S. cerevisiae

FEBS Lett, 1987 May 11, 215(2), 257 - 60
Localization of possible functional domains in sup2 gene product of the yeast Saccharomyces cerevisiae; Kushnirov VV et al.; Primary structures of yeast sup2 gene and polypeptide product coded by the gene are compared with the current nucleotide and amino acid sequence data base . The amino acid sequence of the sup2 product shows homology to elongation factors from different sources . Especially high homology is found in the regions, corresponding to conservative aminoacyl-tRNA- and GTP-binding domains, described in elongation factors and other proteins . The data obtained are discussed in relation to the functions of sup2 polypeptide product in protein synthesis.

Eur J Biochem, 1987 May 4, 164(3), 607 - 12
Isolation of the NPR1 gene responsible for the reactivation of ammonia-sensitive amino-acid permeases in Saccharomyces cerevisiae . RNA analysis and gene dosage effects; Vandenbol M et al.; The NPR1 gene codes for a protein, called the nitrogen permease reactivator protein or Npr1, which appears to promote the activity of several permeases for nitrogenous substances under conditions of nitrogen catabolite derepression, but fails to do so in the presence of ammonium ions . This gene has been cloned . Its transcription seems unaffected by growth on ammonia, so any ammonia regulation of Npr1 function most likely occurs at another level . In order to elucidate further the mechanism of permease inactivation, which requires an intact NPI1 gene product (NPI1 for nitrogen permease inactivator gene, formerly termed MUT2) and the role of Npr1 in counteracting this process, we have studied the effects of NPR1 and NPI1 gene dosage on general amino-acid permease activity . On nitrogen-derepressing media, NPR1 gene dose can be increased from 1 copy in a diploid to 16 plasmid-borne copies in a haploid strain without altering general amino-acid permease activity . On minimal ammonia medium, the plasmid-bearing haploid cells exhibit low but increased general amino-acid permease activity with respect to non-transformed cells . The adverse effect of the NPI1 gene product on general amino-acid permease activity is reduced when NPI1 gene dose is decreased to 1 gene copy in a diploid strain, regardless of the nitrogen source . We hypothesize that this product inactivates the permease by stoichiometric binding and that the Npr1 protein or a product of its catalytic action opposes this binding under conditions of nitrogen derepression.

Eur J Biochem, 1987 May 4, 164(3), 601 - 6
Nitrogen catabolite regulation of proline permease in Saccharomyces cerevisiae . Cloning of the PUT4 gene and study of PUT4 RNA levels in wild-type and mutant strains; Jauniaux JC et al.; The proline permease gene PUT4 has been cloned . Nitrogen-source regulation ('ammonia sensitivity') of this and at least two other amino-acid permeases is believed to occur at two distinct levels, i.e . permease synthesis and permease activity . Therefore, PUT4 transcription/messenger stability was examined in the ammonia- and proline-grown wild type as well as in mutant strains supposedly affected at only one or at both of these levels . We report transcript-level repression of proline permease synthesis in ammonia-grown cells . Repression is lifted at this level in gdhCR, gln1ts and gdhA mutants which exhibit pleiotropically derepressed permease and catabolic enzyme activities . On the other hand, the npi1 and npi2 mutations, formerly called mut2 and mut4, relieve an inactivation process which seems only to affect permeases . These mutations do not affect the detected PUT4 RNA level . The only known positive factor in proline permease regulation, the nitrogen permease reactivator protein Npr1, is believed to counteract the inactivation process on derepressing media . This protein appears to have an additional, indirect effect on PUT4 transcription/messenger stability: it would actually mediate repression via its activating effect on ammonia uptake.

Eur J Biochem, 1987 May 4, 164(3), 559 - 63
A nuclear mutation affecting mitochondrial transcription in Saccharomyces cerevisiae; Lisowsky T et al.; Mitochondrial transcription was studied in a nuclear temperature-sensitive pet mutant of Saccharomyces cerevisiae . The mitochondrial RNA levels in vivo and the in vitro transcriptional activities of isolated mitochondria were analysed . In comparison to the wild-type an overall reduction of mitochondrial gene expression together with a changed expression pattern was observed for the mutant, indicating a defect in mitochondrial RNA synthesis . These findings were supported by studies with a purified DNA-protein complex from yeast mitochondria . This complex was able to synthesize ribosomal and messenger RNAs in an in vitro system . Proteins from wild-type and mutant transcription complexes were tested for their DNA-binding abilities; one of the proteins identified in the wild type had either lost this ability or was absent in the mutant.

Mutat Res, 1987 May, 191(1), 9 - 12
UV response of the temperature-conditional rad 54 mutant of the yeast Saccharomyces cerevisiae; Kiefer J; The survival of the yeast mutant rad 54-3, which is temperature-conditional for the repair of double-strand breaks, was measured after exposure to UV-light (254 nm) and incubation at 23 degrees C and 36 degrees C . It was found that survival was drastically reduced with incubation at the restrictive temperature . Temperature-shift experiments indicated that repair of UV-induced damage which is controlled by the rad 54 gene proceeds with a half-value-time of about 7 h.

Mutat Res, 1987 May, 178(1), 43 - 7
High mutagenic activity of 3-azido-1,2-propanediol (azidoglycerol, AG) in strain D7 of Saccharomyces cerevisiae; Juricek M et al.; 3-Azido-1,2-propanediol (azidoglycerol, AG) showed a high mutagenicity in strain D7 of Saccharomyces cerevisiae . At 5 mM it increased the spontaneous frequency of isoleucine revertants 3500 times and the frequency of gene convertants 3000 times during 24 h of growth, reducing the growth rate to 30% . In non-growth conditions, treatment with 150 mM of AG for 3 h reduced cell survival to 60% and enhanced the frequency of isoleucine revertants 490 times and tryptophan-independent convertants 50 times . At equal survival levels, AG was found to be 3000-fold more mutagenic and 200-fold more convertogenic than sodium azide.

Mutagenesis, 1987 May, 2(3), 187 - 97
Induction of the cytoplasmic 'petite' mutation in pso mutants of Saccharomyces cerevisiae by photoaddition of furocoumarins or by ultraviolet radiation; Da Silva KV et al.; The induction of the cytoplasmic 'petite' mutation (or rho-) after photoaddition of either 8-methoxypsoralen (8-MOP) or 3-carbethoxypsoralen (3-CPs), after 254 nm u.v . and after 6-N-hydroxyaminopurine treatment was examined in three pso mutants in comparison to wild-type Saccharomyces cerevisiae . In three pso mutants which are defective in the induction of nuclear reverse and forward mutations, the photoaddition of 8-MOP enhanced the induction of rho- . This was true for cells in both exponential and stationary phases of growth . After photoaddition of 3-CPs in both growth phases the frequency of rho- was enhanced in pso3-1 whereas pso1-1 showed the same response as the wild-type . In pso2-1 the frequency of rho- was reduced . After treatment with 254 nm u.v . in the stationary phase of growth, rho- induction was increased in pso1-1 and pso3-1 cells as compared to wild-type cells . However, when treated in the exponential phase of growth all three pso mutants showed reduced rho- frequency . The data indicate that the defect in the repair of furocoumarins plus light-induced lesions controlled by nuclear genes (pso) interferes to various extents with the fate of mitochondrial lesions . The frequency of rho- mutants induced in the pso mutants by an analogue of adenine, 6-N-hydroxyaminopurine, was similar to that observed in the wild-type strain, suggesting that this drug may also act at the mitochondrial level as a direct mutagen in yeast.

Genes Dev, 1987 May, 1(3), 238 - 46
A novel role for the 3' region of introns in pre-mRNA splicing of Saccharomyces cerevisiae; Rymond BC et al.; To investigate the importance of sequences between the yeast (Saccharomyces cerevisiae) branch point (TACTAAC box) and 3' splice site (AG), we generated a series of pre-mRNA substrates that differed in the length of RNA retained on the 3' side of the TACTAAC box . These pre-mRNAs were compared as substrates for the first step of in vitro splicing (5' cleavage and lariat formation) and in vitro spliceosome assembly (complex formation) in a whole-cell yeast extract . The results indicate that for rp51A pre-mRNA at least 29 nucleotides of RNA on the 3' side of the TACTAAC box are required for 5' cleavage and lariat formation, as smaller substrates fail to manifest any detectable cleavage or ligation events . Analysis of splicing complex assembly indicates that these smaller substrates undergo efficient yet incomplete complex formation; they are blocked at a late stage of spliceosome assembly, the complex I to complex II transition (Pikielny et al . 1986), a result which suggests that the failure to form lariats is due to a specific assembly defect . The lariat formation block (and assembly defect) can be relieved by the addition of ribohomopolymer "tails" to the 3' end of the shortened rp51A pre-mRNAs, and similar results were obtained with shortened actin pre-mRNAs . The results of this study indicate that this region of the pre-mRNA serves a specific function late in in vitro spliceosome assembly.

Mech Ageing Dev, 1987 May, 38(3), 231 - 43
A formal mortality analysis for populations of unicellular organisms (Saccharomyces cerevisiae); Pohley HJ; A theoretical analysis of the reproductive capacity of yeast (Saccharomyces cerevisiae) under different experimental settings reveals interesting patterns and inter-experimental relations of mortality in yeast . The data on yeast lifespan were derived from experimental research by Meisel (Untersuchungen ueber die Korrelation von Abtoetung und Lebensspannenverkuerzung durch mutagene Agentien bei Hefezellen (Thesis) Cologne, 1984) . The analysis is based on the formulation of a "weak senescence principle" . From this the structure of age-specific mortality is defined and employed as the functional constituent of three hierarchical types of mortality models . Model A consists of one such constituent, model B includes many of these, and in model C mortality is given a phase structure in addition . The models incorporate a few basic hypotheses on "damage" and "ageing", on partitions into subpopulations, and on mortality "shifts", according to the particular experimental treatment . It is shown that the theoretical distributions derived from these models and hypotheses yield extremely good fits to the experimental data.

Genetika, 1987 May, 23(5), 784 - 92
{Mutants of Saccharomyces cerevisiae characterized by increased level of induced mutagenesis . I . Isolation and preliminary characterization of mutants}; Ivanov EL et al.; 6 mutants with enhanced nitrous acid-induced reversibility of the ade2-42 allele were isolated and designated hm (high mutagenesis) . Apart from sensitivity to the mutagenic exposure to nitrous acid, hm mutants were also spontaneous mutators and hypermutable under the action of UV-light and 6-N-hydroxyaminopurine . All these effects were detected not only when analysing reversibility of the ade2-42 allele, but also when scoring forward mutations in the ADE1, ADF2 genes . Gamma-mutagenesis, however, was not affected by hm mutations.

Mol Gen Mikrobiol Virusol, 1987 May, (5), 26 - 32
{Expression of human leukocyte interferon type A in Saccharomyces cerevisiae under the control of regulatory elements of the yeast gene URA3}; Nikoshkov AB et al.; Expression of a chromosomal gene for human leucocyte interferon A was obtained due to interaction of 5'-nontranslating region of a cloned interferon gene with the regulatory sequences from UNA3 yeast gene . The sequence of 5'-nontranslating region from interferon gene essential for its expression in yeast is localized within 130 b p . from the initiating codon . Due to increasing of plasmid stability and copy number a 60-fold increase . in interferon yield was obtained in yeast transformants reaching the level of 6 X 10(7) u X 1(-1) . The data are presented supposing the existence of functional polycistronic mRNA in yeast.

Mol Gen Genet, 1987 May, 207(2-3), 273 - 9
Autogenous regulation of the Saccharomyces cerevisiae regulatory gene GAL80; Igarashi M et al.; We have suggested previously from Northern blot analysis that transcription of the negative regulatory gene GAL80 was controlled positively by another regulatory gene GAL4, and negatively by GAL80 itself, in similar way to GAL1, GAL7 and GAL10 genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae . To study further the controlled expression of GAL80, we have exploited the gene fusion technique . We constructed gene fusions consisting of 5' fragments of GAL80 and a 5' truncated lacZ of Escherichia coli, and introduced the GAL80'-'lacZ fusions into wild-type yeast or various GAL4 or GAL80 mutants using multiple-copy or single-copy plasmid vectors . We then studied beta-galactosidase activity in the resultant transformants under uninduced, induced or glucose-repressed conditions . Expression of the GAL80'-'lacZ fusions was clearly under the control of Gal4/Gal80 . Next we constructed GAL7'-'lacZ fusions, whose upstream activating sequence (UAS) from GAL7 was replaced with a GAL80 fragment containing a UAS-like sequence located in the 5' flanking region of GAL80 . Synthesis of beta-galactosidase directed by the hybrid genes was inducible by galactose exactly like the original GAL7'-'lacZ fusion with a UAS from GAL7 . Finally we constructed a GAL7-GAL80 hybrid gene, in which the entire 5' flanking region was derived from GAL7 . When the chromosomal GAL80 gene in wild-type yeast was replaced with the hybrid gene, the uninduced level, but not the induced level, of the GAL10-encoded enzyme (uridine diphosphoglucose-4-epimerase) was significantly increased.

Genetics, 1987 May, 116(1), 9 - 22
Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae; Rine J et al.; Mating type interconversion in Saccharomyces cerevisiae occurs by transposition of copies of the a or alpha mating type cassettes from inactive loci, HML and HMR, to an active locus, MAT . The lack of expression of the a and alpha genes at the silent loci results from repression by trans-acting regulators encoded by SIR (Silent Information Regulator) genes . In this paper we present evidence for the existence of four SIR genes . Inactivation of any of these genes leads to expression of cassettes at both HML and HMR . Unusual complementation properties are observed for a number of sir mutations . Specifically, some recessive mutations in different genes fail to complement . The correspondence between SIR1, SIR2, SIR3, SIR4 and other genes with similar roles (MAR, CMT, STE8 and STE9) is presented.

Microbiol Sci, 1987 May, 4(5), 150 - 3
Multiplicity of regulatory mechanisms controlling amino acid biosynthesis in Saccharomyces cerevisiae; Messenguy F; Transcriptional, post-transcriptional and translational regulatory mechanisms control gene expression of amino acid biosynthetic pathways in yeast . All three mechanisms are involved in the control of arginine metabolism.

