned KEX2 gene was introduced into the kex2 mutant cells and the KEX2 gene product expressed in these cells was partially purified from their membrane fraction . The enzyme preparation exhibits a calcium-dependent endopeptidase activity with a substrate specificity toward the carboxyl side of Lys-Arg, Arg-Arg and Pro-Arg sequences . The enzyme activity was inhibited by serine-protease inhibitors, such as DFP and PMSF, indicating that the KEX2 endopeptidase belongs to a serine-protease family . The optimal pH was determined to be around 5.5 . Thus, the KEX2 endopeptidase was found to be a unique calcium-dependent serine-protease distinct from calpain and trypsin.

FEBS Lett, 1989 Feb 27, 244(2), 427 - 32
The isolation and reconstitution of the ADP/ATP carrier from wild-type Saccharomyces cerevisiae . Identification of primarily one type (AAC-2); Knirsch M et al.; Methods for isolation of the ADP/ATP carrier (AAC) from yeast (Saccharomyces cerevisiae) are described which allow separation of the carrier from the initially copurified porin which poses a specific problem in yeast . The procedure varies according to whether one wishes to obtain a stable CAT-AAC complex, the free and active AAC for reconstitution, or the SDS-denatured pure AAC peptide . CNBr cleavage of AAC enabled us to differentiate clearly between isogenes AAC-1 and AAC-2 recently found in yeast, due to the exclusive occurrence of a methionine (M-115) residue at the end of the first domain in AAC-2 . Thus the AAC isolated from wild-type yeast is primarily or exclusively AAC-2 . The isolated AAC is active in ADP/ATP exchange in reconstituted liposomes with a Vmax of 1100 mumol/min per g protein and Km = 15 microM for ADP, and a Vmax of 900 mumol/min per g protein and Km = 9 microM for ATP.

FEBS Lett, 1989 Feb 27, 244(2), 397 - 401
Functional molecular masses of vacuolar membrane H+-ATPase from Saccharomyces cerevisiae as studied by radiation inactivation analysis; Hirata R et al.; The functional molecular masses of the vacuolar membrane H+-ATPase in Saccharomyces cerevisiae under two kinetic conditions for ATP hydrolysis were measured by radiation inactivation . When vacuolar membrane vesicles were exposed to gamma-rays from 60Co, the activities catalyzing a single-cycle and multi-cycles of ATP hydrolysis both decreased as single-exponential functions of the radiation dosage . By applying the target theory, the functional molecular masses for single- and multi-cycle hydrolyses of ATP were determined to be approx . 0.9-1.1 X 10(5) and 4.1-5.3 X 10(5) Da, respectively . N,N'-Dicyclohexylcarbodiimide (DCCD) did not inhibit the former reaction but strongly inhibited the latter . It is suggested that the ATPase with a minimal composite of subunits a and b, in which subunit c is not necessarily involved operationally, can catalyze single-cycle hydrolysis of ATP, whereas for multi-cycle hydrolysis of ATP, the ATPase requires a properly organized oligomeric structure with subunits a-c, which may direct a positive cooperative mechanism of ATP hydrolysis and coupled H+ translocation in a DCCD-sensitive manner.

J Biol Chem, 1989 Feb 25, 264(6), 3116 - 21
Activation of phosphatidylinositol kinase and phosphatidylinositol-4-phosphate kinase by cAMP in Saccharomyces cerevisiae; Kato H et al.; In Saccharomyces cerevisiae, cAMP-dependent phosphorylation plays an essential role at the start of the cell cycle . It has also recently been demonstrated that the breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol is a requisite process for cell proliferation (Uno, I., Fukami, K., Kato, H., Takenawa, T., and Ishikawa, T . (1988) Nature 333, 188-190) . To clarify the relationship between the cAMP- and inositol phospholipid-mediated signal transduction systems, alterations in the inositol phospholipid metabolism of cAMP mutants were examined . The incorporation of {32P}Pi into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was markedly reduced in ras2, which produces low levels of cAMP, and increased in bcy1, which produces cAMP-independent protein kinase . The incorporation of {32P}Pi into ATP and phosphatidylinositol (PI) was almost the same in wild type, ras1, ras2, and bcy1 yeast strains . The addition of exogenous cAMP to cyr1-2 caused a tremendous increase in {32P}Pi incorporation into PIP and PIP2 without any effect on incorporation into ATP and PI, suggesting that cAMP plays an important role in polyphosphoinositide synthesis . We therefore examined the activities of PI and PIP kinases, the enzymes that catalyze the sequential steps from PI to PIP2 via PIP . The activities of both kinases were found to be very low in the membranes of cry1-2 and ras2 but very high in the membranes of bcy1 and ras1 ras2 bcy1 strain cells . The addition of cAMP to cyr1-2 cells caused the activation of PI and PIP kinases . Furthermore, the treatment of membranes with cAMP or dibutyryl cAMP caused the activation of PI kinase in wild type, ras1, cry1-2, and ras2 strains, but not in bcy1 strain cells . The effect was most prominent in membranes from cyr1-2 and ras2 cells . These results show that cAMP-dependent phosphorylation enhances polyphosphoinositide synthesis through activation of PI and PIP kinase, an effect which may lead to the enhanced production of inositol 1,4,5-trisphosphate and diacylglycerol.

Biochim Biophys Acta, 1989 Feb 23, 994(3), 200 - 9
Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae; Pugh TA et al.; Glucoamylase (SGA) was purified approximately 250-fold from sporulating Saccharomyces cerevisiae cells . The partially purified enzyme was active against glycogen, starch, maltotriose and maltose . It exhibited maximum catalytic activity against glycogen at pH 5.5 . The enzyme appears to be glycosylated, because it bound to lentil-lectin Sepharose . SGA was expressed in vegetatively growing cells under the control of the GAL1 promoter, and the cellular location of the enzymatic activity determined by fractionation techniques . SGA was preferentially recovered in fractions which were enriched for the vacuolar hydrolases, carboxypeptidase Y and alpha-mannosidase.

J Mol Biol, 1989 Feb 20, 205(4), 647 - 58
FLP recombinase of the 2 microns circle plasmid of Saccharomyces cerevisiae bends its DNA target . Isolation of FLP mutants defective in DNA bending; Schwartz CJ et al.; The FLP recombinase is encoded by the yeast plasmid 2 microns circle and catalyses a site-specific recombination reaction that results in inversion of a segment of the 2 micron plasmid . We describe a method for the isolation of inactivating mutations in the FLP gene . The analysis of the recombination and binding activity of defective FLP proteins in vitro resulted in the identification of two classes of mutations: those that completely abolish FLP function by interfering with DNA binding and others that block recombination after the binding step . We have shown that FLP-mediated recombination is accompanied by bending of the DNA target and that mutations in the FLP recombinase that block bending also eliminate recombination.

J Biol Chem, 1989 Feb 15, 264(5), 2593 - 8
Molecular cloning and primary structure of the uracil-DNA-glycosylase gene from Saccharomyces cerevisiae; Percival KJ et al.; The structural gene for the Saccharomyces cerevisiae repair enzyme uracil-DNA-glycosylase (UNG1) was selected from a yeast genomic library in the multicopy vector YEp24 by complementation of the ung1-1 mutant in in vitro enzyme assays . The sequenced gene has an open reading frame which codes for a protein with molecular weight of 40,471 . The measured size of the mRNA of 1.25 kb is in agreement with the predicted molecular weight of the protein . The gene product was overproduced about 100-fold in strains carrying an UNG1 gene containing plasmid at 100-200 copies/cell . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cleared lysates from such an overproducing strain, followed by renaturation of enzyme activity from individual gel slices showed the presence of two enzymatic activities in comparable quantities with Mr values of 39,500 and 33,000, indicating that the full size protein is either readily degraded in vivo or is very sensitive to proteolytic digestion in vitro . The carboxyl-terminal two-thirds of the yeast uracil-DNA-glycosylase is highly homologous to the entire Escherichia coli enzyme (50% amino acid identity) . Genetic mapping experiments have localized the UNG1 gene on the left arm of chromosome XIII at 17 cM from the GAL80 locus proximal to the centromer . Deletions of the UNG1 gene are viable.