Mol Gen Genet, 1987 May, 207(2-3), 421 - 9
Processing of TY1 proteins and formation of Ty1 virus-like particles in Saccharomyces cerevisiae; Muller F et al.; We have analysed functional properties of putative proteins encoded by the yeast transposable element, Ty1, by overexpression of TY genes . High-level expression was achieved by appropriate fusion of a Ty sequence, TY9C, to the yeast ADH1 promoter and transformation of yeast cells with this construction . As shown recently by others (Garfinkel et al . 1985; Mellor et al . 1985c) TY overexpression leads to an increase in particle-bound reverse transcriptase activity and to an intracellular accumulation of virus-like particles (Ty-VLPs) . We have used a number of deletions in the second open reading frame (TYB) to identify functional domains required for processing and assembly of Ty proteins . Deletions in the TYB region with homology to acid proteases result in overproduction of an unprocessed form of the TYA protein (pro-TYA) which represents the major protein of Ty-VLPs . One particular mutant construction, TY9C-delta 36, led to the accumulation of a particle-bound, 160 kDa protein which cross-reacted with a mouse antiserum raised against purified pro-TYA protein . This supports the hypothesis that TYB is expressed as a TYA/TYB fusion protein which is processed by a TYB-encoded protease activity . Ty-VLPs are formed in the absence of protein processing and even when the TYB gene is not expressed . Thus, we assume that the assembly of Ty particles occurs prior to processing of Ty proteins.

Mol Cell Biol, 1987 May, 7(5), 1906 - 16
Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae; Slater MR et al.; The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes . We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression . Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock . Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region . The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG) . Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100 . The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS) . This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock . YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression . This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.

Mol Cell Biol, 1987 May, 7(5), 1764 - 75
Structure and expression of the Saccharomyces cerevisiae CRY1 gene: a highly conserved ribosomal protein gene; Larkin JC et al.; The Saccharomyces cerevisiae CRY1 gene encodes ribosomal protein rp59, a component of the 40S ribosomal subunit . Mutations in CRY1 can confer resistance to the alkaloid cryptopleurine, an inhibitor of the elongation step of translation . The nucleotide sequence of the cloned CRY1 gene was determined . The predicted amino acid sequence shows that CRY1 encodes a 14,561-dalton polypeptide that has 88% amino acid sequence homology to the hamster or human S14 ribosomal protein responsible for emetine resistance and 45% homology to Escherichia coli ribosomal protein S11 . Analysis of the DNA sequences upstream from CRY1 revealed the presence of three sequences, HOMOL1 (consensus, A/TACATCC/TG/ATA/GCA), RPG (consensus, ACCCA/GTACATT/CT/A), and a thymine-rich sequence, found upstream of more than 20 other cloned yeast genes encoding components of the translational apparatus . We exploited the ability to assay the expression of CRY1 in vivo by using the cryptopleurine resistance phenotype to demonstrate that these three consensus sequences are necessary for the transcription of CRY1 . We previously showed that the upstream promoter element of the yeast RP39A gene consists of these identical sequence motifs . Therefore, we suggest that these three sequences define a consensus promoter element for the genes encoding the yeast translational apparatus . CRY1 is one of several hundred yeast genes, including ribosomal protein genes, whose expression is transiently decreased 10-fold upon heat shock . We found that the HOMOL1 and RPG consensus sequences are not necessary for the heat shock response of CRY1.

J Biol Chem, 1987 Apr 25, 262(12), 5732 - 9
Two chitin synthases in Saccharomyces cerevisiae; Orlean P; Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C . E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W . L., and Robbins, P . W . (1986) Cell 46, 213-225) . It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity . Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain . Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues) . The enzymes are equally sensitive to the competitive inhibitor Polyoxin D . The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+ . In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E . (1986) Fed . Proc . 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis . Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change . Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures . Specific chitin synthase II activities do not change in pheromone-treated cultures . It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.

Nucleic Acids Res, 1987 Apr 24, 15(8), 3581 - 93
Compilation and comparison of the sequence context around the AUG startcodons in Saccharomyces cerevisiae mRNAs; Hamilton R et al.; The nucleotide sequence of the translation initiation regions of 96 Saccharomyces cerevisiae mRNAs was compiled and compared . The entire 5' untranslated sequence of most mRNAs is very rich in A-residues . G-residues are underrepresented in the untranslated region . The AUG startcodon context appeared to be distinctly different from that of animal mRNAs, although an A-residue at -3 also occurs very frequently (81 percent) in yeast mRNAs . The prevailing codon 3' adjacent to the AUG is the UCU serine codon . All these features are more extreme in the highly expressed genes . Fifty percent of all highly expressed genes use the UCU serine codon as second triplet . In this group G-residues are completely absent in the 7 bases preceding the startcodon and an A-residue occurs at position -1 and -3 at a frequency of 89 percent and 100 percent, respectively . The abundance of A-residues throughout the leader suggests that unstructured mRNA is required for efficient translation initiation in yeast . The consensus sequence for the AUG context in highly expressed genes can be summarized as follows: (Sequence: see text).

Nucleic Acids Res, 1987 Apr 24, 15(8), 3515 - 29
The untranslated leader of nuclear COX4 gene of Saccharomyces cerevisiae contains an intron; Schneider JC et al.; The nuclear gene for subunit IV of cytochrome oxidase (COX4) in Saccharomyces cerevisiae contains a 342 bp intron which is contained entirely within the 5' leader of the message . Splicing of the intron results in removal of several small open reading frames; subsequently, the COX4 AUG becomes the 5' proximal initiation codon . A strain with an rna2- mutation fails to splice mRNA efficiently at restrictive temperature and was used to map the intron splice junctions by RNase protection . Two major mRNA initiation sites were mapped by primer extension of synthetic oligodeoxynucleotides . The splice junctions and internal TACTAAC box conform to consensus sequences previously determined from other yeast introns . One gene for subunit V of cytochrome oxidase (COX5b) has also been shown to contain an intron . The significance of introns in two nuclear genes encoding subunits of cytochrome oxidase is discussed.

Eur J Biochem, 1987 Apr 15, 164(2), 369 - 73
Changes in the concentration of cAMP, fructose 2,6-bisphosphate and related metabolites and enzymes in Saccharomyces cerevisiae during growth on glucose; Francois J et al.; Changes in the concentration of several metabolites and enzymes related to carbohydrate metabolism were measured during the growth of Saccharomyces cerevisiae on a mineral medium containing glucose as the limiting nutrient . When about 50% of the original glucose was used the exponential phase ended and the culture entered a 'transition' phase before the complete exhaustion of glucose . In this transition phase several metabolic changes occurred . cAMP, that decreased along growth, reached a constant value of about 0.7 nmol/g dry weight . A pronounced drop in fructose-6-phosphate-2-kinase activity and in the concentration of fructose 2,6-bisphosphate and fructose 1,6-bisphosphate was observed accompanied by a less marked decrease in hexose monophosphates . Trehalase activity also dropped and reached a minimal value at the onset of the stationary phase when synthesis of trehalose began . Glycogen concentration and glycogen synthase activity increased sharply during the transition phase . Plasma membrane ATPase began to increase at the middle of the exponential phase and then, coincident with the glucose exhaustion, a 90% decrease in the measurable activity was observed.

J Theor Biol, 1987 Apr 7, 125(3), 269 - 81
Mathematical models for a G0 phase in Saccharomyces cerevisiae; Britton NF et al.; Three models for the cell cycle of the budding yeast, Saccharomyces cerevisiae, which include a reversible G0 phase during proliferation, are presented . The analysis gives estimates for various quantities of biological interest, such as the probability of entry to and rate of exit from the G0 phase, the average duration of a cell in the G0 phase, the cycle time of cycling daughters and the population doubling time of cycling cells, none of which is easily measurable.

J Biol Chem, 1987 Apr 5, 262(10), 4876 - 81
Primary structure and disruption of the phosphatidylinositol synthase gene of Saccharomyces cerevisiae; Nikawa J et al.; The wild-type yeast nuclear gene, PIS, encodes phosphatidylinositol synthase (CDPdiacylglycerol-inositol 3-phosphatidyltransferase, EC 2.7.8.11) (Nikawa, J., and Yamashita, S . (1984) Eur . J . Biochem . 143, 251-256) . We now report the sequence of the cloned 2, 129-base pair DNA and the location of the PIS coding region within the sequence . The PIS coding frame is capable of encoding 220 amino acid residues with a calculated molecular weight of 24,823 . On Northern blot analysis, an RNA species that hybridized with the coding region was detected in the total poly(A)+ RNA of the wild-type yeast . The primary translation product contains a region showing local sequence homology with yeast phosphatidylserine synthase (EC 2.7.8.8) and Escherichia coli 3-phosphatidyl-1'-glycerol-3'-phosphate synthase (EC 2.7.8.5), suggesting that these three enzymes are evolutionarily related . The PIS gene was disrupted in vitro through insertion of the yeast HIS3 gene into the coding region . A heterozygous diploid, PIS/pis::HIS3, constructed from a PIS/PIS his3/his3 diploid by replacing one of the wild-type PIS genes with the disrupted PIS gene, showed no segregation of viable His+ spores on tetrad analysis, indicating that disruption of the PIS gene is lethal . The nonviable spores were in an arrested state with a characteristic terminal phenotype, suggesting that the function of the PIS gene is essential for progression of the yeast cell cycle.

Genetics, 1987 Apr, 115(4), 627 - 36
Temperature-sensitive lethal pseudorevertants of ste mutations in Saccharomyces cerevisiae; Katz ME et al.; A procedure was devised to isolate mutations that could restore conjugational competence to temperature sensitive ste mutants and simultaneously confer temperature-sensitive lethal growth phenotypes . Three such mutations, falling into two complementation groups, were identified on the basis of suppression of ste5 alleles . These same mutations were later shown to be capable of suppressing ste4 and ste7 alleles . Five mutations in a single complementation group were isolated as suppressors of ste2 alleles . None of the mutations described in this study conferred a homogeneous cell cycle arrest phenotype, and all were shown to define complementation groups distinct from those previously identified in studies of cell division cycle (cdc) mutations . In no instance did pseudoreversion appear to be achieved by mutational G1 arrest of ste mutant cells . Instead, it is proposed that the mutations restore conjugation by reestablishing the normal pheromone response.

Mutat Res, 1987 Apr, 187(4), 209 - 17
Mutagenic, recombinogenic and antimitochondrial effects of nitracrine analogues in Saccharomyces cerevisiae; Ferguson LR et al.; The mutagenic and recombinogenic potential of 9-{(3-dimethylaminopropyl)amino}acridine and its 1-, 2-, 3- and 4-nitro derivatives was studied using 3 different strains of Saccharomyces cerevisiae . The parent compound slightly enhanced the frequency of total aberrant colonies in diploid strains D5 and D7, but showed no evidence of recombinogenic effects . Each of the nitroacridines enhanced the frequency of total aberrant colonies in strains D5 and D7, but only the 1- and 4-nitro compounds significantly enhanced mitotic crossing-over (measured as twin spotted colonies in strains D5 and D7) or gene conversion in D7 . The 3- and 4-nitro derivatives were effective mitochondrial mutagens, substantially increasing the frequency of 'petite' mutants in strains D5 and 5178B.

J Bacteriol, 1987 Apr, 169(4), 1684 - 90
Regulation of allantoate transport in wild-type and mutant strains of Saccharomyces cerevisiae; Chisholm VT et al.; Accumulation of intracellular allantoin and allantoate is mediated by two distinct active transport systems in Saccharomyces cerevisiae . Allantoin transport (DAL4 gene) is inducible, while allantoate uptake is constitutive (it occurs at full levels in the absence of any allantoate-related compounds from the culture medium) . Both systems appear to be sensitive to nitrogen catabolite repression, feedback inhibition, and trans-inhibition . Mutants (dal5) that lack allantoate transport have been isolated . These strains also exhibit a 60% loss of allantoin transport capability . Conversely, dal4 mutants previously described are unable to transport allantoin and exhibit a 50% loss of allantoate transport . We interpret the pleiotropic behavior of the dal4 and dal5 mutations as deriving from a functional interaction between elements of the two transport systems.

J Bacteriol, 1987 Apr, 169(4), 1656 - 62
The SNF3 gene is required for high-affinity glucose transport in Saccharomyces cerevisiae; Bisson LF et al.; Glucose uptake mutants have not been previously obtained in Saccharomyces cerevisiae, possibly because there seem to be at least two transport systems, of low and high affinities . We showed that snf3 (sucrose nonfermenting) mutants did not express high-affinity glucose uptake . Furthermore, their growth was completely impaired on low concentrations of glucose in the presence of antimycin A (which blocks respiration) . Several genes which complemented the original snf3 gene were obtained on multicopy plasmids . Some of them, as well as plasmid-carried SNF3 itself, conferred a substantial increase in high-affinity glucose uptake in both snf3 and wild-type hosts . The effects of glucose on the expression of such a plasmid-determined high-affinity uptake resembled those in the wild type . Other genes complementing snf3 seemed to cause an increase in low-affinity glucose uptake . We suggest that SNF3 may function specifically in high-affinity glucose uptake, which is needed under some conditions of growth on low glucose concentrations . SNF3 itself or the other complementing genes may specify components of the glucose uptake system.

J Bacteriol, 1987 Apr, 169(4), 1571 - 8
RRP1, a Saccharomyces cerevisiae gene affecting rRNA processing and production of mature ribosomal subunits; Fabian GR et al.; The Saccharomyces cerevisiae mutant ts351 had been shown to affect processing of 27S pre-rRNA to mature 25S and 5.8S rRNAs (C . Andrew, A . K . Hopper, and B . D . Hall, Mol . Gen . Genet . 144:29-37, 1976) . We showed that this strain contains two mutations leading to temperature-sensitive lethality . The rRNA-processing defect, however, is a result of only one of the two mutations . We designated the lesion responsible for the rRNA-processing defect rrp1 and showed that it is located on the right arm of chromosome IV either allelic to or tightly linked to mak21 . This rrp1 lesion also results in hypersensitivity to aminoglycoside antibiotics and a reduced 25S/18S rRNA ratio at semipermissive temperatures . We cloned the RRP1 gene and provide evidence that it encodes a moderately abundant mRNA which is in lower abundance and larger than most mRNAs encoding ribosomal proteins.