Nucleic Acids Res, 1989 Feb 11, 17(3), 1103 - 20
Organization and expression of the COX6 genetic locus in Saccharomyces cerevisiae: multiple mRNAs with different 3' termini are transcribed from COX6 and regulated differentially; Wright RM et al.; COX6 and its surrounding genetic locus have been characterized for the yeast Saccharomyces cerevisiae . Flanking genes are found closely spaced upstream and downstream of COX6 . The upstream gene and COX6 are transcribed from opposite strands and are separated by no more than 300 bp . COX6 is transcribed into three different size classes of mRNA (1000b, 830b, and 700b) differing in length in their 3' untranslated regions . All three classes of mRNAs are found on polysomes and, hence, are most likely translated . The different COX6 mRNAs vary in abundance during growth in rich media and are affected differentially as cells are shifted into media containing high or low glucose concentrations . The largest mRNA is much more susceptible to glucose repression/derepression than are the two smaller mRNAs, whereas the smallest RNA is preferentially accumulated during growth in rich media . These findings demonstrate that COX6 mRNAs with different 3'-termini are either synthesized differentially or differ in stability and suggest the existence of a complex system regulating COX6 expression.

J Biol Chem, 1989 Feb 5, 264(4), 1946 - 50
Glycoprotein biosynthesis in Saccharomyces cerevisiae . Characterization of alpha-1,6-mannosyltransferase which initiates outer chain formation; Romero PA et al.; A particulate fraction from the Saccharomyces cerevisiae mnn1 mutant was obtained after extracting a 115,000 x g pellet with 0.75% Triton X-100 . Incubation of this preparation with labeled Man8GlcNAc and Man9GlcNAc in the presence of GDP-mannose followed by high pressure liquid chromatography showed the formation of Man9GlcNAc and Man10GlcNAc, respectively . Analysis by high resolution 1H NMR of the products indicates that, in each case, the mannose residue added is alpha-1,6-linked to the alpha-1,6-mannose residue of the substrate as follows (where M represents mannose and Gn represents N-acetylglucosamine): (Formula: see text) . The mannosyltransferase therefore catalyzes the first step specific to the biosynthesis of the outer chain of yeast mannoproteins . The apparent Km values for both substrates are similar: 0.39 mM for Man8GlcNAc and 0.35 mM for Man9GlcNAc . The alpha-1,6-mannosyltransferase exhibits maximum activity between pH 7.1 and 7.6 in Tris maleate buffer, has an absolute requirement for Mn2+, and also requires Triton X-100 . These results indicate that removal of the alpha-1,2-linked mannose residue from Man9GlcNAc is not essential for the alpha-1,6-mannosyltransferase which initiates outer chain synthesis, at least when oligosaccharides are used as substrates in a cell-free system.

Biochem Int, 1989 Feb, 18(2), 429 - 38
Cloning of human cytochrome P-450 cDNA and its expression in Saccharomyces cerevisiae; Ohgiya S et al.; A human cytochrome P-450 cDNA clone, lambda hPA6, was isolated from a human adult liver cDNA library . The expression plasmid pHM3 delta 2 was constructed by insertion of lambda hPA6 and a synthetic DNA into the yeast mRNA and reduced carbon monoxide-difference spectra of the yeast microsomes revealed that only the yeast transformed by the expression plasmid pHM3 delta 2 produced foreign cytochrome P-450 . In addition, the expressed protein reacted with antiserum to P-450-HM2, a form probably identical with P-450MP, and showed a detectable level of mephenytoin 4-hydroxylase activity.

Curr Genet, 1989 Feb, 15(2), 129 - 34
Enhanced canavanine uptake is associated with nucleotide permeability in a thymidylate auxotroph of Saccharomyces cerevisiae; Kohalmi SE et al.; The recovery of spontaneous canavanine-resistant mutants is reduced dramatically in a strain of Saccharomyces cerevisiae that carries a suppressed can 1-100 allele and is permeable to and auxotrophic for thymidylate . This effect does not occur in an isogenic strain that neither takes up nor requires the nucleotide . However, it is observed for another isogenic strain which is permeable to but not auxotrophic for thymidylate, indicating that the effect is related to thymidylate permeability . Apparently, increased sensitivity of the permeable cells to growth inhibition by canavanine accounts for the diminished mutant recovery . In turn, enhanced uptake of canavanine in these cells seems to be responsible for the increased sensitivity . The experimental findings suggest that the elevated transport of canavanine in the thymidylate auxotroph is unlikely to be due to enhanced suppression of the can 1-100 allele or to activation of the yeast general amino acid permease.

Curr Genet, 1989 Feb, 15(2), 113 - 20
A galactose-dependent cmd1 mutant of Saccharomyces cerevisiae: involvement of calmodulin in nuclear division; Ohya Y et al.; The coding region of a yeast calmodulin gene was fused to a galactose-inducible GAL1 promoter, and a conditional-lethal mutant of Saccharomyces cerevisiae, in which the expression of calmodulin was regulated by galactose, was constructed . The mutant grew normally in galactose medium, but in glucose medium, in which the promoter was repressed, it ceased growing after 12-15 h . The growth arrest was associated with a decrease in intracellular calmodulin levels: after 12 h, no intracellular calmodulin protein was detectable . Analysis of the terminal phenotype showed that when the cell stopped growing, it had a bud, a nucleus after S-phase and a short mitotic spindle . Thus, the defect was mainly in nuclear division . Bud growth was partially inhibited in these cells: 27% of the cells stopped growing with a small bud . Furthermore, calmodulin-deficient cells showed elevated rates of chromosome loss, possibly as the result of a defect in the precise segregation of chromosomes.

Curr Genet, 1989 Feb, 15(2), 107 - 12
FLP-FRT mediated intrachromosomal recombination on a tandemly duplicated YEp integrant at the ILV2 locus of chromosome XIII in Saccharomyces cerevisiae; Rank GH et al.; A YEp chimaeric plasmid carrying SMR1 and URA3 genetic markers was integrated into chromosome XIII at the ilv2-delta 1 locus in a {cir (o)} background . The 1.5 kb BglII deletion of ilv2-delta 1 allowed the clear identification of an integrant structure which consisted of a direct tandem duplication (TD) of the chimaeric plasmid . Within the integrant structure, a single copy of the plasmid sequence was flanked by a direct duplication of the 2 microns site-specific recombinase (FLP) recognition target (FRT) . Isogenic {cir (o)} and {cir+} diploids formed by crossing the {cir (o)} TD strain to complementary haploids were analyzed for plasmid marker loss and chromosomal DNA alterations in the presence and absence of selection pressure for the URA3 and SMR1 plasmid borne markers . {cir (o)} diploids showed no plasmid marker loss and maintained the TD structure . In the absence of selection pressure, the {cir+} diploid underwent FLP-FRT mediated unequal interchromatid recombination, resulting in the breakage-fusion-bridge cycle and homozygotization of chromosome XIII (Rank et al . 1988) . Maintenance of selection pressure for the centromere distal plasmid URA3 marker selected against FLP-FRT interchromatid recombinants so that the effects of site specific recombinase on intrachromatid recombination could be evaluated . Intrachromatid recombination at the directly duplicated FRT sites of the TD structure resulted in the loss of a diagnostic internal fragment . These results show that in the presence of FLP, FRT sites separated by up to 13.3 kb of chromosomal DNA function as substrates for intra and interchromatid recombination.

Genetics, 1989 Feb, 121(2), 223 - 36
The MF alpha 1 gene of Saccharomyces cerevisiae: genetic mapping and mutational analysis of promoter elements; Flessel MC et al.; The activity and cell-type specificity of the promoter of the MF alpha 1 gene of Saccharomyces cerevisiae were examined by measuring expression of an MF alpha 1-SUC2 gene fusion in MATa, MAT alpha, and MATa/MAT alpha cells . A high level of invertase activity was observed only in MAT alpha cells . Weak expression occurred in MATa cells when the hybrid gene was carried on a multicopy plasmid or on a centromere-containing plasmid, but not when the hybrid gene was integrated at the normal MF alpha 1 locus . Analysis of a set of 5'-deletions of the promoter region of the MF alpha 1-SUC2 gene on the multicopy plasmid indicated that sequences from -354 to -274 upstream of the translational start site were required for high level expression in MAT alpha cells . Smaller internal deletions and insertions within the promoter region of the MF alpha 1-SUC2 gene were inserted into the genome at the normal MF alpha 1 locus . These mutations further delineated four promoter domains important for expression: (1) two 26 bp elements (-365 to -340 and -312 to -287) with imperfect dyad symmetry; (2) a 40 bp segment (-264 to -226) that lies about 120 bp upstream of the TATA box; and (3) the TATA box itself (-128 to -122) . The transcriptional start sites of the normal MF alpha 1 promoter and of a mutant lacking the TATA box were determined . The MF alpha 1 locus was mapped to the left arm of chromosome XVI, about 22 cM centromere-proximal to the PEP4 gene.