J Gen Microbiol, 1987 Apr, 133 ( Pt 4), 843 - 8
Changes in 45Ca and 109Cd uptake, membrane potential and cell pH in Saccharomyces cerevisiae provoked by Cd2+; Kessels BG et al.; The effect of Cd2+ poisoning of Saccharomyces cerevisiae on 45Ca, 109Cd and {14C}tetraphenylphosphonium (TPP) uptake and cell pH was examined . At Cd2+ concentrations that produced substantial K+ efflux the rates of uptake of 45Ca, 109Cd and {14C}TPP increased progressively during incubation of the cells with Cd2+, and the cell pH was lowered concomitantly . The initial rates of uptake of the divalent cations and of TPP were increased in cells pre-loaded with Cd2+, which shows that stimulation of the ion fluxes was exerted by the Cd2+ that accumulated in the cells . The distribution ratio of TPP between cells and medium, however, was decreased by Cd2+ . Although hyperpolarization of the cell membrane by Cd2+ cannot be excluded, it is argued that Cd2+ primarily stimulated divalent cation uptake by increasing the cation permeability of the cell membrane allowing the cations to enter the cells more easily.

Antimicrob Agents Chemother, 1987 Apr, 31(4), 512 - 7
Killing of Saccharomyces cerevisiae by the lysosomotropic detergent N-dodecylimidazole; Hussain M et al.; The lysosomotropic detergent N-dodecylimidazole (C12-Im) has previously been found to kill mammalian cells by concentrating in lysosomes, followed by lysosomal disruption and release of cytotoxic enzymes into the cytoplasm . The action of C12-Im on Saccharomyces cerevisiae is described in this report . C12-Im prevented growth of colonies when present in 1% yeast extract-2% Bacto-Peptone-2% glucose plates at concentrations of 5 micrograms/ml or above, or when present in a soft agar overlay at 20 micrograms/ml . Treatment of cells suspended in glucose-containing buffer (pH 8.0, 37 degrees C) with C12-Im (6 micrograms/ml) caused greater than 95% cell death within 6 min . Dependence of killing on C12-Im concentration was sigmoidal, suggesting a cooperative mode of action . Killing was pH dependent, being much more effective at pH 8.0 than at pH 5.0 . Ammonium sulfate and imidazole protected against killing if added before, but not after, the addition of C12-Im . Sensitivity to C12-Im was strongly growth dependent: the cells were most sensitive at early to mid-logarithmic phase of growth and became progressively less sensitive during progression through late logarithmic and stationary phase . Vacuolar disruption by C12-Im was demonstrated by using cells loaded with lucifer yellow CH or fluoresceinated dextran in their vacuoles; vacuoles of logarithmically growing cells were more sensitive than those of stationary-phase cells . These results suggest that vacuolar disruption by C12-Im may underlie its cytotoxic effects.

Mol Gen Genet, 1987 Apr, 207(1), 165 - 70
Molecular genetics of met 17 and met 25 mutants of Saccharomyces cerevisiae: intragenic complementation between mutations of a single structural gene; D'Andrea R et al.; We cloned the MET 17 gene of Saccharomyces cerevisiae by functional complementation after transformation of a yeast met 17 mutant . Restriction mapping and nucleotide sequencing of the MET 17 clones revealed that these were from the same genomic region as clones isolated previously and shown to contain the MET 25 gene encoding the enzyme O-acetylhomoserine, O-acetylserine sulphydrylase (OAH-OAS sulphydrylase) . Transformation studies with MET 25 clones showed that the MET 17 and MET 25 functions were both endoced in a single transcription unit . We conclude that met 17 and met 25 are both mutations in the structural gene for the OAH-OAS sulphydrylase subunit and that each affects a different functional domain of the enzyme allowing subunit complementation in the met 17 X met 25 diploid . Enzyme assays indicated that the diploid, although not requiring methionine, had a low OAH-OAS sulphydrylase activity (10% of wild type) . This is consistent with MET 17 and MET 25 being the same gene . We found that both met 17 and met 25 mutants were devoid of 3' phospho-adenosine 5' phospho-sulphite (PAPS) reductase activity and that this activity was fully restored in the met 17 X met 25 diploid . The possible interactions between OAH-OAS sulphydrylase and PAPS reductase are discussed.

Can J Microbiol, 1987 Apr, 33(4), 331 - 5
Cell surface specific immunoglobulin inhibits alpha factor mediated morphogenesis in Saccharomyces cerevisiae; Merkel GJ et al.; Immunoglobulins raised from Saccharomyces cerevisiae a and alpha mating type cell envelope preparations inhibited alpha factor mediated morphogenesis of the a cell without inhibiting normal cell division . The Ig responsible for this inhibition was absorbed to both a and alpha whole cells and heat-killed cells, indicating that the immunoglobulin binding sites were exposed on the cell surface and not mating type specific . Additionally, alpha factor mediated cell cycle arrest was not affected by the immunoglobulin preparations, implying that the immunoglobulin was not preventing alpha factor from binding to its receptor.

Int J Radiat Biol Relat Stud Phys Chem Med, 1987 Apr, 51(4), 655 - 64
Contribution of a photoreactivable component to the oxygen effect observed in certain rad mutants of Saccharomyces cerevisiae after exposure to high energy electrons; Tsyb TS et al.; It is shown that a fraction of damage induced by high energy electrons (25 MeV) in certain rad mutants of the yeast Saccharomyces cerevisiae can be photoreactivated . The photoreactivable damage contributes to the lethal effect of this type of irradiation and modifies the oxygen effect . Using photoreactivating light or nigrosin, the amount of photoreactivable damage is reduced and the oxygen enhancement ratio (OER) for yeast mutants increases approximately to the OER found in wild-type cells.

Mol Cell Biol, 1987 Apr, 7(4), 1311 - 9
Saccharomyces cerevisiae mutants unresponsive to alpha-factor pheromone: alpha-factor binding and extragenic suppression; Jenness DD et al.; Mutations in six genes that eliminate responsiveness of Saccharomyces cerevisiae a cells to alpha-factor were examined by assaying the binding of radioactively labeled alpha-factor to determine whether their lack of responsiveness was due to the absence of alpha-factor receptors . The ste2 mutants, known to be defective in the structural gene for the receptor, were found to lack receptors when grown at the restrictive temperature; these mutations probably affect the assembly of active receptors . Mutations in STE12 known to block STE2 mRNA accumulation also resulted in an absence of receptors . Mutations in STE4, 5, 7, and 11 partially reduced the number of binding sites, but this reduction was not sufficient to explain the loss of responsiveness; the products of these genes appear to affect postreceptor steps of the response pathway . As a second method of distinguishing the roles of the various STE genes, we examined the sterile mutants for suppression . Mating of the ste2-3 mutant was apparently limited by its sensitivity to alpha-factor, as its sterility was suppressed by mutation sst2-1, which leads to enhanced alpha-factor sensitivity . Sterility resulting from each of four ste4 mutations was suppressed partially by mutation sst2-1 or by mutation bar1-1 when one of three other mutations (ros1-1, ros2-1, or ros3-1) was also present . Sterility of the ste5-3 mutant was suppressed by mutation ros1-1 but not by sst2-1 . The ste7, 11, and 12 mutations were not suppressed by ros1 or sst2 . Our working model is that STE genes control the response to alpha-factor at two distinct steps . Defects at one step (requiring the STE2 gene are suppressed (directly or indirectly) by mutation sst2-1, whereas defects at the other step (requiring the STE5 gene) are suppressed by the ros1-1 mutation . The ste4 mutants are defective for both steps . Mutation ros1-1 was found to be allelic to cdc39-1 . Map positions for genes STE2, STE12, ROS3, and FUR1 were determined.

Arch Microbiol, 1987 Apr, 147(3), 231 - 4
Catabolite inactivation of isocitrate lyase from Saccharomyces cerevisiae; Lopez-Boado YS et al.; A reversible carbon catabolite inactivation step is described for isocitrate lyase from Saccharomyces cerevisiae . This reversible inactivation step of isocitrate lyase is similar to that described for fructose 1,6-bisphosphatase . Addition of 2,4-dinitrophenol, nystatin or glucose to cultures, grown in ethanol as carbon source, caused a rapid loss of the isocitrate lyase and fructose 1,6-bisphosphatase activities at pH 5.5 but not at pH 7.5 . These results suggest that intracellular acidification and thus a cAMP increase is involved in the catabolite inactivation mechanism of both enzymes . From results obtained by addition of glucose to yeast cultures at pH 7.5 it was concluded that others factors than cAMP can play a role in the catabolite inactivation mechanism of both enzymes.

Genetics, 1987 Apr, 115(4), 637 - 47
PET111, a Saccharomyces cerevisiae nuclear gene required for translation of the mitochondrial mRNA encoding cytochrome c oxidase subunit II; Poutre CG et al.; Mutations in the nuclear gene PET111 are recessive and specifically block accumulation of cytochrome c oxidase subunit II (coxII), the product of a mitochondrial gene . However, the coxII mRNA is present in pet111 mutants at a level approximately one-third that of wild type . The simplest explanation for this phenotype is that PET111 is required for translation of the coxII mRNA . The reduced steady-state level of this mRNA is probably a secondary effect, caused by increased degradation of the untranslated transcript . Mitochondrial suppressors of pet111, carried on rho-mtDNAs, bypass the requirement for PET111 in coxII translation . Three suppressors are fusions between the coxII structural gene and other mitochondrial genes, that encode chimeric proteins consisting of the N-terminal portions of other mitochondrially coded proteins fused to the coxII precursor protein . When present together with rho+ mtDNA in a heteroplasmic state, these suppressors allow coxII synthesis in pet111 mutants . Thus in wild type, the PET111 product, or something under its control, probably acts at a site coded in the proximal portion of the gene for coxII to promote translation of the mRNA . PET111 was isolated by molecular cloning and genetically mapped to a position approximately midway between rna1 and SUP8 on chromosome XIII.

Proc Natl Acad Sci U S A, 1987 Apr, 84(8), 2140 - 4
Occurrence in Saccharomyces cerevisiae of a gene homologous to the cDNA coding for the alpha subunit of mammalian G proteins; Nakafuku M et al.; From cross-hybridization studies with cDNAs that code for the alpha subunits of rat brain guanine nucleotide-binding regulatory (G) proteins, we have isolated a gene from yeast Saccharomyces cerevisiae encoding an amino acid sequence that is highly homologous to the alpha subunit of the G protein that mediates inhibition of adenylate cyclase (Gi alpha) from rat brain . The gene, tentatively designated as GPA1, contains a contiguous, single open reading frame of 1416 nucleotides that codes for a protein of 472 amino acids with a calculated Mr of 54,075 . The predicted amino acid sequence of the protein encoded by the GPA1 gene (tentatively designated as G protein 1 alpha or GP1 alpha) is remarkably homologous to the amino acid sequence of rat brain Gi alpha and the alpha subunit of the G protein of unknown function (Go alpha); the primary structure of the sites for GTP hydrolysis as well as GTP interaction are nearly identical . The main difference in the molecular sizes of yeast GP1 alpha (472 amino acids) and rat brain Gi alpha (355 amino acids) is due to the presence of a stretch of 110 extra amino acid residues in yeast GP1 alpha, which are inserted near the NH2-terminal one-third of mammalian Gi alpha . From blot-hybridization analysis, the size of the GP1 alpha mRNA was estimated as 1.7 kilobases.

J Gen Microbiol, 1987 Apr, 133 ( Pt 4), 857 - 65
Effect of ethanol on activity of the plasma-membrane ATPase in, and accumulation of glycine by, Saccharomyces cerevisiae; Cartwright CP et al.; The pH optimum of the ATPase activity in plasma membranes from Saccharomyces cerevisiae NCYC 431 from 8 h cultures was around 6.5 and that in membranes from organisms from 16 h cultures near 6.0 . The Km{ATP} of the enzyme was virtually unaffected by the age of the culture from which organisms were harvested, although the Vmax of the enzyme in membranes from organisms from 8 h cultures was higher than that for organisms from 16 h cultures . Ethanol non-competitively inhibited ATPase activity in membranes, although the inhibition constant for the enzyme from organisms from 8 h cultures was lower than that from organisms from 16 h cultures . Glycine accumulation by the general amino acid permease was non-competitively inhibited by ethanol . Inhibition constants were virtually the same for glycine uptake by deenergized organisms from 8 h and 16 h cultures, but under energized conditions the value was greater for organisms from 16 h rather than 8 h cultures . The data indicate that inhibition of plasma-membrane ATPase activity by ethanol could account, at least in part, for inhibition of glycine accumulation by ethanol.

Mol Gen Genet, 1987 Apr, 207(1), 38 - 46
Genetic and molecular mapping of the pma1 mutation conferring vanadate resistance to the plasma membrane ATPase from Saccharomyces cerevisiae; Ulaszewski S et al.; In the yeast Saccharomyces cerevisiae, the pma1 mutations confers vanadate-resistance to H+-ATPase activity when measured in isolated plasma membranes . In vivo, the growth of pma1 mutants is resistant to Dio-9, ethidium bromide and guanidine derivatives . This phenotype was used to map the pma1 mutation adjacent to LEU1 gene on chromosome VII . From a cosmid library of a wild-type Saccharomyces cerevisiae genome, a large 30 kb DNA fragment was isolated by complementation of a leu1-pma1 double mutant . A 5kb HindIII fragment was subcloned and it restored both Leu+ and Pma+ phenotypes after integrative transformation . The restriction map of the 5 kb HindIII fragment and Southern blot analysis reveal that the cloned fragment contains the entire structural gene for the plasma membrane ATPase and the 5' end of the adjacent LEU1 gene . The pma1 mutation conferring vanadate-resistance is thus located in the structural gene for the plasma membrane ATPase.

Nucleic Acids Res, 1987 Mar 25, 15(6), 2417 - 29
Messenger RNA stability in Saccharomyces cerevisiae: the influence of translation and poly(A) tail length; Santiago TC et al.; A comparison between the half-lives of 10 specific yeast mRNAs and their distribution within polysomes (fractionated on sucrose density gradients) was used to test the relationship between mRNA translation and degradation in the eukaryote Saccharomyces cerevisiae . Although the mRNAs vary in their distribution across the same polysome gradients, there is no obvious correlation between the stability of an mRNA and the number of ribosomes it carries in vivo . This suggests that ribosomal protection against nucleolytic attack is not a major factor in determining the stability of an mRNA in yeast . The relative lengths of the poly(A) tails of 9 yeast mRNAs were analysed using thermal elution from poly(U)-Sepharose . No dramatic differences in poly(A) tail length were observed amongst the mRNAs which could account for their wide ranging half-lives . Minor differences were consistent with shortening of the poly(A) tail as an mRNA ages.

J Biol Chem, 1987 Mar 15, 262(8), 3909 - 17
Mutants of Saccharomyces cerevisiae defective in sn-1,2-diacylglycerol cholinephosphotransferase . Isolation, characterization, and cloning of the CPT1 gene; Hjelmstad RH et al.; A colony autoradiographic assay for the sn-1,2-diacylglycerol cholinephosphotransferase activity in Saccharomyces cerevisiae was developed . Twenty-two mutants defective in cholinephosphotransferase activity were isolated . Genetic analysis revealed that all of these mutations were recessive, and three complementation groups were identified . The cholinephosphotransferase activities in membranes prepared from cpt1 mutants were reduced 2-10-fold compared to wild-type activity . The cholinephosphotransferase activities of two cpt1 isolates differed from wild-type activity with respect to their apparent KM for CDP-choline . The residual cholinephosphotransferase activities of cpt1 isolates were more sensitive to inhibition by CMP than the wild-type activity . The CPT1 gene was cloned by genetic complementation of cpt1 using a yeast genomic library . In strains transformed with the CPT1-bearing plasmid, a 5-fold overproduction of cholinephosphotransferase activity with wild-type kinetic properties was observed . The CPT1 gene was localized to a 1.2-2.4-kilobase region of DNA by transposon Tn5 mutagenesis and deletion mapping . An insertional mutant of the CPT1 gene was constructed and introduced into the chromosome by integrative transformation . The resulting cpt insertional mutant fell into the cpt1 complementation group . The cholinephosphotransferase activity in membranes prepared from the cpt1 insertional mutant was reduced 5-fold and exhibited CMP sensitivity . The sn-1,2-diacylglycerol ethanolaminephosphotransferase activities in membranes from all of the cpt1 isolates including the insertional mutant were normal . The data indicate that the cloned CPT1 gene represents the yeast cholinephosphotransferase structural gene, that the yeast choline- and ethanolaminephosphotransferase activities are encoded by different genes, and that the CPT1 gene is nonessential for growth.

Science, 1987 Mar 6, 235(4793), 1218 - 21
CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae; Robinson LC et al.; The yeast Saccharomyces cerevisiae contains two functional homologues of the ras oncogene family, RAS1 and RAS2 . These genes are required for growth, and all evidence indicates that this essential function is the activation of adenylate cyclase . In contrast, ras in mammalian cells does not appear to influence adenylate cyclase activity . To clarify the relation between ras function in yeast and in higher eukaryotes, and the role played by yeast RAS in growth control, it is necessary to identify functions acting upstream of RAS in the adenylate cyclase pathway . The evidence presented here indicates that CDC25, identified by conditional cell cycle arrest mutations, encodes such an upstream function.

J Mol Biol, 1987 Mar 5, 194(1), 41 - 58
Frameshift suppressor mutations affecting the major glycine transfer RNAs of Saccharomyces cerevisiae; Mendenhall MD et al.; Mutations have been identified in Saccharomyces cerevisiae glycine tRNA genes that result in suppression of +1 frameshift mutations in glycine codons . Wild-type and suppressor alleles of genes encoding the two major glycine tRNAs, tRNA(GCC) and tRNA(UCC), were examined in this study . The genes were identified by genetic complementation and by hybridization to a yeast genomic library using purified tRNA probes . tRNA(UCC) is encoded by three genes, whereas approximately 15 genes encode tRNA(GCC) . The frameshift suppressor genes suf1+, suf4+ and suf6+ were shown to encode the wild-type tRNA(UCC) tRNA . The suf1+ and suf4+ genes were identical in DNA sequence, whereas the suf6+ gene, whose DNA sequence was not determined, was shown by a hybridization experiment to encode tRNA(UCC) . The ultraviolet light-induced SU F1-1 and spontaneous SU F4-1 suppressor mutations were each shown to differ from wild-type at two positions in the anticodon, including a +1 base-pair insertion and a base-pair substitution . These changes resulted in a CCCC four-base anticodon rather than the CCU three-base anticodon found in wild-type . The RNA sequence of tRNA(UCC) was shown to contain a modified uridine in the wobble position . Mutant tRNA(CCCC) isolated from a SU F1-1 strain lacked this modification . Three unlinked genes that encode wild-type tRNA(GCC), suf20+, trn2, and suf17+, were identical in DNA sequence to the previously described suf16+ frameshift suppressor gene . Spontaneous suppressor mutations at the SU F20 and SU F17 loci were analyzed . The SU F20-2 suppressor allele contained a CCCC anticodon . This allele was derived in two serial selections through two independent mutational events, a +1 base insertion and a base substitution in the anticodon . Presumably, the original suppressor allele, SU F20-1, contained the single base insertion . The SU F17-1 suppressor allele also contained a CCCC anticodon resulting from two mutations, a +1 insertion and a base substitution . However, this allele contained an additional base substitution at position 33 adjacent to the 5' side of the four-base anticodon . The possible origin and significance of multiple mutations leading to frameshift suppression is discussed.

Genetika, 1987 Mar, 23(3), 421 - 30
{Genetic study of plasmid integration into yeast chromosomes . III . Selection for a phenotype of Saccharomyces cerevisiae (Meyen ex Hansen) clones which lost the 2-micron DNA and their genetic testing}; Bulat SA et al.; We used the coloured adeI (cir+) haploid strain containing an episomal plasmid integrated into the chromosome I for visual detection and genetic testing of Saccharomyces cerevisiae clones having lost 2 microns DNA . During incubation, colonies of this strain were covered with numerous papillae of the same genotype . Stable clones which did not generate such papillae were isolated . Hybrids of these clones with (cir0) partner were not shown to exhibit destabilization of the chimeric chromosome . The stable clones isolated proved to lack 2 microns DNA, as shown by colony hybridization technique . We conclude therefore that the loss of the cryptic yeast plasmid may be phenotypically detected.

Genetics, 1987 Mar, 115(3), 451 - 60
Allosuppressors that enhance the efficiency of omnipotent suppressors in Saccharomyces cerevisiae; Song JM et al.; Two recessive Mendelian-allosuppressors have been isolated and have been shown to enhance the efficiency of omnipotent suppressors thought to be translational ambiguity mutations . These allosuppressors are unlinked to each other or to the omnipotent suppressors on which they act . They also increase the efficiency of the serine-inserting UAA-suppressor, SUP16 . One allosuppressor is allelic or tightly linked to the previously isolated sal2 . Another allosuppressor, called sal6, represents a new locus, unlinked to the previously isolated sal1-sal5 that enhance the efficiency of the UAA-suppressors . When present singly in the absence of suppressors or other modifiers the sal2 and sal6 mutations do not have suppressor activity . However, when sal2 and sal6 are combined together in a haploid cell they do suppress weakly . In addition sal2 becomes a weak suppressor in the presence of the {eta +} modifying factor.

Genetics, 1987 Mar, 115(3), 441 - 9
STE16, a new gene required for pheromone production by a cells of Saccharomyces cerevisiae; Wilson KL et al.; Genes required for mating by a and alpha cells of Saccharomyces cerevisiae (STE, "sterile," genes) encode products such as peptide pheromones, pheromone receptors, and proteins responsible for pheromone processing . a-specific STE genes are those required for mating by a cells but not by alpha cells . To identify new a-specific STE genes, we have employed a novel strategy that enabled us to determine if a ste mutant defective in mating as a is also defective in mating as alpha without the need to do crosses . This technique involved a strain (K12-14b) of genotype mata1 HML alpha HMR alpha sir3ts, which mates as a at 25 degrees and as alpha at 34 degrees . We screened over 40,000 mutagenized colonies derived from K12-14b and obtained 28 a-specific ste mutants . These strains contained mutations in three known a-specific genes--STE2, STE6 and STE14--and in a new gene, STE16 . ste16 mutants are defective in the production of the pheromone, a-factor, and exhibit slow growth . Based on the distribution of a-specific ste mutants described here, we infer that we have identified most if not all nonessential genes that can give rise to a-specific mating defects.

Mol Cell Biol, 1987 Mar, 7(3), 998 - 1003
mRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae; Altmann M et al.; We have isolated genomic and cDNA clones encoding protein synthesis initiation factor eIF-4E (mRNA cap-binding protein) of the yeast Saccharomyces cerevisiae . Their identity was established by expression of a cDNA in Escherichia coli . This cDNA encodes a protein indistinguishable from purified eIF-4E in terms of molecular weight, binding to and elution from m7GDP-agarose affinity columns, and proteolytic peptide pattern . The eIF-4E gene was isolated by hybridization of cDNA to clones of a yeast genomic library . The gene lacks introns, is present in one copy per haploid genome, and encodes a protein of 213 amino acid residues . Gene disruption experiments showed that the gene is essential for growth.

Mol Cell Biol, 1987 Mar, 7(3), 1233 - 41
Transcription of the ADH2 gene in Saccharomyces cerevisiae is limited by positive factors that bind competitively to its intact promoter region on multicopy plasmids; Irani M et al.; Transcription of the ADH2 gene in the yeast Saccharomyces cerevisiae was inhibited by excess copies of its own promoter region . This competition effect was promoter specific and required the upstream activation sequence of ADH2 as well as sequences 3' to the TATA box . Introducing excess copies of ADR1, an ADH2-specific regulatory gene, did not alleviate the competition that was observed in these circumstances during both constitutive and derepressed ADH2 expression . Excess copies of the upstream region did not release ADH2 from glucose repression, consistent with the view that ADH2 is regulated by positive trans-acting factors.

Mol Cell Biol, 1987 Mar, 7(3), 1198 - 207
Concerted deletions and inversions are caused by mitotic recombination between delta sequences in Saccharomyces cerevisiae; Rothstein R et al.; Deletions of a tyrosine tRNA suppressor gene, SUP4-o, are mediated by recombination between short repeated delta sequences in Saccharomyces cerevisiae . The arrangement of the five solo delta sequences that surround the SUP4 locus was established by DNA sequence analysis . Seven deletion classes were identified by genomic blotting . DNA sequence analysis also showed that the delta sequences within a 6.5-kilobase region of the SUP4 locus were the endpoints of these events . In three of these classes, an adjacent interval surrounded by delta sequences was inverted in concert with the deletion . The frequency of all deletion classes decreased in strains that contained mutations in the recombination and repair gene RAD52 . We present two gene conversion mechanisms by which these rearrangements could have been generated . These models may also explain deletions between repeated sequences in other systems.

Mol Cell Biol, 1987 Mar, 7(3), 1180 - 92
RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli; Fleer R et al.; In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J . Mol . Biol . 183:31-42, 1985) . We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al . (Mol . Cell . Biol . 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2 . When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10% . However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants . Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid . The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost . We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping . The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene . Both genes are transcribed in the same direction . RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide . The site of inactivation of RAD4 in a particular plasmid propagated in E . coli was localized to a 100-base-pair region by gene disruption and gap repair experiments . In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

Mol Cell Biol, 1987 Mar, 7(3), 1078 - 84
Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage; Cole GM et al.; The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination . RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not . To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli beta-galactosidase gene . Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed beta-galactosidase activity constitutively . In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock . The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate . Typical induction levels for RAD54 in cells exposed to such agents were from 3- to 12-fold, in good agreement with previous mRNA analyses . When MATa cells were arrested in G1 with alpha-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle . Using a yeast vector containing the EcoRI structural gene fused to the GAL1 promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.

Mol Cell Biol, 1987 Mar, 7(3), 1012 - 20
Nucleotide sequence and functional analysis of the RAD1 gene of Saccharomyces cerevisiae; Reynolds P et al.; The RAD1 gene of Saccharomyces cerevisiae is involved in excision repair of damaged DNA . The nucleotide sequence of the RAD1 gene presented here shows an open reading frame of 3,300 nucleotides . Two ATG codons occur in the open reading frame at positions +1 and +334, respectively . Since a deletion of about 2.7 kilobases of DNA from the 5' region of the RAD1 gene, which also deletes the +1 ATG and 11 additional codons in the RAD1 open reading frame, partially complements UV sensitivity of a rad1 delta mutant, we examined the role of the +1 ATG and +334 ATG codons in translation initiation of RAD1 protein . Mutation of the +1 ATG codon to ATC affected the complementation ability of the RAD1 gene, whereas mutation of the +334 ATG codon to ATC showed no discernible effect on RAD1 function . These results indicate that translation of RAD1 protein is initiated from the +1 ATG codon . Productive in-frame RAD1-lacZ fusions showed that the RAD1 open reading frame is expressed in yeasts . The RAD1-encoded protein contains 1,100 amino acids with a molecular weight of 126,360.

Yeast, 1987 Mar, 3(1), 11 - 21
Identification of polypeptides of the carbon metabolism machinery on the two-dimensional protein map of Saccharomyces cerevisiae . Location of 23 additional polypeptides; Bataille N et al.; Using a modification of the basic two-dimensional polyacrylamide gel electrophoresis technique, we have undertaken a systematic identification of the polypeptides of the protein map of Saccharomyces cerevisiae corresponding to components of the carbon metabolism machinery . To the previous location of nine glycolytic enzyme polypeptides on the yeast protein map we add the location of 23 polypeptides . Ten of them were identified as corresponding to cytoplasmic enzymes of the carbon metabolism machinery and 13 were characterized as mitochondrial proteins . The criteria used to establish the identification of these polypeptides spots include migration with purified proteins, immunodetection, overproduction by plasmid-carrying strains and physiological behaviour.