FEMS Microbiol Lett, 1989 Feb, 48(3), 271 - 4
O-linked mannose composition of secreted invertase of Saccharomyces cerevisiae; Mormeneo S et al.; The secreted invertase (EC 3.2.1.26) of Saccharomyces cerevisiae is a glycoenzyme that contains N- and O-linked mannoses in 40/1 proportion . The small amount of mannose chains O-linked to invertase is distributed as follows: mannose (20%), mannobiose (50%), mannotriose (6%), mannotetraose (7%) and mannopentaose (17%).

FEMS Microbiol Lett, 1989 Feb, 48(3), 265 - 9
Role of glycosylation in the incorporation of intrinsic mannoproteins into cell walls of Saccharomyces cerevisiae; Sanz P et al.; Cell wall mannoproteins from Saccharomyces cerevisiae are completely or partially incorporated into their final location when N-glycosylation is inhibited by tunicamycin . These include a 90-100 kDa species still containing O-linked oligomannose chains, derived from a N-glycosylated material larger than 120 kDa; and a 30.5 kDa peptide lacking mannose residues, derived from a 33 kDa species . For both species, the growth temperature influences the level of incorporation of the non N-glycosylated molecules . Secretion of the peptides lacking N-linked saccharide chains follows the route defined by sec mutants.

Mol Cell Biol, 1989 Feb, 9(2), 602 - 8
Identification of sequences responsible for transcriptional activation of the allantoate permease gene in Saccharomyces cerevisiae; Rai R et al.; DAL5 is a constitutively expressed allantoin system gene whose product is required for allantoate transport . Its simple pattern of expression prompted us to use this gene for identifying the element(s) that mediates transcriptional activation of allantoin system genes . Deletion analysis of the DAL5 5'-flanking sequences resulted in identification of two small regions required for DAL5 expression . Analysis of these two regions with synthetic oligonucleotides localized the sequences supporting transcriptional activation to two DNA fragments of 10 to 12 base pairs, each containing one copy of the pentanucleotide 5'-GATAA-3' . The 5'-flanking region of DAL5 contained eight copies of this sequence . Synthetic constructions containing single copies of these fragments were unable to support transcriptional activation, while those containing two or more copies supported high-level activation . The 5'-GATAA-3' sequence was also found beneath the footprint of a DNA-binding protein . These observations are consistent with the suggestion that DNA fragments containing the sequence 5'-GATAA-3' play an important role in DAL5 gene expression, probably representing a portion of the binding site for a transcriptional activation factor.

Mol Cell Biol, 1989 Feb, 9(2), 442 - 51
Identification of an upstream activating sequence and an upstream repressible sequence of the pyruvate kinase gene of the yeast Saccharomyces cerevisiae; Nishizawa M et al.; To clarify carbon source-dependent control of the glycolytic pathway in the yeast Saccharomyces cerevisiae, we have initiated a study of transcriptional regulation of the pyruvate kinase gene (PYK) . By deletion analysis of the 5'-noncoding region of the PYK gene, we have identified an upstream activating sequence (UASPYK1) located between 634 and 653 nucleotides upstream of the initiating ATG codon . The promoter activity of the PYK 5'-noncoding region was abolished when the sequence containing the UASPYK1 was deleted from the region . Synthetic UASPYK1 (26mer), in either orientation, was able to restore the transcriptional activity of UAS-depleted mutants when placed upstream of the TATA sequence located at -199 (ATG as +1) . While the UASPYK1 was required for basal to intermediate levels of transcriptional activation, a sequence between -714 and -811 was found to be necessary for full activation . On the other hand, a sequence between -344 and -468 was found to be responsible for transcriptional repression of the PYK gene when yeast cells were grown on nonfermentable carbon sources . This upstream repressible sequence also repressed transcription, although to a lesser extent, when glucose was present in the medium . The possible mechanism for carbon source-dependent regulation of PYK expression through these cis-acting regulatory elements is discussed.

Mol Cell Biol, 1989 Feb, 9(2), 390 - 5
Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae; Powers S et al.; Two mutant alleles of RAS2 were discovered that dominantly interfere with wild-type RAS function in the yeast Saccharomyces cerevisiae . An amino acid substitution which caused the dominant interference was an alanine for glycine at position 22 or a proline for alanine at position 25 . Analogous mutations in human H-ras also dominantly inhibited RAS function when expressed in yeast cells . The inhibitory effects of the mutant RAS2 or H-ras genes could be overcome by overexpression of CDC25, but only in the presence of wild-type RAS . These results suggest that these mutant RAS genes interfere with the normal interaction of RAS and CDC25 proteins and suggest that this interaction is direct and has evolutionarily conserved features.

Mol Cell Biol, 1989 Feb, 9(2), 365 - 76
DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae; Budd ME et al.; We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo . Previously, we showed that DNA polymerase I is required for mitotic DNA replication . Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage . We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur . Commitment to meiotic recombination is only 2% of wild-type levels . Thus, DNA polymerase I is essential for these steps . However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions . These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination . These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

Mol Gen Genet, 1989 Feb, 215(3), 517 - 28
Novel class of nuclear genes involved in both mRNA splicing and protein synthesis in Saccharomyces cerevisiae mitochondria; Asher EB et al.; We have cloned three distinct nuclear genes, NAM1, NAM7, and NAM8, which alleviate mitochondrial intron mutations of the cytochrome b and COXI (subunit I of cytochrome oxidase) genes when present on multicopy plasmids . These nuclear genes show no sequence homology to each other and are localized on different chromosomes: NAM1 on chromosome IV, NAM7 on chromosome XIII and NAM8 on chromosome VIII . Sequence analysis of the NAM1 gene shows that it encodes a protein of 440 amino acids with a typical presequence that would target the protein to the mitochondrial matrix . Inactivation of the NAM1 gene by gene transplacement leads to a dramatic reduction of the overall synthesis of mitochondrial protein, and a complete absence of the COXI protein which is the result of a specific block in COXI pre-mRNA splicing . The possible mechanisms by which the NAM1 gene product may function are discussed.

Mol Gen Genet, 1989 Feb, 215(3), 490 - 500
Dependence of inessential late gene expression on early meiotic events in Saccharomyces cerevisiae; Kao G et al.; SPR3 is one of at least nine genes which are expressed in sporulating Saccharomyces cerevisiae cells at the time of meiosis I . We show below that strains homozygous for null alleles of SPR3 are capable of normal meiosis and the production of viable ascospores . We have also monitored SPR3 expression in a series of strains that are defective in meiotic development, using an SPR3:lacZ fusion carried on a single copy plasmid . beta-Galactosidase activity occurred at wild-type levels in diploid strains homozygous for mutations in spo13, rad50, rad57 and cdc9, but was greatly reduced in strains carrying cdc8 or spo7 defects . We conclude that SPR3 expression is a valid monitor of early meiotic development, even though the gene is inessential for the sporulation process.

J Cell Biol, 1989 Feb, 108(2), 309 - 25
Processing pathway for protease B of Saccharomyces cerevisiae; Moehle CM et al.; The vacuolar protease B of Saccharomyces cerevisiae is a subtilisin-like protease encoded by the PRB1 gene . Antibodies raised against a synthetic peptide and an Escherichia coli-derived PRB1 open reading frame (ORF) protein cross-react with authentic protease B from yeast . By using these antibodies, the posttranslational biosynthetic pathway of protease B has been elucidated . Preproprotease B is a 76-kD unglycosylated precursor that enters the endoplasmic reticulum (ER), where it receives one asparagine-linked (Asn-linked) and an undetermined number of non-Asn-linked carbohydrate side chains . The large glycosylated intermediate is proteolytically processed to a 39-kD form before exiting the ER . In the Golgi complex, the 39-kD form becomes 40 kD, due to elaboration of the Asn-linked side chain . The carboxyterminal end of the 40-kD proprotease B undergoes protease A-mediated processing to a 37-kD intermediate, which in turn is quickly processed to 31-kD mature protease B . The ultimate processing step removes a peptide containing the Asn-linked chain; mature PrB has only non-Asn-linked carbohydrate.

Curr Genet, 1989 Feb, 15(2), 121 - 7
Increased diuron resistance in the joint expression of mutations located at the DIU2, DIU3 and DIU4 loci of Saccharomyces cerevisiae; Meunier B et al.; In Saccharomyces cerevisiae, diuron blocks the respiratory pathway at the level of the bc1 complex . Two mitochondrially inherited loci, DIU1 and DIU2, located in the cytochrome b gene, and two nuclearly inherited loci, DIU3 and DIU4, have previously been identified . The present work genetically characterizes two double mutants . One mutant, Diu-217, carries two nuclearly inherited mutations, diu3-217a and diu-217b; the second mutant, Diu-783, carries the previously described nuclear mutation diu3-783 and a mitochondrial mutation diu2-783 . Each mutation, independent of its location, exhibits a weak diuron resistance . The joint expression of two or three mutations leads to a cumulative or a cooperative enhanced diuron-resistant phenotype.

Mol Gen Mikrobiol Virusol, 1989 Feb, (2), 46 - 9
{Isolation of DNA fragments of the barley genome, supporting autonomous regulation of plasmids in Saccharomyces cerevisiae}; Anan'ev EV et al.; BamHI fragments of the barley genomic DNA were cloned in Escherichia coli cells on the vector plasmid YIp5 carrying the URA3 gene of Saccharomyces cerevisiae . Yeast cells were transformed by individual plasmid DNA preparations from each clone selected . Approximately 10% of the studied plasmids are able to replicate in yeast cells . Five barley DNA fragments which could support replication of recombinant plasmids in yeast cells belong to moderately repeated DNA and are dispersed through the chromosomes.

Mol Cell Biol, 1989 Feb, 9(2), 757 - 68
IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae; Tanaka K et al.; A mutation in the gene IRA1 (formerly called PPD1) was originally characterized as a deficiency of a phosphoprotein phosphatase . The IRA1 gene has been cloned and sequenced . A large open reading frame (8,817 base pairs) which can encode a protein of 2,938 amino acids was found . Northern (RNA) blot analysis detected a message of about 10 kilobases, and nuclease S1 protection demonstrated mRNA start points at 97 and 98 base pairs upstream from the putative initiator ATG codon . Disruption of the IRA1 gene resulted in sensitivity to nitrogen starvation and heat shock . Diploids homozygous for the disrupted IRA1 gene were deficient in sporulation . Disruption of the IRA1 gene suppressed the lethality of the cdc25 mutation but did not suppress the lethality of either the ras1 ras2 or the cyr1 mutations . Deficiency of the phosphoprotein phosphatase was not reproducible in the disruption mutant of the IRA1 gene . Moreover, the ira1 mutant showed an increased level of cyclic AMP . Our results suggest that the IRA1 protein inhibits the function of the RAS proteins in a fashion antagonistic to the function of the CDC25 protein in the RAS-cyclic AMP pathway in Saccharomyces cerevisiae.

Mol Cell Biol, 1989 Feb, 9(2), 484 - 91
Control of the Saccharomyces cerevisiae regulatory gene PET494: transcriptional repression by glucose and translational induction by oxygen; Marykwas DL et al.; The product of the Saccharomyces cerevisiae nuclear gene PET494 is required to promote the translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII) . The level of cytochrome c oxidase activity is affected by several different environmental conditions, which also influence coxIII expression . We have studied the regulation of PET494 to test whether the level of its expression might modulate coxIII translation in response to these conditions . A pet494::lacZ fusion was constructed and used to monitor PET494 expression . PET494 was regulated by oxygen availability: expression in a respiration-competent diploid strain grown anaerobically was one-fifth the level of expression in aerobically grown cells . However, since PET494 mRNA levels did not vary in response to oxygen deprivation, regulation by oxygen appears to occur at the translational level . This oxygen regulation was not mediated by heme, and PET494 expression was independent of the heme activator protein HAP2 . The regulation of PET494 expression by carbon source was also examined . In cells grown on glucose-containing medium, PET494 was expressed at levels four- to sixfold lower than in cells grown on ethanol and glycerol . However, addition of ethanol to glucose-containing medium induced PET494 expression approximately twofold . PET494 transcript levels varied over a fourfold range in response to different carbon sources . The effects on PET494 expression of mutations in the SNF1, SNF2, SSN6, and HXK2 genes were also determined and found to be minimal.

Mol Gen Genet, 1989 Feb, 215(3), 425 - 30
Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of rad18 deletion mutants; Fabre F et al.; The RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair . We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid . The insert was subcloned into YCp50 and into the multicopy YRp7 plasmid . RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive . A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRNA sup61 known to be tightly linked to RAD18 . Chromosomal deletions of RAD18 were constructed . The gene is not essential and the deleted strains have the properties of single site mutants . Thus, RAD18 appears to be essentially involved in DNA repair metabolism.

J Photochem Photobiol B, 1989 Feb, 3(1), 33 - 52
UVA-induced binding of 8-methoxypsoralen to cells of Saccharomyces cerevisiae: separation and characterization of DNA photoadducts; Bankmann M et al.; We present methods for the determination of UVA-induced binding of 8-methoxypsoralen (8-MOP) to nucleic acids and protein and for a quantitative assay of radioactively labelled 8-MOP plus UVA induced DNA photoproducts in the yeast Saccharomyces cerevisiae . For the dose range up to 60 kJ m-2, with a wild-type survival of 1% or higher, binding to DNA is 100-fold and to RNA 10- to 20-fold more efficient than that to protein . Between 20% and 65% of the 8-MOP binds to macromolecules that are neither nucleic acids nor protein . The number of DNA-bound 8-MOP molecules for the haploid genome rises from 14 (unirradiated control) to 349 at the highest UVA exposure dose (60 kJ m-2) . Gel chromatography reveals three types of DNA thymine photoproduct, the pyrone-side monoadducts, the furan-side monoadducts and the diadducts . Among these, pyrone-side monoadducts always constitute the smallest fraction, regardless of whether the treatment is with in vitro or in vivo 8-MOP plus UVA.

Mol Cell Biol, 1989 Feb, 9(2), 384 - 9
Context affects nuclear protein localization in Saccharomyces cerevisiae; Nelson M et al.; Proteins destined for the nucleus contain nuclear localization sequences, short stretches of amino acids responsible for targeting them to the nucleus . We show that the first 29 amino acids of GAL4, a yeast DNA-binding protein, function efficiently as a nuclear localization sequence when fused to normally cytoplasmic invertase, but not when fused to Escherichia coli beta-galactosidase . Moreover, the nuclear localization sequence from simian virus 40 T antigen functions better when fused to invertase than when fused to beta-galactosidase . A single amino acid change in the T-antigen nuclear localization sequence inhibits the nuclear localization of simian virus 40-invertase and simian virus 40-beta-galactosidase in Saccharomyces cerevisiae . From these results, we conclude that the relative ability of a nuclear localization sequence to act depends on the protein to which it is linked.

Proc Natl Acad Sci U S A, 1989 Feb, 86(3), 858 - 62
Phosphorylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae; Cobitz AR et al.; RAS1 and RAS2 proteins of Saccharomyces cerevisiae are guanine nucleotide-binding proteins involved in the regulation of adenylate cyclase . In this paper, we report that these proteins are phosphorylated . The phosphorylation of RAS1 protein is demonstrated by treating with alkaline phosphatase as well as by labeling with {32P}orthophosphate . The phosphorylation occurs exclusively on serine residues and phosphorylated RAS1 protein is predominantly membrane localized . The phosphorylation of RAS2 protein is demonstrated by similar 32P-labeling experiments . The phosphorylation occurs exclusively on serine residues and phosphopeptide analyses suggest that only two major phosphorylated tryptic peptides are generated from the RAS2 protein . These results provide evidence for the phosphorylation of RAS proteins in vivo . Furthermore, our demonstration that the phosphorylation occurs exclusively on serine residues and that the RAS2 protein contains only two major phosphorylated tryptic peptides argues that the phosphorylation may be physiologically significant.

Mol Cell Biol, 1989 Feb, 9(2), 415 - 20
Selection and characterization of ricin toxin A-chain mutations in Saccharomyces cerevisiae; Frankel A et al.; A DNA sequence encoding the A chain of ricin toxin (RTA) from the castor bean plant, Ricinus communis, was placed under GAL1 promoter control and transformed into Saccharomyces cerevisiae . Induction of expression of RTA was lethal . This lethality was the basis for a selection of mutations in RTA which inactivated the toxin . A number of mutant alleles which encoded cross-reactive material were sequenced . Eight of the first nine mutant RTAs studied showed single-amino-acid changes involving residues located in the proposed active-site cleft.

Biochim Biophys Acta, 1989 Jan 23, 1007(1), 80 - 3
Yeast, Saccharomyces cerevisiae, cell-free translation: the inhibition of translation by high temperature is reversible; Mandel T et al.; Yeast, Saccharomyces cerevisiae, extracts are inactive for translation at 37 degrees C . Two unexplained, simultaneously occurring phenomena appear to be responsible for this effect: (i) rapid inhibition of translation, and (ii) time-dependent inactivation of (a) translational component(s) at 37 degrees C . After short incubation of an extract at 37 degrees C, protein synthesis recovers efficiently after transfer of the extract to 23 degrees C . This behaviour of yeast cell-free systems enables the in vitro inactivation of temperature-sensitive translational components and therefore facilitates studies with extracts derived from temperature-sensitive strains.