Yeast, 1987 Mar, 3(1), 1 - 4
Cell division age dependency of meiosis in an apomictic variant of Saccharomyces cerevisiae; Bilinski CA et al.; The cell division age dependency of sporulation was investigated in a diploid strain of Saccharomyces cerevisiae (19el) which undergoes a single equational nuclear division during sporulation with consequent formation of asci containing two uninucleate diploid spores (apomictic dyads) . Under modified nutritional conditions which partially restore meiosis and hence normal tetrad formation, newly formed (age 0) daughter cells were observed to be capable of formation of apomictic dyads but not of meiotic tetrads . Even under conditions in which only apomictic dyads developed, approximately 20% of the asci resulted from differentiation of newborn 'inexperienced' cells . Thus, the data indicated production of at least one bud to be a prerequisite for meiosis but not for apomixis; however, occurrence of at least one complete mitotic cell division cycle was evidently insufficient for the morphogenetic switch from diploid to haploid spore formation, since older cells bearing several bud scars often underwent apomictic dyad development, and some produced no spores.

Plasmid, 1987 Mar, 17(2), 171 - 2
A vector for construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae; Khan MI et al.; We have constructed a convenient new vector, YEp-DE, for the construction of gene libraries and the expression of heterologous genes in Saccharomyces cerevisiae . The vector contains the yeast LEU2 gene, the 2 mu origin of replication, and a region from pUC18 that includes the ampr gene, the Escherichia coli origin of replication (ori), and the LacZ gene with multiple cloning sites . Five sites (Sac1, Sma1, BamH1, Sal1, Sph1) in this region are unique . This vector has advantages over similar yeast-E . coli shuttle vectors: small size (7291 bp, entirely sequenced), convenient cloning sites, and lacZ selection for detecting recombinant plasmids.

Biochem Int, 1987 Mar, 14(3), 569 - 80
Adenosine deaminase from Saccharomyces cerevisiae: purification and characterization; Marmocchi F et al.; Adenosine deaminase from Saccharomyces Cerevisiae was purified about 1600 fold by salt fractionation, ion exchange and affinity chromatography . Some physico-chemical properties have been determined: the molecular weight of the enzyme by gel filtration is 85,000 daltons; one -SH is readily titrated by paramercuribenzoate per 78,000 mol . weight; optimum pH is 7; Km for adenosine is 40.7 microM; 2'-deoxyadenosine is not a substrate . Deazaadenosine analogues are good inhibitors, while erythro-9-(2-hydroxy-3-nonyl) adenine binds with low affinity . These properties are compared with those of other adenosine deaminases.

Arch Microbiol, 1987 Mar, 147(2), 105 - 8
Effect of ozone on ATP, cytosolic enzymes and permeability of Saccharomyces cerevisiae; Hinze H et al.; Treatment of a yeast suspension with ozone inactivates a number of cytosolic enzymes . Among 15 studied, the most drastic inactivation was found for glyceraldehyde-3-phosphate dehydrogenase and to lesser extents: NAD-glutamate dehydrogenase, pyruvate decarboxylase, phosphofructokinase-1 and NAD-alcohol dehydrogenase . Ozone treatment also effects the quantity of ATP and of other nucleoside triphosphates, reducing to about 50% of the initial value . The ATP missing in the cells appears in the medium . NAD and protein also accumulate in the medium suggesting that the yeast cells have been permeabilized . Permeabilization of the yeast cells by treatment with ozone precedes the inactivation of glyceraldehyde-3-phosphate dehydrogenase and other cytosolic enzymes.

Tsitol Genet, 1987 Mar-Apr, 21(2), 127 - 31
{Genetic activity of sim-triazine herbicides on Saccharomyces cerevisiae yeast strains}; Emnova EE et al.; Triazine chloro derivatives: atrazine, simazine manifest no mutagenic and recombinogenic properties in yeasts; triazine methylthio derivatives: prometryne, semeron (desmetryne) generate both genetic events with low concentrations of 0.5 and 5 mg/l . It is found that prometryne is more able to generate point mutations, while semeron--to generate mitotic recombinations . In this case frequency of experimental prototrophs is twice higher than the control level.

Appl Environ Microbiol, 1987 Mar, 53(3), 509 - 13
Transport of lactate and other short-chain monocarboxylates in the yeast Saccharomyces cerevisiae; Cassio F et al.; Saccharomyces cerevisiae IGC4072 grown in lactic acid medium transported lactate by an accumulative electroneutral proton-lactate symport with a proton-lactate stoichiometry of 1:1 . The accumulation ratio measured with propionate increased with decreasing pH from ca . 24-fold at pH 6.0 to ca . 1,400-fold at pH 3.0 . The symport accepted the following monocarboxylates (Km values at 25 degrees C and pH 5.5): D-lactate (0.13 mM), L-lactate (0.13 mM), pyruvate (0.34 mM), propionate (0.09 mM), and acetate (0.05 mM), whereas apparently a different proton symport accepted formate (0.13 mM) . The lactate system was inducible and was subject to glucose repression . Undissociated lactic acid entered the cells by simple diffusion . The permeability of the plasma membrane for undissociated lactic acid increased exponentially with pH, and the diffusion constant increased 40-fold when the pH was increased from 3.0 to 6.0.

Mol Cell Biol, 1987 Mar, 7(3), 1300 - 3
Effect of deletion and insertion on double-strand-break repair in Saccharomyces cerevisiae; Struhl K; I investigated double-strand-break repair in Saccharomyces cerevisiae cells by measuring the frequencies and types of integration events at the PET56-HIS3-DED1 chromosomal region associated with the introduction of linearized plasmid DNAs containing homologous sequences . In general, the integration frequencies observed in strains containing a wild-type region, a 1-kilobase (kb) deletion, or a 5-kb insertion were similar, provided that the cleavage site in the plasmid DNA was present in the host genome . Cleavage at a plasmid DNA site corresponding to a region deleted in the chromosome caused a 10-fold reduction in the integration frequency even when the site was close to regions of homology . However, although the integration frequency was normal even when cleavage occurred only 25 base pairs (bp) outside the deletion breakpoint, 98% of the events were associated not with the usual heterogenote structure, but instead with a homogenote structure containing two copies of the deletion allele separated by vector sequences . Similarly, when cleavage occurred 80 bp outside the 5-kb substitution breakpoint, 40% of the integration events were associated with homogenote structures . From these observations, I suggest that exonuclease and polymerase activities are not rate-limiting steps in double-strand-break repair, exonuclease activity is coupled to the initiation step, the integration frequency is strongly influenced by the amount of homology near the recombinogenic ends, both ends of a linear DNA molecule might interact with the host chromosome before significant exonuclease or polymerase action, and the average repair tract is about 600 bp.

Mol Cell Biol, 1987 Mar, 7(3), 1208 - 16
Cloning and characterization of LOS1, a Saccharomyces cerevisiae gene that affects tRNA splicing; Hurt DJ et al.; Saccharomyces cerevisiae strains carrying los1-1 mutations are defective in tRNA processing; at 37 degrees C, such strains accumulate tRNA precursors which have mature 5' and 3' ends but contain intervening sequences . Strains bearing los1-1 and an intron-containing ochre-suppressing tRNA gene, SUP4(0), also fail to suppress the ochre mutations ade2-1(0) and can1-100(0) at 34 degrees C . To understand the role of the LOS1 product in tRNA splicing, we initiated a molecular study of the LOS1 gene . Two plasmids, YEpLOS1 and YCpLOS1, that complement the los1-1 phenotype were isolated from the YEp24 and YCp50 libraries, respectively . YEpLOS1 and YCpLOS1 had overlapping restriction maps, indicating that the DNA in the overlapping segment could complement los1-1 when present in multiple or single copy . Integration of plasmid DNA at the LOS1 locus confirmed that these clones contained authentic LOS1 sequences . Southern analyses showed that LOS1 is a single copy gene . The locations of the LOS1 gene within YEpLOS1 and YCpLOS1 were determined by deletion and gamma-delta mapping . Two genomic disruptions of the LOS1 gene were constructed, i.e., an insertion of a 1.2-kilobase fragment carrying the yeast URA3 gene, los1::URA3, and a 2.4-kilobase deletion from the LOS1 gene, los1-delta V . Disruption or deletion of most of the LOS1 gene was not lethal; cells carrying the disrupted los1 alleles were viable and had phenotypes similar to those of cells carrying the los1-1 allele . Thus, it appears that the los1 gene product expedites tRNA splicing at elevated temperatures but is not essential for this process.

Virology, 1987 Mar, 157(1), 252 - 6
Gene disruption indicates that the only essential function of the SKI8 chromosomal gene is to protect Saccharomyces cerevisiae from viral cytopathology; Sommer SS et al.; We have cloned the SKI8 gene, one of the chromosomal genes that repress replication of M, L-A, and L-BC double-stranded RNA viruslike particles in yeast . The clone was used to map SKI8 to chromosome VII near ade5 and to construct a deletion mutant . The deletion mutant was unable to grow at 8 degrees if and only if M1 double-stranded RNA was present.

Biochim Biophys Acta, 1987 Feb 27, 908(2), 179 - 87
Assembly of the mitochondrial ribosomes in a temperature-conditional mutant of Saccharomyces cerevisiae defective in the synthesis of the var1 protein; Hibbs AR et al.; An investigation of the role of the var1 protein in the assembly of the yeast mitochondrial ribosomes was carried out in a temperature conditional mutant, strain h56, which contains a mutation (tsv1) just upstream of the structural gene for the var1 protein . The mutation results in a marked decrease in the synthesis of the var1 protein at the permissive temperature of 28 degrees C and an apparently complete absence of var1 synthesis at the restrictive temperature of 36 degrees C . Long-term growth of strain h56 at the non-permissive temperature was found to result in the loss of the small (37 S) ribosomal subunit and the appearance of a novel 30 S ribonucleoparticle . Both the small (37 S) and the large (54 S) mitochondrial ribosomal subunits were found to be assembled in strain h56 for at least 3 h after transfer to the non-permissive temperature.

J Biol Chem, 1987 Feb 25, 262(6), 2549 - 53
Isolation and nucleotide sequence of a Saccharomyces cerevisiae protein kinase gene suppressing the cell cycle start mutation cdc25; Lisziewicz J et al.; We have isolated two unlinked yeast genes complementing the cell division cycle mutant cdc25-1, one containing the wild type allele CDC25 and the other acting as an extragenic suppressor of the cdc25-1 lesion if present on a multicopy plasmid . Nucleotide sequence analysis of the suppressor gene has revealed an open reading frame that encodes a 45,000-dalton protein belonging to the protein kinase family . The cdc25-suppressing protein kinase (PK-25) shows 48% sequence similarity to the catalytic subunit (CA) of mammalian cAMP-dependent protein kinase and 27-31% similarity to cyclic nucleotide-independent enzymes, including the yeast CDC28 gene product . The PK-25 gene was targeted by integrative transformation into a chromosomal region unlinked to the CYR2 site, the structural gene of CA . The cdc25-suppressing protein kinase is also functionally different from CA, since cyr2 strains deficient in the free catalytic subunit remain temperature sensitive if transformed with a multicopy plasmid containing the PK-25 gene . Furthermore, a deficiency of the cAMP-binding regulatory subunit (RA) caused by the bcy1 mutation fails to suppress the cdc25 mutation, indicating that PK-25 does not interact with the cAMP receptor protein . Our data suggest that the cdc25 suppressor gene encodes a cAMP-independent protein kinase involved in the control of the cell cycle start.

Biochim Biophys Acta, 1987 Feb 20, 923(2), 214 - 21
Studies on the regulation of enolases and compartmentation of cytosolic enzymes in Saccharomyces cerevisiae; Entian KD et al.; Three enolase isoenzymes can be distinguished after electrophoresis of yeast crude extracts . After adding glucose to derepressed cells, there was a coordinated increase in the activity of enolase I and decrease in enolase II activity . Enolase I was found to be repressed and enolase II simultaneously induced by glucose . The third enolase activity remained unchanged and was identified as that of a hybrid enzyme . Enolase catalyses the first common step of glycolysis and gluconeogenesis . Gluconeogenic enolase I shows substrate inhibition for 2-phosphoglycerate (glycolytic substrate) and glycolytic enolase II is substrate-inhibited by phosphoenolpyruvate (gluconeogenic substrate) . The gluconeogenic reaction was inhibited up to 45% by physiological concentrations of fructose 1,6-bisphosphate . To test for cytological compartmentation, a method was developed for isolating microsomes . Effective enrichment of rough and smooth endoplasmic reticulum was demonstrated by electron microscopy . No evidence was obtained for any compartmentation of either enolases or other glycolytic enzymes.

J Biol Chem, 1987 Feb 15, 262(5), 2056 - 61
Phosphorylated fructose-1,6-bisphosphatase dephosphorylating protein phosphatase from Saccharomyces cerevisiae; Horn D et al.; Phosphorylation of fructose-1,6-bisphosphatase with cyclic AMP-dependent protein kinase from yeast is accompanied by a 50% decrease in the catalytic activity (Pohlig, G . and Holzer, H . (1985) J . Biol . Chem . 260, 13818-13823) . Using reactivation of phoshorylated fructose-1,6-bisphosphatase as assay, a protein phosphatase was about 2,000-fold purified to electrophoretic homogeneity from Saccharomyces cerevisiae . Upon incubation with phosphorylated fructose-1,6-bisphosphatase the purified protein phosphatase not only reverses the 50% inactivation caused by phosphorylation, but also the previously observed change in the pH optimum and in the ratio of activity with Mg2+ or Mn2+ . The phosphatase is strongly inhibited by heparin and fluoride . L-Carnitine, orthophosphate, pyrophosphate, and succinate inhibit to 50% at concentrations from 1 to 10 mM . The molecular mass of the native phosphatase was found to be 180,000 Da . Sodium dodecyl sulfate-gel electrophoresis suggested four subunits with a molecular mass of 45,000 Da each . Half-maximal activity was observed with 5 mM Mg2+ or Mn2+, the pH optimum of activity was found at pH 7 . Using polyclonal antibodies, disappearance of 32P-labeled fructose-1,6-bisphosphatase and concomitant liberation of the expected amount of inorganic {32P} phosphate was demonstrated.