J Mol Biol, 1989 Jan 20, 205(2), 275 - 89
Protein encoded by the third intron of cytochrome b gene in Saccharomyces cerevisiae is an mRNA maturase . Analysis of mitochondrial mutants, RNA transcripts proteins and evolutionary relationships; Lazowska J et al.; We have established the nucleotide sequence of the wild-type and that of a trans-acting mutant located in the third (bi3) intron of the Saccharomyces cerevisiae mitochondrial cytochrome b gene . The intron, 1691 base-pairs long, has an open reading frame 1045 base-pairs long, in phase with the preceding exon and the mutation replaces the evolutionarily conserved Gly codon of the second consensus motif by an Asp codon and blocks the formation of mature cytochrome b mRNA . Splicing intermediates of 5300 and 3900 bases with unexcised bi3 intron and a characteristic novel polypeptide (p50), the size of which corresponds to the chimeric protein encoded by upstream exons and the bi3 intronic open reading frame (ORF), accumulate in this and other bi3 splicing-deficient mutants . We conclude that the protein encoded by the bi3 ORF is a specific mRNA maturase involved in the splicing of the cytochrome b mRNA . The open reading frame of the third intron is remarkably similar to that of the unique intron of the cytochrome b gene (cob A) of Aspergillus nidulans . Both are located in exactly the same position and possibly derive from a recent common ancestor by a horizontal transfer . We have established the nucleotide sequence of an exonic mutant located in the B3 exon . This missense mutation changes the Phe codon 151 into a Cys codon and leads to the absence of functional cytochrome b but does not affect splicing . Finally, we have studied the splicing pathway leading to the synthesis of cytochrome b mRNA by analysing, in a comprehensive manner, the 22 splicing intermediates of several mutants located in bi3.

J Biol Chem, 1989 Jan 15, 264(2), 1092 - 9
Purification and characterization of Saccharomyces cerevisiae transcription factor IIIA; Wang CK et al.; Saccharomyces cerevisiae Transcription Factor IIIA (TFIIIA) has been purified to apparent homogeneity . Two polypeptides copurified with TFIIIA activity . Yeast TFIIIA is a DNA-binding protein which exhibits a high affinity for the internal control region of the homologous 5 S ribosomal RNA gene . Characterization of the yeast protein indicates that it shares most, but not all, of the molecular properties of its Xenopus TFIIIA counterpart.

Cancer Res, 1989 Jan 15, 49(2), 279 - 83
Mutational specificities of 1,3-bis(2-chloroethyl)-1-nitrosourea and nitrogen mustard in the SUP4-o gene of Saccharomyces cerevisiae; Kunz BA et al.; A collection of 346 mutations arising in the SUP4-o gene of the yeast Saccharomyces cerevisiae following treatment with 1,3-bis(2-chloro-ethyl)-1-nitrosourea (BCNU) or nitrogen mustard was analyzed by DNA sequencing . Both agents induced all possible types of base-pair substitution as well as deletions and double mutations . The base-pair changes consisted primarily of events at G.C pairs and were distributed throughout the gene . However, the distributions differed for the two drugs, and a prominent substitution hotspot was detected for nitrogen mustard . BCNU induced a substantial fraction of deletions the majority of which were recovered at a hotspot encompassing a tract of five G.C pairs . In contrast, nitrogen mustard generated relatively few deletions, but substantially more double mutations were recovered than with treatment with BCNU . Neither agent exhibited a preference for contiguous G.C sites, and more than one quarter of the mutations occurred at G.C sites, flanked by A.T pairs or at A.T pairs indicating that mutagenesis was not restricted to G.C runs . The data indicate that for BCNU and nitrogen mustard, monoadducts may play a role in mutagenesis, and site-specific mutability is influenced by factors in addition to the G.C richness of the sequence involved.

FEBS Lett, 1989 Jan 2, 242(2), 341 - 5
Glucose-induced cAMP signaling in Saccharomyces cerevisiae is mediated by the CDC25 protein; Munder T et al.; Functional mapping of the cell cycle START gene CDC25 has revealed two domains which are dispensable for viability (germination and growth in glucose media), but are essential for sporulation and differentially involved in glucose-induced cAMP signaling . The transient rise of cAMP is completely prevented by various deletions within the amino-terminal half (alpha domain) of the CDC25 gene product . In contrast, the deletion of the carboxy-terminal 38 residues (beta 2 domain) results in a rapid, but persisting, rise of cAMP . Our data suggest that the alpha domain of the CDC25 protein is involved in glucose signal transduction, whereas the beta 2 domain is required for downregulating the cAMP control chain.

J Cell Sci Suppl, 1989, 12, 145 - 8
Control of G2 delay by the rad9 gene of Saccharomyces cerevisiae; Weinert T et al.; In response to DNA damage, Saccharomyces cerevisiae cells arrest the cell cycle in the G2 phase . Arrest is defective in rad9 mutants; rad9 cells divide and die without repairing the damage . Several cell cycle mutants that are defective in DNA replication arrest in G2 at the restrictive temperature; this arrest is due to the RAD9 control function . Thus RAD9 is responsible for the fact that mitosis is normally dependent upon DNA replication, a function we term a 'checkpoint' . Four additional genes have been identified that are also components of the RAD9 checkpoint.

Folia Biol (Praha), 1989, 35(5), 315 - 27
Expression of hepatitis B virus large envelope protein in Escherichia coli and Saccharomyces cerevisiae; Korec E et al.; The gene coding for hepatitis B large envelope protein was cloned under the lac promoter in bacterial vector pUC-8 and under the ADH1 promoter in yeast expression shuttle vector pVT103-U, and expression of HBsAg in bacteria and yeast was determined . The strongest expression of large envelope protein was obtained after transformation of the protease-deficient yeast strain BJ1991 . The recombinant large envelope protein did not form complex 22-nm particles and was not secreted into medium.

Genome, 1989, 31(2), 684 - 9
Expression of members of the Saccharomyces cerevisiae hsp70 multigene family; Werner-Washburne M et al.; The hsp70 multigene family of Saccharomyces cerevisiae is a complex multigene family, composed of members exhibiting complex patterns of regulation . Expression of some members is induced after a heat shock, whereas expression of others is repressed . Some members of the family are expressed during exponential growth . One gene, SSA3, shows an unusual pattern of expression during approach to stationary phase . While most RNAs decrease in abundance, SSA3 RNA levels dramatically increase . The constitutive expression of SSA3 in cells lacking adenylate cyclase activity suggests that cAMP modulates SSA3 expression.

Genome, 1989, 31(2), 597 - 600
The structure and function of RAD6 and RAD18 DNA repair genes of Saccharomyces cerevisiae; Prakash L; The RAD6 and RAD18 genes of Saccharomyces cerevisiae are required for postreplication repair of discontinuities occurring in newly synthesized DNA following exposure to uv light . In addition, rad6 mutants are highly defective in mutagenesis induced by uv and other DNA damaging agents and in sporulation . RAD6 encodes a protein of 172 amino acids with a highly acidic carboxyl terminus . Deletion of the carboxyl terminal 23 residues, 20 of which are acidic, has little or no effect on uv sensitivity or uv mutagenesis, but sporulation is greatly reduced . Addition of the first four residues of the polyacidic tail restores sporulation to 50% the level observed in RAD+/RAD+ diploids . RAD6 protein has been previously shown to be a ubiquitin-conjugating (E2) enzyme that attaches ubiquitin to histones H2A and H2B in vitro . Our experiments show that deletion of varying lengths of the polyacidic tail of RAD6 protein greatly reduces its ubiquitin-conjugating activity . The RAD18 encoded protein contains features which suggest that it binds DNA and nucleotides . Ten of the 12 cysteine residues occur in regions that could form zinc finger domains for nucleic acid binding . The other interesting feature in RAD18 protein is the presence of a putative nucleotide binding sequence . The possible in vivo functions of the RAD6 and RAD18 proteins are discussed.