J Biol Chem, 1987 Feb 15, 262(5), 1989 - 95
Messenger RNA guanylyltransferase from Saccharomyces cerevisiae . Large scale purification, subunit functions, and subcellular localization; Itoh N et al.; Messenger RNA capping enzyme (GTP:mRNA guanylyltransferase) purified from yeast Saccharomyces cerevisiae consisted of two polypeptides (45 and 39 kDa) and possessed two enzymatic activities, i.e . mRNA guanylyltransferase and RNA 5'-triphosphatase (Itoh, N., Mizumoto, K., and Kaziro, Y . (1984) J . Biol . Chem . 259, 13923-13929) . In this paper, we describe an improved procedure suitable for the large scale purification of the enzyme . The steps include glass beads disruption of the cells and several ion-exchange and affinity column chromatographies . The enzyme was purified from kilogram quantities of yeast cells to apparent homogeneity . The purified enzyme had an approximate Mr of 180,000 and consisted of two heterosubunits of 80 and 52 kDa and had the same two enzymatic activities as above . We consider that this is the more intact form of the enzyme . Using the in situ assays on sodium dodecyl sulfate-polyacrylamide gels, RNA 5'-triphosphatase, and mRNA guanylyltransferase activities were located on the 80- and 52-kDa chains, respectively . In agreement with this, the 52-kDa enzyme-{32P}GMP complex was formed on incubation of the enzyme with {alpha-32P}GTP . Guinea pig antisera against purified yeast capping enzyme recognized both 80- and 52-kDa chains in Western blot analysis . The antibody did not cross-react with the enzymes from rat liver . Artemia salina, or vaccinia virus . Nuclear localization of the enzyme was demonstrated by immunofluorescence microscopy.

J Biol Chem, 1987 Feb 5, 262(4), 1836 - 41
Characterization of the fusion of enveloped viruses with the plasma membrane of Saccharomyces cerevisiae spheroplasts; Makarow M et al.; Vesicular stomatitis virus (VSV) was associated at low pH with Saccharomyces cerevisiae spheroplasts . In the cold, the association was characterized as reversible binding to the spheroplast surface . At 37 degrees C, the association became irreversible due to fusion of the viral envelope with the yeast plasma membrane according to the following data . Proteinase K digestion degraded the viral envelope glycoprotein G but left the internal N and M proteins of VSV intact and associated with the spheroplasts . The plasma membrane could be stained by indirect immunofluorescent labeling using antiserum against VSV . By immunoelectron microscopy, no VSV particles could be detected at the spheroplast surface . Instead, the G protein could be visualized at the external aspect of the plasma membrane using specific antiserum and protein A-gold . Fusion of VSV with spheroplasts occurred below pH 4.75 at temperatures of 30-42 degrees C . It was strictly dependent on the prior removal of the yeast cell wall . The fusion process was fast, calcium-independent, and nonleaky, leaving the spheroplasts viable for at least 4 h . On the average, less than 100 VSV particles could be fused per one spheroplast . Similar data were obtained with Semliki Forest virus.

Eur J Biochem, 1987 Feb 2, 162(3), 635 - 42
Subcellular location of enzymes involved in the N-glycosylation and processing of asparagine-linked oligosaccharides in Saccharomyces cerevisiae; Tillmann U et al.; A particulate translation system isolated from the yeast Saccharomyces cerevisiae was shown to translate faithfully in-vitro-transcribed mRNA coding for a mating hormone precursor (prepro-alpha-factor mRNA) and to N-glycosylate the primary translation product after its translocation into the lumen of the microsomal vesicles . Glycosylation of its three potential sugar attachment sites was found to be competitively inhibited by acceptor peptides containing the consensus sequence Asn-Xaa-Thr, supporting the view that the glycan chains are N-glycosidically attached to the prepro-alpha-factor polypeptide . The accumulation in the presence of acceptor peptides of a membrane-specific, unglycosylated translation product (pp-alpha-F0) differing in molecular mass from a cytosolically located, protease-K-sensitive alpha-factor polypeptide (pp-alpha-Fcyt) by about 1.3 kDa, suggests that, in contrast to previous reports, a signal sequence is cleaved from the mating hormone precursor on/after translocation . This conclusion is supported by the observation that the multiply glycosylated alpha-factor precursor is cleaved by endoglucosaminidase H to a product with a molecular mass smaller than the primary translation product pp-alpha-Fcyt but larger than the membrane-specific pp-alpha-F0 . Translation and glycosylation experiments carried out in the presence of various glycosidase inhibitors (e.g . 1-deoxynojirimycin, N-methyl-1-deoxynojirimyin and 1-deoxymannojirimycin) indicate that the N-linked oligosaccharide chains of the glycosylated prepro-alpha-factor species are extensively processed under the in vitro conditions of translation . From the specificity of the glycosidase inhibitors applied and the differences in the molecular mass of the glycosylated translation products generated in their presence, we conclude that the glycosylation-competent microsomes contain trimming enzymes, most likely glucosidase I, glucosidase II and a trimming mannosidase, which process the prepro-alpha-factor glycans down to the (Man)8(GlcNAc)2 stage . Furthermore, several arguments strongly suggest that these three enzymes, which apparently represent the full array of trimming activities in yeast, are exclusively located in the lumen of microsomal vesicles derived from endoplasmic reticulum membranes.

Genetics, 1987 Feb, 115(2), 255 - 63
Protease B of Saccharomyces cerevisiae: isolation and regulation of the PRB1 structural gene; Moehle CM et al.; We have isolated the structural gene, PRB1, for the vacuolar protease B of Saccharomyces cerevisiae from a genomic library by complementation of the prb1-1122 mutation . Deletion analysis localized the complementing activity to a 3.2-kilobase pair XhoI-HindIII restriction enzyme fragment . The fragment was used to identify a 2.3-kilobase mRNA . S1 endonuclease mapping indicated that the mRNA and the gene were colinear . No introns were detected . The mRNA is of sufficient size to encode a protein of about 69,000 molecular weight, a number much larger than either the mature enzyme (congruent to 30,000 protein molecular weight) or the sole reported precursor (congruent to 39,000 protein molecular weight) . These results suggest that proteolytic processing steps beyond that thought to be catalyzed by protease A may be required to convert the initial glycosylated translation product into mature protease B . The PRB1 mRNA is made in substantial amounts only when the cells have exhausted the glucose supply and enter the diauxic plateau . There is an extended time lag between PRB1 transcription and expression of protease B activity . A deletion that removes about 83% of the coding region was constructed as a diploid heterozygote . Spores bearing the deletion germinate, grow at normal rates into colonies, and have no obvious phenotype beyond protease B deficiency.

Genetics, 1987 Feb, 115(2), 233 - 46
Meiotic gene conversion and crossing over between dispersed homologous sequences occurs frequently in Saccharomyces cerevisiae; Lichten M et al.; We have examined meiotic recombination between two defined leu2 heteroalleles present at the normal LEU2 locus and in leu2-containing plasmids inserted at four other genomic locations . In diploids where the two leu2 markers were present at allelic locations on parental homologs, the frequency of Leu2+ spores varied 38-fold, in a location-dependent manner . These results indicate that recombination in a genetic interval can be modulated by sequences at least 2.7 kb outside that interval . Leu2+ meiotic segregants were also recovered from diploids where LEU2 was marked with one heteroallele, and the other leu2 heteroallele was inserted at another genomic location . These products of ectopic interactions, between dispersed copies of leu2 sharing only 2.2 kb of homology, were recovered at a frequency comparable to that observed in corresponding allelic crosses . This high frequency of ectopic meiotic recombination was observed in crosses where both recombining partners could potentially pair with sequences at an allelic position . In addition, a significant fraction (22-50%) of these ectopic recombinants were associated with crossing over of flanking sequences.

DNA, 1987 Feb, 6(1), 31 - 9
Expression in Saccharomyces cerevisiae of chimeric cytochrome P450 cDNAs constructed from cDNAs for rat cytochrome P450c and P450d; Sakaki T et al.; Three chimeric cytochrome P450 cDNAs were constructed by replacing the central region, carboxy-terminal region, or both central and carboxy-terminal regions of cytochrome P450c cDNA with the corresponding regions of cytochrome P450d cDNA . These were inserted between the alcohol dehydrogenase I promoter and terminator of yeast expression vector pAAH5 to form expression plasmids pACDC2, pACCD1, and pACDD2 . On introduction of each of these plasmids into Saccharomyces cerevisiae AH22 cells, chimeric cytochrome P450 proteins were expressed in AH22/pACDC2, AH22/pACCD1, and AH22/pACDD2 cells at the level of at least 10(5), 4 X 10(5) molecules per cell, respectively . The reduced CO-difference spectra showed that AH22/pACCD1 and AH22/pACDD2 cells contained 4 X 10(5) and 10(5) molecules per cell of the corresponding chimeric cytochrome P450 hemoproteins, designated as cytochrome P450ccd and cytochrome P450cdd, respectively . Cytochrome P450ccd exhibited higher monooxygenase activities toward 7-ethoxycoumarin, acetanilide, and benzo{alpha}pyrene than cytochrome P450c, although the substrate specificity of cytochrome P450ccd seemed to be the same as that of cytochrome P450c . Cytochrome P450cdd exhibited lower activities toward 7-ethoxycoumarin and benzo{alpha}pyrene, and a higher activity toward acetanilide as compared with those of cytochrome P450c and cytochrome P450ccd . Therefore, the substrate specificity of cytochrome P450cdd seemed to be the same as that of cytochrome P450d . These results suggest that the central one-third region of cytochrome P450c and cytochrome P450d is responsible for substrate-binding, and that the carboxy-terminal third of both cytochromes P450 plays an important role in electron transport.

Mol Cell Biol, 1987 Feb, 7(2), 813 - 20
The GCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces cerevisiae; Holland MJ et al.; The intracellular concentrations of the polypeptides encoded by the two enolase (ENO1 and ENO2) and three glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2, and TDH3) genes were coordinately reduced more than 20-fold in a Saccharomyces cerevisiae strain carrying the gcr1-1 mutation . The steady-state concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA was shown to be approximately 50-fold reduced in the mutant strain . Overexpression of enolase and glyceraldehyde-3-phosphate dehydrogenase in strains carrying multiple copies of either ENO1 or TDH3 was reduced more than 50-fold in strains carrying the gcr1-1 mutation . These results demonstrated that the GCR1 gene encodes a trans-acting factor which is required for efficient and coordinate expression of these glycolytic gene families . The GCR1 gene and the gcr1-1 mutant allele were cloned and sequenced . GCR1 encodes a predicted 844-amino-acid polypeptide; the gcr1-1 allele contains a 1-base-pair insertion mutation at codon 304 . A null mutant carrying a deletion of 90% of the GCR1 coding sequence and a URA3 gene insertion was constructed by gene replacement . The phenotype of a strain carrying this null mutation was identical to that of the gcr1-1 mutant strain.

Mol Cell Biol, 1987 Feb, 7(2), 672 - 8
SSN20 is an essential gene with mutant alleles that suppress defects in SUC2 transcription in Saccharomyces cerevisiae; Neigeborn L et al.; Dominant and recessive mutations at the SSN20 locus were previously isolated as extragenic suppressors of mutations in three genes (SNF2, SNF5, and SNF6) that are required in trans to derepress invertase expression . All ssn20 alleles cause recessive, temperature-sensitive lethality . In this study we cloned the SSN20 gene, identified a 4.6-kilobase poly(A)-containing RNA, and showed that disruption of the gene is lethal in a haploid cell . Genetic mapping of SSN20 to a locus on chromosome VII 10 centimorgans distal to cly8 led to the finding that SSN20 is the same gene as SPT6, which affects expression of delta insertions in the 5' noncoding region of HIS4 (F . Winston, D . T . Chaleff, B . Valent, and G . R . Fink, Genetics 107:179-197, 1984) . We also showed that an ssn20 mutation restored expression of secreted invertase from deletions of the SUC2 upstream regulatory region; ssn20 restored derepression of SUC2 mRNA in strains with a SUC2 upstream region deletion or a snf2 mutation . Increased or decreased gene dosage of SSN20 also suppressed defects that are suppressed by ssn20 missense mutations . These findings suggest that SSN20 plays a role in general transcriptional processes.

Mol Cell Biol, 1987 Feb, 7(2), 614 - 21
Role of transcriptional and posttranscriptional regulation in expression of histone genes in Saccharomyces cerevisiae; Lycan DE et al.; We analyzed the role of posttranscriptional mechanisms in the regulation of histone gene expression in Saccharomyces cerevisiae . The rapid drop in histone RNA levels associated with the inhibition of ongoing DNA replication was postulated to be due to posttranscriptional degradation of histone transcripts . However, in analyzing the sequences required for this response, we showed that the coupling of histone RNA levels to DNA replication was due mostly, if not entirely, to transcriptional regulatory mechanisms . Furthermore, deletions which removed the negative, cell cycle control sequences from the histone promoter also uncoupled histone transcription from DNA replication . We propose that the arrest of DNA synthesis prematurely activates the regulatory pathway used in the normal cell cycle to repress transcription . Although posttranscriptional regulation did not appear to play a significant role in coupling histone RNA levels to DNA replication, it did affect the levels of histone RNA in the cell cycle . Posttranscriptional regulation could apparently restore much of the periodicity of histone RNA accumulation in cells which constitutively transcribed the histone genes . Unlike transcriptional regulation, periodic posttranscriptional regulation appears to operate on a clock which is independent of events in the mitotic DNA cycle . Posttranscriptional recognition of histone RNA must require either sequences in the 3' end of the RNA or an intact three-dimensional structure since H2A- and H2B-lacZ fusion transcripts, containing only 5' histone sequences, were insensitive to posttranscriptional controls.

Mol Cell Biol, 1987 Feb, 7(2), 578 - 85
Sequence and nuclear localization of the Saccharomyces cerevisiae HAP2 protein, a transcriptional activator; Pinkham JL et al.; Activation of the CYC1 upstream activation site (UAS2) and other Saccharomyces cerevisiae genes encoding respiratory functions requires the products of the regulatory loci HAP2 and HAP3 . We present here the DNA sequence of the yeast HAP2 gene and an initial investigation into the function of its product . The DNA sequence indicated that HAP2 encoded a 265-amino-acid protein whose carboxyl third was highly basic . Also found in the sequence was a polyglutamine tract spanning residues 120 to 133 . Several experiments described herein suggest that HAP2 encodes a direct activator of transcription . First, a bifunctional HAP2-beta-galactosidase fusion gene was localized to the yeast nucleus . Second, a lexA-HAP2 fusion gene was capable of activating transcription when bound to a lexA operator site . The additional requirement for the HAP3 product in activation is discussed.