Proteins, 1989, 6(3), 324 - 37
Recombinant HIV1 protease secreted by Saccharomyces cerevisiae correctly processes myristylated gag polyprotein; Pichuantes S et al.; The protease of the human immunodeficiency virus type I (HIV1) was expressed both intracellularly and extracellularly in Saccharomyces cerevisiae . Intracellular expression of the protease was achieved by fusing a 179 amino acid precursor form of the protease to human superoxide dismutase (hSOD) . Self-processing of the viral enzyme from the hybrid precursor was demonstrated to occur within the yeast host . Secretion of the protease was achieved by fusing the leader sequence of yeast alpha-factor to the precursor form of the protease or to the 99 amino acid mature form of the protease . Authentic and active forms of the retroviral enzyme were detected in yeast supernatants of cells expressing the precursor or the mature form of the protease . A D25E active site variant of the retroviral enzyme exhibited diminished autocatalytic activity when expressed intracellularly or secreted from yeast . The wild-type protease was active in an in vitro assay on the natural substrate, myristylated gag precursor, Pr53gag . Correct processing of Pr53gag at the Tyr 138-Pro 139 junction was confirmed by amino terminal sequence analysis of the resulting capsid protein (CA, p24) . The secreted protease was purified to homogeneity from yeast media using preparative isoelectric focusing and reverse-phase HPLC . Amino terminal sequence analysis showed a sequence beginning at amino acid 1 of the mature enzyme (Pro) and another sequence beginning at amino acid 6 (Trp) . This shorter sequence may represent a natural autolytic product of the protease.

J Cell Sci Suppl, 1989, 11, 109 - 13
The use of gene-fusions to determine membrane protein topology in Saccharomyces cerevisiae; Green GN et al.; We have used protein-fusions to study in Saccharomyces cerevisiae the topology and integration of arginine permease . Since this membrane protein does not contain a cleavable signal sequence, we sought to identify the first internal signal by, initially, fusing the cytoplasmic enzyme, galactokinase, to various positions along the amino-terminal region, and then measuring in vitro the translocation of galactokinase across the membrane of the endoplasmic reticulum . Five fusion proteins were examined that contained, progressively, zero to four hydrophobic segments . The galactokinase moiety of fusion 5, but not fusions 1-4, was translocated . Fusion 4 differed from 5 by only the fourth hydrophobic segment, indicated that this region contains the first internal signal . From this we conclude that hydrophobic segment IV spans the membrane, and that the hydrophilic domain amino-terminal to it lies on the cytoplasmic side of the membrane . If any of the first three segments actually span the membrane, then they probably integrate by a mechanism that differs from segment IV.

Folia Microbiol (Praha), 1989, 34(4), 273 - 8
Inhibition of hexose transport by glucose in a glucose-6-phosphate isomerase mutant of Saccharomyces cerevisiae; Alonso A et al.; The rate of hexose transport was approximately 60% lower for both the high- and the low-affinity components of hexose uptake when a glucose-6-phosphate isomerase mutant of Saccharomyces cerevisiae was preincubated with glucose, as compared with preincubation with water . Similarly the Jmax value of the high-affinity system of the mutant was 25-35% of the corresponding Jmax value for normal cells incubated with glucose . Accumulation of glucose 6-phosphate or of some other metabolite, such as fructose 6-phosphate or trehalose, may be responsible for this striking inhibition.

Genome, 1989, 31(1), 95 - 9
Genetic regulation of differentiation towards meiosis in the yeast Saccharomyces cerevisiae; Simchen G et al.; Normally, meiosis and sporulation in Saccharomyces cerevisiae occur only in diploid strains and only when the cells are exposed to starvation conditions . Diploidy is determined by the mating-type system (the genes MAT, RME1, IME1), whereas the starvation signal is transmitted through the adenylate cyclase - protein kinase pathway (the genes CDC25, RAS2, CDC35 (CYR1), BCY1, TPK1, TPK2, TPK3) . The two regulatory pathways converge at the gene IME1, which is a positive regulator of meiosis and whose early expression in sporulating cells correlates with the initiation of meiosis . Sites upstream (5') of IME1 appear to mediate in the repression of the gene by repressors originating from both the mating-type and the cyclase--kinase pathways.

Microbios, 1989, 59(239), 101 - 11
Effects of sterol alterations on nystatin sensitivity in Saccharomyces cerevisiae; Richman-Boytas CM et al.; A systematic study of the qualitative and quantitative effects of sterol on nystatin sensitivity has been made in a single organism . The use of a sterol auxotroph of Saccharomyces cerevisiae offered a convenient way to control the sterol content of the yeast cell . There was a correlation between the ergosterol content of the cell and sensitivity to nystatin, as monitored by both potassium leakage from the cell and viability . When the sterol auxotroph contained high levels of ergosterol, the cells were sensitive to the effects of nystatin . When the ergosterol content was low or when ergosterol was replaced by cholesterol or cholestanol, sensitivity to nystatin was markedly decreased . Although resistant to nystatin, cholestanol enriched cells showed an enhanced background of potassium ion loss.

Proteins, 1989, 5(3), 218 - 23
The 15 N-terminal amino acids of hexokinase II are not required for in vivo function: analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae; Ma H et al.; The function of the N-terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form) . This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons . As expected, the in vitro analysis of the short form protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild-type protein dimerizes . Kinetic studies show that the enzymatic activities are very similar to wild-type behavior . The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form of the enzyme is similar to the wild-type both in terms of phosphorylation of hexoses and glucose repression . We conclude that the N-terminal amino acids of hexokinase II are not required in vivo either for phosphorylation of hexoses or for glucose repression.

Arch Microbiol, 1989, 152(3), 263 - 8
Cloning of the LEU2 gene of Saccharomyces cerevisiae by in vivo recombination; Valinger R et al.; We describe a convenient method for the in vivo construction of large plasmids that possess a multitude of restriction sites . A large (23 kbases) circular self-replicating plasmid carrying a partial LEU2-d gene was cotransformed with a circular non-replicating plasmid carrying the entire LEU2 gene . In vivo recombination results preferentially in a plasmid that carries both the LEU2-d and the entire LEU2 gene . In addition we also found one plasmid with a tandem LEU2 insertion and one plasmid where the LEU2-d gene was replaced by the entire LEU2 gene.

Prog Clin Biol Res, 1989, 311, 327 - 48
Meiotic segregation of normal and deletion chromosomes in Saccharomyces cerevisiae; Surosky RT et al.; We explored the behavior of meiotic chromosomes in Saccharomyces cerevisiae by examining the effects of chromosomal rearrangements on recombination and disjunction . Chromosome III derivatives in which the entire left arm or the entire right arm was deleted (telocentric) segregated with fidelity from a normal chromosome III . Recombination between either of these two deletion chromosomes and the intact chromosome also appeared normal . In a strain containing a right arm telocentric, a left arm telocentric and one normal chromosome both telocentrics disjoined from the normal chromosome . Homology on one arm was sufficient for proper recombination and segregation of these chromosomes . In strains containing two normal chromosomes and one telocentric chromosome the two normal chromosomes preferentially disjoined . In a few cases however, the two normal chromosomes cosegregated . Recombination between the two normal chromosomes or between one normal chromosome and the deletion chromosome increased the probability that they would disjoin, although cosegregation of recombinants was observed . A chromosome III derivative which contained a large centromeric deletion and an insertion of the centromere from chromosome V into a nonhomologous position segregated with fidelity from a normal chromosome III . These studies demonstrate that it is not pairing of the centromeres, but pairing and recombination along the arms of the homologs that directs meiotic chromosome disjunction.

Ann Ist Super Sanita, 1989, 25(1), 99 - 113
Excision repair genes of Saccharomyces cerevisiae; Prakash L et al.; In the yeast Saccharomyces cerevisiae, at least ten genes are involved in excision repair of DNA damaged by UV radiation and by other agents that distort the DNA helix . Mutations in the RAD1, RAD2, RAD3, RAD4 and RAD10 genes render cells highly defective in the incision of damaged DNA, whereas mutations in the RAD7, RAD14, RAD16, RAD23 and MMS19 genes reduce the level of damage excision . This review summarizes the evidence for the involvement of these genes in excision repair and highlights the important features in the structures of the proteins encoded by the various RAD genes . The RAD3 protein has been purified and characterized in our laboratory, and it possesses single stranded DNA dependent ATPase and DNA helicase activities . The RAD3 helicase moves along the single-stranded DNA in the 5'----3' direction . We suggest that this activity plays a role in strand displacement synthesis during excision repair and in DNA replication.

Curr Genet, 1989 Jan, 15(1), 31 - 8
Two nuclearly inherited loci conferring increased diuron resistance to NADH oxidase in Saccharomyces cerevisiae; Meunier B et al.; In Saccharomyces cerevisiae, diuron blocks the respiration pathway at the level of the bc1 complex . Nuclear diuron-resistant mutations which confer in vitro resistance to mitochondrial NADH oxidase have been identified . Five mutations were found to be clustered at two distinct nuclear loci DIU3 and DIU4 . The distance between the two loci was estimated to be about 36.7 cM . These loci do not appear to be centromere-linked and did not show a linkage to any of the genes coding for bc1 complex subunits . DIU3 and DIU4 loci might, therefore code for other components of the respiratory chain.