Arch Biochem Biophys, 1987 Feb 1, 252(2), 339 - 47
A 5'----3' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity; Stevens A et al.; The purification scheme for a 5'----3' exoribonuclease of Saccharomyces cerevisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography . As determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or physical characterization, the enzyme has a molecular weight of about 160,000 . Further studies of its substrate specificity show that poly(C) and poly(U) preparations require 5' phosphorylation for activity and that poly(A) with a 5'-triphosphate end group is hydrolyzed at only 12% of the rate of poly(A) with a 5'-monophosphate end group . DNA is not hydrolyzed, but synthetic polydeoxyribonucleotides are strong competitive inhibitors of the hydrolysis of noncomplementary ribopolymers . Poly(A).poly(U) and poly(A).poly(dT) are hydrolyzed at 60 and 50%, respectively, of the rate of poly(A) at 37 degrees C . The RNase H activity of the enzyme can also be demonstrated using an RNA X M13 DNA hybrid as a substrate . When poly(dT).poly(dA) with a 5'-terminal poly(A) segment on the poly(dA) is used as a substrate, the enzyme hydrolyzes the poly(A) "tail," removing the last ribonucleotide, but does not hydrolyze the poly(dA).

Proc Natl Acad Sci U S A, 1987 Feb, 84(3), 779 - 83
Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae; Madaule P et al.; The rho genes comprise an evolutionarily conserved family with significant homology to the ras oncogene family . Two members of the rho family were isolated from the yeast Saccharomyces cerevisiae and characterized by DNA sequence analysis . The yeast genes RHO1 and RHO2 are 70% and 57% identical, respectively, to the rho gene of the marine snail Aplysia, and they are 53% identical to each other . Inactivation of these genes showed that RHO1 is required for cell viability, while RHO2 is not an essential gene . A mutant allele of RHO1 (RHO1-His68) was constructed with a mutation analogous to one that activates the transforming potential of the human HRAS gene . Diploid strains containing RHO1-His68 in either low or high copy number are unable to sporulate, and the mutant allele is dominant over wild-type RHO1 . The requirement for RHO1 cannot be circumvented by introduction of high copy number plasmids containing either the gene encoding the catalytic subunit of cAMP-dependent protein kinase or the mutant allele RAS2-Val19 . Despite the conservation between the rho and ras gene families, the finding that RHO1 functions independently of the adenylate cyclase cAMP-dependent protein kinase cascade suggests that rho and ras are involved in distinct biochemical pathways.

Mutat Res, 1987 Feb, 187(2), 79 - 89
A comparison of the genetic activity of pyrvinium pamoate with that of several other anthelmintic drugs in Saccharomyces cerevisiae; Hennig UG et al.; Several anthelmintic drugs that are used routinely in oxyuriasis therapy were analyzed for genotoxicity in a diploid mitotic recombination and gene conversion assay (strain D5 of Saccharomyces cerevisiae), and in a haploid yeast reversion assay (strain XV185-14C) . Piperazine citrate, piperazine adipate, mebendazole and thiabendazole did not appear to be genotoxic in either yeast strain . Pyrvinium pamoate induced the reversion of the missense, nonsense and frameshift alleles in strain XV185-14C, whereas pyrantel pamoate induced only the reversion of the frameshift allele . Pyrvinium pamoate was recombinogenic in strain D5, and there is an indication that pyrantel pamoate, at the lowest dose that was tested, might induce gene conversion or aneuploidy.

J Bacteriol, 1987 Feb, 169(2), 483 - 8
Interaction of alpha-agglutinin with Saccharomyces cerevisiae a cells; Lipke PN et al.; Binding of Saccharomyces cerevisiae alpha-agglutinin to target a cells was assayed by agglutination inhibition and 125I-alpha-agglutinin binding . The assays showed characteristics of equilibrium binding, namely saturability, competability, and the establishment of a kinetic endpoint in the presence of free alpha-agglutinin and free receptor . The binding was heterogeneous, displaying strong binding (10(9) liters/mol) and a weaker interaction . There were about 2 X 10(4) strong binding sites per a cell . Denaturing gels displayed identical labeled species binding to the a cells in the weak and strong interactions . Furthermore, weakly bound material could subsequently bind tightly to fresh a cells, implying that the same species of alpha-agglutinin was bound in the two states.

J Bacteriol, 1987 Feb, 169(2), 475 - 82
Identification of glycoprotein components of alpha-agglutinin, a cell adhesion protein from Saccharomyces cerevisiae; Terrance K et al.; Several glycoproteins which inhibit the agglutinability of Saccharomyces cerevisiae mating type a cells were partially purified from extracts of mating type alpha cells . These proteins, called alpha-agglutinin, were labeled with 125I-Bolton-Hunter reagent . The labeled alpha-agglutinin showed specific binding to a cells . Such specific binding approached saturation with respect to agglutinin or cells and was inhibited in the presence of excess unlabeled alpha-agglutinin . Nonspecific binding was similar in a and alpha cells, was neither saturable nor competable, and was three- to fourfold less than the specific binding to a cells at maximum tested agglutinin concentrations . The major a-specific binding species had a low electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels and had an apparent molecular weight of 155,000 by rate zonal centrifugation . Endo-N-acetylglucosaminidase H digestion of the purified glycoprotein complex converted the low-mobility material to four major and several minor bands which were resolved by polyacrylamide gel electrophoresis . All but two minor peptides bound specifically to a cells . Analyses of agglutinin from mnn mutants confirmed the deglycosylation results in suggesting that the N-linked carbohydrate portion of alpha-agglutinin was not necessary for activity.

J Biochem (Tokyo), 1987 Feb, 101(2), 535 - 44
Characterization of nystatin-resistant mutants of Saccharomyces cerevisiae and preparation of sterol intermediates using the mutants; Nakanishi S et al.; Analysis of sterols of Saccharomyces cerevisiae mutants N3, N15, N26, and N3H, defective in sterol biosynthesis, was performed . Strains N3, N15, and N26 were isolated from their mother strain, M10, by screening with nystatin (Nagai et al . (1980) Mie Med . J . 30, 215-224), and strain N3H was isolated from N3 as a doubly-mutated strain . The main sterols of N3, N15, N26, and N3H were ergosta-7,22-dienol, ergost-8-enol, cholesta-5,7,24-trienol, and ergosta-7,22,24(28)-trienol, respectively . The former three strains were characterized as defective in delta 5-desaturation, delta 8--delta 7 isomerization, and C-24 transmethylation . Strain N3H was found to be defective in delta 5-desaturation as well as in delta 24(28)-reduction . However, the defect of N26 and N3H was suggested to be leaky, since small amounts of ergosterol and ergosta-7,22-dienol were found in these mutants, respectively . In N15, an accumulation (2% in total sterols) of the compound likely to be hydroxylated sterol was found . By aerobic adaptation of these strains, the accumulation of these strains, the accumulations of ergosta-7,22-dienol (22 mg/g dry cells), ergosta-7,22,24(28)-trienol (24 mg), ergosta-8,24(28)-dienol (18 mg), and cholesta-8,24-dienol (22 mg) reached a maximum in N3, N3H, N15, and N26 after 20, 20, 30, and 30 h, respectively . These strains appear to be useful for making 14C-labeled and non-labeled preparations of the above sterols.

Can J Microbiol, 1987 Feb, 33(2), 93 - 7
Effect of ethanol on the glucose-induced movements of protons across the plasma membrane of Saccharomyces cerevisiae NCYC 431; Juroszek JR et al.; The study of glucose-induced proton fluxes in Saccharomyces cerevisiae NCYC 431 showed a decrease of proton net efflux by ethanol across the plasma membrane of energized cells . Furthermore a negative net proton efflux (an influx) occurred from a given ethanol concentration (between 1.3 and 1.5 M) whatever the experimental conditions used, thus allowing the definition of a nil-net exchange step where no net movement of protons across the plasma membrane could be observed . A new technique of ethanol tolerance determination in yeast based upon a correlation for the same ethanol concentration between both the collapse of the proton gradient and the growth cessation in cultures supplemented with ethanol after 8 h incubation was proposed . The defined method also showed a cumulated effect of temperature and ethanol on Saccharomyces cerevisiae NCYC 431.

Mol Cell Biol, 1987 Feb, 7(2), 679 - 86
The SPT6 gene is essential for growth and is required for delta-mediated transcription in Saccharomyces cerevisiae; Clark-Adams CD et al.; Mutations in the Saccharomyces cerevisiae SPT6 gene were originally identified as one class of extragenic suppressors of Ty and delta insertion mutations in the 5' noncoding regions of HIS4 and LYS2 . We cloned SPT6 and constructed a null allele by gene disruption . Haploid spores carrying the spt6 null allele were inviable, indicating that the SPT6 gene is essential for mitotic growth . SPT6 was mapped to the right arm of chromosome VII, 44 centimorgans (cM) from ADE6 and 9 cM from CLY8 . We showed that spt6 mutations suppress delta insertion mutations at the level of transcription but have no qualitative or quantitative effect on Ty transcription . In addition, we observed interesting SPT6 gene dosage effects . An SPT6 strain containing a high-copy-number plasmid clone of SPT6 showed suppression of delta insertion mutations, and a diploid strain with half its normal dose of SPT6 (SPT6/spt6 null) also exhibited suppression of delta insertion mutations . Therefore, having either too many or too few copies of SPT6 causes a mutant phenotype . Finally, this study and that in the accompanying paper (L . Neigeborn, J . L . Celenza, and M . Carlson, Mol . Cell . Biol . 7:679-686, 1986) showed that spt6 and ssn20 mutations (isolated as suppressors of snf2 and snf5 {sucrose nonfermenting} mutations) identify the same gene . SPT6 and SSN20 have the same genetic map position and share an identical restriction map . Furthermore, spt6 and ssn20 mutations fail to complement each other, and ssn20 mutations suppress solo delta insertion mutations at HIS4 and LYS2 . These results, taken in conjunction with the SPT6 dosage effects and the fact that SPT6 is an essential gene, suggest that SPT6 plays a fundamental role in cellular transcription, perhaps by interaction with other transcription factors.

J Bacteriol, 1987 Feb, 169(2), 533 - 9
Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae; Homann MJ et al.; The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined . Maximum activities were found in the exponential phase of cells grown in complete synthetic medium . As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold . The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase . When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline . Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline . The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed . Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture . The phospholipid composition of cells in the exponential and stationary phase of growth was also examined . The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells . The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.

Protein Eng, 1987 Feb-Mar, 1(2), 95 - 9
Replacement of cysteine-107 of Saccharomyces cerevisiae iso-1-cytochrome c with threonine: improved stability of the mutant protein; Cutler RL et al.; Site-directed mutagenesis has been used to change the codon for cysteine-107 of Saccharomyces cerevisiae iso-1-cytochrome c to a threonine codon . The resulting protein is active in vivo, is methylated as in the wild-type protein and has optical properties indistinguishable from those of the wild-type protein . The threonine-107 iso-1-cytochrome c demonstrated fully reversible electrochemical behaviour and a mid-point reduction potential of 272 mV versus NHE . In addition, this mutant does not demonstrate a tendency to autoreduce or to dimerize as does the wild-type protein . These properties of the threonine-107 mutant establish that it will provide a useful background in which to make subsequent mutations for mechanistic and physical studies of yeast iso-1-cytochrome c.

Biochem Pharmacol, 1987 Jan 15, 36(2), 229 - 35
Interaction of azole antifungal agents with cytochrome P-45014DM purified from Saccharomyces cerevisiae microsomes; Yoshida Y et al.; Mechanism of action of azole antifungal agents was studied by analyzing interaction of ketoconazole, itraconazole, triadimefon and triadimenol with a purified yeast cytochrome P-450 which catalyzes lanosterol 14 alpha-demethylation (P-45014DM) . These antifungal agents formed low-spin complexes with P-45014DM, indicating the interaction of their azole nitrogens with the heme iron . Affinity of these antifungal agents for the cytochrome was extremely high compared with usual nitrogenous ligands . Upon reduction with sodium dithionite, the azole complexes of ferric P-45014DM were converted to the corresponding ferrous derivatives . Spectral analysis of these complexes suggested that geometric orientation of the azole moiety of an antifungal agent to the ferrous heme iron was regulated by the interaction between the N-1 substituent and the heme environment . CO could not readily replace ketoconazole or itraconazole co-ordinating to the heme iron of ferrous P-45014DM while triadimefon and triadimenol complexes of the cytochrome were promptly converted to the CO complexes . The inhibitory effects of ketoconazole and itraconazole on the P-45014DM-dependent lanosterol 14 alpha-demethylation were higher than that of triadimenfon . The substituents at N-1 of the azole moieties of ketoconazole and itraconazole are extremely large while those of triadimefon and triadimenol are relatively small . Accordingly, observations described above suggest that the N-1 substituent of an azole antifungal agent regulates the mobility of the molecule in the heme crevice of ferrous P-45014DM and determines the inhibitory effect of the compound.

J Biol Chem, 1987 Jan 15, 262(2), 546 - 8
Amino acid sequences of a-factor mating peptides from Saccharomyces cerevisiae; Betz R et al.; The molecular structure of a-factor, the mating hormone produced by mating type a cells of Saccharomyces cerevisiae, has been investigated . In culture filtrates of a cells four oligopeptides (a1 to a4) exhibiting a-factor activity have been found . These peptides have been isolated and their amino acid sequences have been determined . The a-factor peptides comprise two (apparently identical) pairs, a1/a2 and a3/a4, which differ in an interchange at position 6 of a valine in a1/a2 for a leucine in a3/a4 . a1 and a4, which can be obtained by oxidation with H2O2 of purified a2 and a3, respectively, obviously represent oxidation artifacts formed under the conditions of culture . The amino acid sequences determined for the a-factor peptides are Tyr-Ile-Ile-Lys-Gly-Val Leu-Phe-Trp-Asp-Pro-Ala-Cys . Several lines of evidence suggest that the carboxyl-terminal cysteine residue is S-alkylated by a hydrophobic moiety.