Biopolymers, 1989 Jan, 28(1), 487 - 97
Biologically significant conformation of the Saccharomyces cerevisiae alpha-factor; Naider F et al.; The conformation of the tridecapeptide alpha-factor of the yeast Saccharomyces cerevisiae was examined in both solution and in the presence of lipid vesicles . CD, differential scanning calorimetry, and phosphorus nmr all indicate that this mating pheromone interacts with lipid vesicles . In both aqueous and organic solution the alpha-factor is a flexible molecule that exhibits features of a type II beta-turn spanning the center of the peptide . Two-dimensional Nuclear Overhauser enhancement spectroscopy gives evidence that the beta-turn is stabilized on interaction of the peptide with lipid vesicles . Our current belief is that the beta-turn may play an important role in the biologically active conformation of the alpha-factor.

Arch Microbiol, 1989, 151(2), 154 - 8
Effects of nucleotides and divalent cations on phospholipase activity in Saccharomyces cerevisiae; Witt W et al.; Divalent cations activate the lysophospholipase and transacylase reactions catalyzed by the same enzymes in the yeast Saccharomyces cerevisiae . The activation was observed at neutral pH, but not at the pH optimum of lysophospholipase/transacylase, near 3.5 . Adenine nucleotides, especially AMP and ADP, are strong inhibitors of the same group of enzymes . Half maximal inhibition by AMP was found at a concentration of about 20 microM . The inhibition by nucleotides in low concentrations is enhanced by divalent cations.

FEMS Microbiol Lett, 1989 Jan 1, 48(1), 25 - 8
Effects of 2-deoxyglucose on Saccharomyces cerevisiae as observed by in vivo 31P-NMR; Loureiro-Dias MC et al.; Saccharomyces cerevisiae cells were treated with 2-deoxyglucose (1 mM) and the effects induced in the levels of phosphorus compounds and in the internal pH were monitored using 31P-NMR . Upon incubation with 2-deoxyglucose a strong decrease in the polyphosphate level was observed and the cytoplasmic pH decreased by about 0.4 units . This shows that 2-deoxyglucose strongly interferes with the cell conditions and consequently, the results of experiments in which 2-deoxyglucose was used to obtain deenergized cells should be carefully reanalysed.

EMBO J, 1989 Jan, 8(1), 247 - 54
Stabilization of dicentric chromosomes in Saccharomyces cerevisiae by telomere addition to broken ends or by centromere deletion; Jager D et al.; We introduced CEN6 DNA via integrative transformation into the right arm of chromosome II in a haploid Saccharomyces cerevisiae strain thus creating a dicentric chromosome . The majority of the transformed cells did not grow into colonies as concluded from control transformations with mutated CEN6 DNA . Five percent of the initial transformants with the wild-type centromere gave rise to well growing cells . We analysed the probable fate of the dicentric chromosome in two transformants by electrophoretic separation of chromosome sized DNA and by hybridizations with chromosome II DNA probes . We found two different mechanisms which generated cells lacking dicentric chromosomes . The first mechanism is breakage of the chromatid between the two-centromeres and healing of the new ends to functional telomeres thus creating progeny cells with the chromosome II information split into two genetically stable new chromosomes one carrying CEN2 and the other CEN6 . The second mechanism is loss of the resident CEN2 by a 30-50 kb deletion event which resulted in a genetically stable but shortened chromosome II . Both mechanisms operated in the two transformants studied.

Arch Microbiol, 1989, 151(3), 198 - 202
Genetic analysis of inducible sexual agglutination ability in the yeast Saccharomyces cerevisiae; Nakagawa Y; Genetic regulation of the inducibility of sexual agglutination ability in the yeast Saccharomyces cerevisiae was studied . Detailed analysis of the degree of sexual agglutination was carried out; it showed that a greater number of genes are involved in the regulation of inducible sexual agglutination in strain H1-0 than previously assumed . Although dominancy of inducible phenotype over constitutive was confirmed, the effectiveness of one gene changing the constitutive phenotype to the inducible seemed to be somewhat low . Quantity per cell of agglutination substances responsible for sexual agglutination increased as the agglutination ability became greater.

Yeast, 1989 Jan-Feb, 5(1), 55 - 72
Sequence and mutational analysis of ESS1, a gene essential for growth in Saccharomyces cerevisiae; Hanes SD et al.; A newly isolated gene, ESS1, was shown to encode a protein required for vegetative growth in Saccharomyces cerevisiae . The nucleotide sequence of ESS1 revealed a 172 amino acid open reading frame predicting a highly basic, 19.5 kilodalton product . Although the gene was isolated by cross-hybridization with the vertebrate v-sis oncogene, the primary amino acid sequence bears only a slight resemblance to the p28sis protein . ESS1 was shown to be single copy in the yeast genome and transcriptionally active during logarithmic growth . It is located on the right arm of chromosome X, 6 centimorgans distal to ilv3 . The genetic map location indicates it is not allelic to any previously characterized mutation in this organism . Both inactivation of ESS1 by gene disruption and overexpression by fusion to a heterologous promoter were detrimental to growth in both haploid and diploid cell types . Under non-permissive conditions, the terminal phenotype of strains containing a suppressible amber mutation within ESS1 was one of aberrant multibudded structures . Examination of this morphology indicates that loss of ESS1 function may lead to a defect in cytokinesis or cell separation.

Yeast, 1989 Jan-Feb, 5(1), 51 - 3
Extraction and rapid inactivation of proteins from Saccharomyces cerevisiae by trichloroacetic acid precipitation; Wright AP et al.; Methods currently used for the extraction of proteins from yeast involve relatively long time periods between sampling cells from a culture and analysis of their proteins by polyacrylamide gel electrophoresis-sodium dodecylsulphate . Often it is desirable to inactivate cellular metabolism rapidly after sampling and here we show that trichloroacetic acid precipitation techniques, often used for rapid extraction and inactivation of proteins from higher eukaryotes, can be adapted for use with organisms which have cell walls.

Mol Cell Biol, 1989 Jan, 9(1), 34 - 42
Adjacent upstream activation sequence elements synergistically regulate transcription of ADH2 in Saccharomyces cerevisiae; Yu J et al.; A 22-base-pair (bp) inverted repeat present in the ADH2 promoter is an upstream activation sequence (UAS1) which confers ADR1-dependent activation upon a heterologous Saccharomyces cerevisiae promoter . UAS1 was nonfunctional when placed within an intron 3' to the transcription start site . The 11-bp sequence which constitutes one-half of the UAS1 palindrome did not activate transcription in a single copy, as direct repeats, or in an inverted orientation opposite to that of ADH2 UAS1 . Furthermore, two pairs of symmetrical point mutations within UAS1 significantly reduced activation . This result suggests that a specific orientation of sequences within UAS1 is necessary for ADR1-dependent activation . We determined that an ADR1-dependent complex was formed with UAS1 and, to a lesser extent, with the nonfunctional 11-bp half palindrome . However, the 11 bp did not confer UAS activity, suggesting that ADR1 binding is not sufficient for activation in vivo . ADR1 did not bind to mutant UAS1 sequences in vitro, indicating that their decreased activation is attributable to a reduced affinity of ADR1 for these sequences . We also identified an additional 20-bp ADH2 element (UAS2) that increased the expression of CYC1-lacZ 20-fold when combined with UAS1 . UAS2 permitted ADR1-independent, glucose-regulated expression of the hybrid gene . Consistent with this observation, ADR1 did not form a detectable complex with UAS2 . Deletion of UAS2 at the chromosomal ADH2 locus virtually abolished ADH2 derepression and had no effect on glucose repression.

Comp Biochem Physiol B, 1989, 92(2), 297 - 301
Saccharomyces cerevisiae porphobilinogenase: some physical and kinetic properties; Araujo LS et al.; 1 . Properties of porphobilinogenase (PBGase), the enzyme complex converting porphobilinogen (PBG) into uroporphyrinogens, were studied in a wild strain, D273-10B and a mutant, B231, of Saccharomyces cerevisiae . 2 . A well-defined maximum of enzyme activity was observed for the mutant strain after 20 hr of growth; whilst the activity in the wild strain did not vary significantly during growth . 3 . Neither PBG consumption nor uroporphyrinogen formation were modified by the presence of air either in the wild or in the mutant strain . 4 . In both the wild and mutant strains uroporphyrinogen formation increased linearly with both protein concentration and incubation time . 5 . The addition of a mixture of sodium and magnesium salts to the assay system inhibited the enzyme activity of both strains by 50% without modifying the isomer composition . 6 . The same optimum pH (7.4) and mol . wt (50,000 +/- 5000) was found for the enzyme from both strains . 7 . The enzyme from both the wild and mutant strains shows Michaelis-Menten kinetics when isolated from cells at either the exponential or the stationary phases of growth . Accumulation of porphyrins and delta-aminolevulinic acid occurring during the exponential phase in the mutant strain, did not modify the kinetics.

J Bacteriol, 1989 Jan, 171(1), 37 - 42
Evidence for the involvement of a cytoplasmic factor in the aging of the yeast Saccharomyces cerevisiae; Egilmez NK et al.; The life spans of individual Saccharomyces cerevisiae cells were determined microscopically by counting the number of buds produced by each cell to provide a measure of the number of cell generations (age) before death . As the cells aged, their generation times increased five- to sixfold . The generation times of daughter cells were virtually identical to those of their mothers throughout the life spans of the mothers . However, within two to three cell divisions after the daughters were detached from their mothers by micromanipulation, their generation times reverted to that characteristic of their own age . Recovery from the mother cell effect was also observed when the daughters were left attached to their mothers . The results suggest that senescence, as manifested by the increase in generation time, is a phenotypically dominant feature in yeast cells and that it is determined by a diffusible cytoplasmic factor(s) that undergoes turnover . This factor(s) appeared to be transmitted by a cell not only to its daughter, but also indirectly to its granddaughter . In separate studies, it was determined that the induced deposition of chitin, the major component of the bud scar, in the yeast cell wall had no appreciable effect on life span . We raise the possibility that the cytoplasmic factor(s) that appears to mediate the "senescent phenotype" is a major determinant of yeast life span . This factor(s) may be the product of age-specific gene expression.

Radiat Res, 1989 Jan, 117(1), 145 - 57
An attempt to stimulate cell division in Saccharomyces cerevisiae with weak ultraviolet light; Quickenden TI et al.; Liquid cultures of the yeast Saccharomyces cerevisiae were irradiated with weak light having irradiances ranging from ca . 1 X 10(2) to 5 X 10(9) photons cm-2 s-1 and at wavelengths ranging from 200 to 700 nm . When particular care was taken to control the temperature of the cultures and the flow rate of oxygen, no evidence was obtained for stimulation of either yeast growth or division by the incident light . These results do not support the claims of early workers that very low intensity uv light can stimulate cell division in living organisms.

J Trauma, 1989 Jan, 29(1), 129 - 30
Saccharomyces cerevisiae fungemia in a multiply traumatized patient; Manzella JP et al.; A case of Saccharomyces cerevisiae fungemia in a severely traumatized patient is described . This organism, although usually considered a nonpathogen, may occasionally cause serious illness in debilitated patients.

Mol Cell Biol, 1989 Jan, 9(1), 144 - 51
The general control activator protein GCN4 is essential for a basal level of ARO3 gene expression in Saccharomyces cerevisiae; Paravicini G et al.; The ARO3 gene encodes one of two 3-deoxy-D-arabino-heptulosonate-7-phosphate isoenzymes in Saccharomyces cerevisiae catalyzing the first step in the biosynthesis of aromatic amino acids . The ARO3-encoded 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) is feedback inhibited by phenylalanine; its isoenzyme, the ARO4 gene product, is inhibited by tyrosine . Both genes ARO3 and ARO4 are strongly regulated under the general control regulatory system . Cells carrying only one intact isogene are phenotypically indistinguishable from a wild-type strain when grown on minimal medium . The complete functional ARO3 promoter comprises 231 base pairs and contains only one TGACTA binding site for the general control activator protein GCN4 . Mutating this element to TTACTA inhibits binding of GCN4 and results in a decreased basal level of ARO3 gene product and slow growth of a strain defective in its isogene ARO4 . In addition, ARO3 gene expression cannot be elevated under amino acid starvation conditions . An ARO3 aro4 strain with gcn4 genetic background has the same phenotype of low ARO3 gene expression and slow growth . The amount of GCN4 protein present in repressed wild-type cells therefore seems to contribute to a basal level of ARO3 gene expression . The general control activator GCN4 has thus two functions: (i) to maintain a basal level of ARO3 transcription (basal control) in the presence of amino acids and (ii) to derepress the ARO3 gene to a higher transcription rate under amino acid starvation (general control).

Cell Signal, 1989, 1(6), 577 - 86
Rapid intracellular alkalinization of Saccharomyces cerevisiae MATa cells in response to alpha-factor requires the CDC25 gene product; Perlman R et al.; The alpha-factor mating pheromone induces a transient intracellular alkalinization of MATa cells within minutes after exposure to the pheromone, and is the earliest biochemical event that can be identified subsequent to the exposure . Dissipation of the pheromone induced pH gradient, using 2,4-dinitrophenol or sodium orthovanadate, does not inhibit the biological response of the yeast to the pheromone such as mating and 'schmoo' formation . These findings suggest that the pheromone mediated pH change per se is not a part of the transmembrane signalling but rather the consequence of a biochemical reaction triggered by the alpha-pheromone interaction with its receptor and may have a permissive effect on the pheromonal response . The cdc25ts mutation causes MATa cells to become nonresponsive to alpha-factor subsequent to a shift to the restrictive temperature, suggesting that the CDC25 gene product participates in the pheromone response pathway.

J Cell Sci Suppl, 1989, 12, 117 - 27
Further characterization of a size control gene in Saccharomyces cerevisiae; Cross FR; The DAF1-1 mutation reduces cell size and reduces or eliminates G1 phase in Saccharomyces cerevisiae, and results in alpha-factor resistance . DAF1-1 cells transferred into low cycloheximide express an increased G1 phase in their cycle, suggesting that G1 regulation is present but cryptic in the DAF1-1 cycle in rich medium . DAF1-1 reduces cell size by the criterion of RNA content per cell as well as cell volume . The alpha-factor resistance of DAF1-1 cannot be suppressed by bypassing the pheromone-receptor interaction with 'signalling-constitutive' mutations, suggesting that pheromone binding and initial signalling is normal in DAF1-1 strains, but that division arrest in response to the signal is specifically defective . Consistent with this idea, the cdc28-13 mutation significantly suppresses DAF1-1 alpha-factor resistance at permissive temperature; CDC28 is a gene required specifically for START and the G1/S transition, and does not affect pheromone response . Genetic results additional to those previously reported confirm that the wild-type dafl+/WHI1 gene is non-essential; this result may be surprising since the gene product is apparently rate-limiting for the G1/S transition: its deletion increases cell size, and multiple copies decrease cell size.

Genome, 1989, 31(2), 909 - 19
The biology and exploitation of the retrotransposon Ty in Saccharomyces cerevisiae; Garfinkel DJ et al.; Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate . The element is transcribed into RNA, and this RNA is reverse transcribed into a DNA copy capable of insertion into many different chromosomal locations . Maturation of proteins and reverse transcription take place within noninfectious intracellular viruslike particles . We have studied the element Ty, which is found dispersed in the genome of the yeast Saccharomyces cerevisiae . The frequency of Ty element transposition is normally quite low but can be greatly increased by expressing an element from a strong promoter . We have used the ability to control the level of Ty transposition to investigate the functions of Ty proteins, the regulation of Ty transposition, and the exploitation of Ty elements as insertional mutagens in yeast . The information gained from these experiments should be applicable to the study of retrotransposons found in multicellular organisms.

Growth Factors, 1989, 1(3), 271 - 81
Expression of three recombinant homodimeric isoforms of PDGF in Saccharomyces cerevisiae: evidence for difference in receptor binding and functional activities; Ostman A et al.; Three recombinant homodimeric isoforms of platelet-derived growth factor (PDGF) were produced and purified in milligram quantities by expression of PDGF A- and B-chains in yeast cells . Structural analysis of the purified short and long variants of PDGF-AA (PDGF-AAS and PDGF-AAL) and PDGF-BB showed that they had been properly processed and assembled into dimers . PDGF-AAS and PDGF-AAL were found to bind only to the PDGF A-type receptor on human fibroblasts, with affinities of 0.1 and 0.2 nM, respectively . PDGF-BB bound to cells with A- and B-type receptors and to cells with B-type receptor only with affinities of 0.6 nM in both cases . Each fibroblast appeared to express about 4-5 times more B-type receptors than A-type receptors . The maximal mitogenic response to PDGF-BB of human fibroblasts was almost 2-fold higher than that induced by either of the two PDGF-AA forms . The three isoforms of PDGF also stimulated growth in soft agar of human fibroblasts with PDGF-BB inducing a higher maximal response.

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