Nucleic Acids Res, 1987 Jan 12, 15(1), 233 - 46
The sequence of the Saccharomyces cerevisiae gene PHO2 codes for a regulatory protein with unusual aminoacid composition; Sengstag C et al.; A new centromere vector for the construction of a Saccharomyces cerevisiae gene library, allowing direct selection for DNA insert, will be described . From that library the gene for the regulatory protein PHO2 involved in PHO5 induction has been cloned by complementation of a pho2 mutation . The complementing activity was shown to be located on a 3.6 kb HindIII fragment . This fragment was used to evict the genomic copy and with appropriate genetic crosses we proved, that the cloned gene is PHO2 . The DNA sequence of PHO2 was determined . Analysis of the sequence data uncovered striking homology regions with PHO4, another protein necessary for the induction of PHO5 . The relevance of the observed homology will be discussed.

Vet Med Nauki, 1987, 24(4), 84 - 7
{Effect of a Saccharomyces cerevisiae yeast preparation on the quality of pork}; Rizvanov S; Studies were carried out to ascertain the effect of a preparation produced from yeasts of the Saccharomyces cerevisiae species on some indices characterizing the quality of pork . Studied was the chemical composition, the pH values at the 45th minute and the 24th hour post mortem, the content of tryptophane and oxiproline as well as the colour of meat obtained from musculus longissimus dorsi . It was found that the preparation had no negative effect on the indices, characterizing the quality of meat.

Cytobios, 1987, 49(197), 89 - 97
Exocytosis in Saccharomyces cerevisiae treated with congo red; Vannini GL et al.; When dividing cells of Saccharomyces cerevisiae were exposed to the polysaccharide-binding dye Congo red, the walls and septa became sites of chitin accumulation . In addition, the cytoplasm showed many vesicles that were different from those accumulating in the growing bud and from the lytic vacuoles of the untreated yeasts . To obtain information about these membranous structures, living cells were observed under phase contrast and UV light microscopes . Furthermore, ultrathin sections of Congo red-treated cells were processed by cytochemical techniques to reveal the chitin areas . Observations suggest that the aberrant vesicles were involved in a secretory process, and that pre-assembled chitin was not among the components transported to the cell periphery.

Mol Cell Biol, 1987 Jan, 7(1), 420 - 6
L-A double-stranded RNA viruslike particle replication cycle in Saccharomyces cerevisiae: particle maturation in vitro and effects of mak10 and pet18 mutations; Fujimura T et al.; Previously, we found that log-phase cells of Saccharomyces cerevisiae contain a new type of viruslike particles containing only plus- strand L-A single-stranded RNA (ssRNA) . These particles synthesize minus-strand RNA in an in vitro RNA polymerase reaction to produce L-A double-stranded RNA (dsRNA) . The major class of particles contains L-A dsRNA and synthesizes plus-strand L-A ssRNA by a conservative mechanism . In this paper, we show that mutations in mak10 or the pet18 locus, which result in temperature-dependent replication of L-A dsRNA in vivo, also result in instability of the L-A dsRNA-containing (major class) viruslike particles in vitro . The L-A dsRNA (minus-strand)-synthesizing particles isolated by CsCl density gradient centrifugation synthesize plus-strand L-A ssRNA after completion of dsRNA (minus-strand) synthesis and have the same major coat protein as that of the major-class particles . Furthermore, the density of the dsRNA-synthesizing particles from wild-type cells shifts to that of the major-class dsRNA-containing particles as a result of the in vitro RNA polymerase reaction . Thus, L-A dsRNA-synthesizing particles undergo functional and structural maturation in vitro.

Mol Cell Biol, 1987 Jan, 7(1), 225 - 30
Effects on mRNA splicing of mutations in the 3' region of the Saccharomyces cerevisiae actin intron; Fouser LA et al.; Point mutations, deletions, and a sequence context change were introduced at positions 3' to the internal conserved TACTAAC sequence of the Saccharomyces cerevisiae actin intron . In vivo analysis of yeast mRNA splicing suggests that, in contrast to the importance of the polypyrimidine tract in metazoan introns, specific sequences in this region are not required for efficient excision of a yeast intron . However, a double point mutation near the 3' junction (GG/AC) does severely inhibit splicing . Although this mutagenesis of the 3' junction, as well as deletion of most nucleotides between the TACTAAC and the 3' junction, caused only a slight accumulation of primary transcript, the observed accumulation of lariat intermediate by these mutants demonstrates the significance of this region for a step(s) in the splicing process after lariat formation.

Genetics, 1987 Jan, 115(1), 83 - 90
The RAD24 (= Rs1) gene product of Saccharomyces cerevisiae participates in two different pathways of DNA repair; Eckardt-Schupp F et al.; The moderately UV- and X-ray-sensitive mutant of Saccharomyces cerevisiae originally designated rs1 complements all rad and mms mutants available . Therefore, the new nomination rad24-1 according to the RAD nomenclature is suggested . RAD24 maps on chromosome V, close to RAD3 (1.3 cM) . In order to associate the RAD24 gene with one of the three repair pathways, double mutants of rad24 and various representative genes of each pathway were constructed . The UV and X-ray sensitivities of the double mutants compared to the single mutants indicate that RAD24 is involved in excision repair of UV damage (RAD3 epistasis group), as well as in recombination repair of UV and X-ray damage (RAD52 epistasis group) . Properties of the mutant are discussed which hint at the control of late steps in the pathways.

Genetics, 1987 Jan, 115(1), 65 - 71
Functional alcohol dehydrogenase mutants of Saccharomyces cerevisiae conferring temperature-conditional allyl alcohol resistance; Hall JG et al.; Selection for allyl alcohol resistance in respiratory incompetent yeast is a highly specific method for isolating functional mutations at ADH1, the gene coding for the cytoplasmic alcohol dehydrogenase, ADHI . Because of the nature of this selection scheme, the ADHI activity of such mutants is retained, but the kinetic characteristics of the enzymes are altered . The high specificity for targeting functional mutations at this locus suggested that selection for enzyme variants with more subtle phenotypic effects might be possible . Here, we describe functional ADHI mutants that are temperature-conditional in their allyl alcohol resistance . Haploid cells of one of these mutants grow well on plates at 10 mM allyl alcohol at 19 degrees, but not at 37 degrees, the restrictive temperature . A second mutant grows well at 10 mM at 37 degrees, but its growth is restricted at 19 degrees . What distinguishes these mutants from other temperature-sensitive mutants is that the temperature-conditional growth phenotypes described here must be due to interactions between allyl alcohol levels and ADHI functional properties and cannot be due to lability of the enzyme at the restrictive temperature . This system shows promise for the investigation of functional enzyme variants that differ by only one or two amino acid residues but have significant temperature- and substrate-conditional effects on growth phenotypes in both the haploids and the diploids.

Appl Environ Microbiol, 1987 Jan, 53(1), 33 - 5
Autoconditioning factor relieves ethanol-induced growth inhibition of Saccharomyces cerevisiae; Walker-Caprioglio HM et al.; Viable Saccharomyces cerevisiae suspended in medium containing growth-inhibiting concentrations of ethanol produce a metabolite that relieves growth inhibition . This autoconditioning of the medium by yeasts is due to the formation of small amounts (0.01%, vol/vol) of acetaldehyde . The effect is duplicated precisely in fresh medium by the addition of acetaldehyde . Acetaldehyde does not increase the yield of or accelerate ethanol production by the organism . Ethanol-induced modifications of membrane order in the plasma membranes, as measured by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, were not resolved by exogenously added acetaldehyde.

Genetika, 1987 Jan, 23(1), 41 - 4
{Genetic analysis of mitochondrial rho-mutability in Saccharomyces . IV . Relation between spontaneous rho-mutability and mitotic stability in disomic Saccharomyces cerevisiae}; Smirnova ME et al.; In the yeast Saccharomyces cerevisiae the disomy for chromosome XIV resembles the previously described disomy for chromosome IV in that it leads to a significant decrease in spontaneous rho- mutability . The nuclear srm1 mutation, reducing spontaneous rho- mutability, diminishes significantly the mitotic disome stability . So, the mechanisms of spontaneous rho- mutagenesis and mitotic disome stability seem to compete for the function affected by the srm1 mutation.

Mutat Res, 1987 Jan, 187(1), 21 - 30
Effects of chemical combinations on the induction of aneuploidy in Saccharomyces cerevisiae; Mayer VW et al.; Nocodazole, ethyl acetate, acetone and methyl ethyl ketone all are known to induce aneuploidy . Treatment of yeast strain D61.M with mixtures containing ineffective low levels of nocodazole and ineffective low levels of these solvents was highly effective in inducing aneuploidy . Ineffective low levels of nocodazole mixed with ineffective low levels of methyl 2-benzimidazolecarbamate also gave elevated frequencies of aneuploidy . Dimethyl formamide, a solvent that does not induce aneuploidy, mixed with low levels of nocodazole gave no increase in aneuploidy frequency above those levels seen in controls.

J Basic Microbiol, 1987, 27(10), 603 - 12
Ultrastructure of Saccharomyces cerevisiae cells accumulating Golgi organelles; Svoboda A et al.; Restrictive phenotype of sec 7 mutant of Saccharomyces cerevisiae was examined by freezefracture electron microscopy . In accordance with previous findings (NOVICK et al . 1981) dictyosomes and middle-size (200-600 nm) vesicles (Berkeley bodies) were found to accumulate . Dictyosomes are formed by aggregated flattened or dilated cisternae without associated secretory vesicles . After transfer to permissive conditions the dictyosomes disappear and are not detectable, just like in the wild type or in the permissive phenotype . Associated with the restrictive phenotype is an extended plasma membrane and tonoplasts with particle-free impressions.

Curr Genet, 1987, 12(3), 161 - 6
A DNA sequence which shows genomic variation in a, alpha and HO strains of Saccharomyces cerevisiae; Gupta NJ et al.; HindIII digested DNA from various mutant strains of Saccharomyces cerevisiae probed with a 340 bp nucleotide sequence in M13mp8 derived from a mouse liver cDNA clone p1581 showed strong hybridization to a 4.1 kb DNA fragment class . This was limited to the DNA of cells of alpha mating type but the fragments concerned apparently do not originate from chromosome III . The pattern of hybridization was modified in strains carrying the HO gene consistent with there being extra copies of the Bkm-homologous sequence in these cells . Northern analysis of RNA from cells synchronised in various stages of the mitotic and meiotic cell cycle probed with M13mp8/p1581 indicated related transcripts in meiotic cells.

Curr Genet, 1987, 12(1), 1 - 7
Gene conversion, unequal crossing-over and mispairing at a non-tandem duplication during meiosis of Saccharomyces cerevisiae; Maloney DH et al.; We have developed a novel system to examine conversion, exchange and mispairing involving a non-tandem duplication of the ade8 locus in yeast by monitoring the segregation of heterozygous markers between the duplicated sequence . Plasmid Yrp17 carries the yeast selectable markers URA3+ and TRP1+ . Yrp17 derivatives with a 4 kb insert carrying ade8-18 were used to clone the mutations trp1-1 and ura3-1 by gap repair . Integrants of the resulting plasmids at the Ade8 locus were crossed to yield diploid hybrids with a non-tandem duplication of Ade8 and heterozygosity for the plasmid markers between the duplicated sequences . 1192 complete, unselected asci were analyzed and 270 exhibiting recombination of the markers contributed by the plasmid were analyzed by Southern transfers to detect changes in plasmid sequences . Twenty-seven tetrads had unequal homologous exchanges and five had unequal sister-chromatid exchanges . Seven tetrads carry an additional copy of the integrated plasmid and ten are missing one . We propose that these two classes represent conversions of the entire 11 kb plasmid, which occur after misalignment and formation of an unpaired loop . Mispairing is a frequent event, and occurs in approximately fifty percent of all meioses . The system described provides a means to determine the meiotic rules of conversion, exchange and pairing for duplicated DNA sequences.

Curr Genet, 1987, 11(4), 321 - 6
Induced cellular resistance to ultraviolet light in Saccharomyces cerevisiae is not accompanied by increased repair of plasmid DNA; White CI et al.; Many reports show that resistance of Saccharomyces cerevisiae to a large UV dose can be enhanced by pre-induction with a smaller one given some hours before . This work tests if such increased cell survival is associated with increased DNA repair on UV damaged plasmid transformed into yeast . There was no change in transformation efficiency of UV-damaged plasmid DNA under conditions where RAD cell survival increased 5-fold, and where rad1-1 and rad6-1 survival increased 2-fold . It is concluded that DNA repair activity involving the RAD6 and RAD3 pathways is either not inducible or is unable to work on plasmid DNA . It is suggested that the enhancement of cellular survival detected may be based on changes in cell-cycle behaviour which permit cells generally proficient in repair a greater chance to recover.

Gene, 1987, 60(2-3), 237 - 43
A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector; Rose MD et al.; A set of genomic plasmid banks was constructed using the centromere-containing yeast shuttle vector YCp50 . The centromere-containing vector is useful for the isolation of genes that are toxic to yeast when present in high copy number . Fourteen independent banks were prepared each with an average representation of two to three times the yeast genome . Any individual plasmid from a given bank is guaranteed to be of independent origin from plasmids obtained from each of the other banks . The banks were constructed from three different size classes of DNA fragments that resulted from varying conditions of partial digestion with Sau3A . This avoided the bias caused by differential sensitivity of sites to cleavage with Sau3A . Insert DNA is sufficiently large that most genes will be present in the set of plasmid banks at a frequency of about 0.1%.

Prep Biochem, 1987, 17(4), 435 - 46
Improved purification and some molecular and kinetic properties of sn-glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae; Chen SM et al.; The purification procedure for isolating sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from Saccharomyces cerevisiae was improved by the introduction of an ion-exchange step . Enzyme yields were doubled and the specific activity was increased as compared to the original procedure . A new value of 42,000 was obtained for the molecular weight by several denaturing methods . By native gel chromatography the molecular weight appears to be 31,000 as reported earlier . Michaelis constants were found to be 0.37 mM with dihydroxyacetone phosphate as the variable substrate and 0.018 mM for NADH as the variable substrate.

Gene, 1987, 59(2-3), 151 - 9
Expression of human pancreatic secretory trypsin inhibitor in Saccharomyces cerevisiae; Izumoto Y et al.; Human pancreatic secretory trypsin inhibitor (PSTI) cDNA was expressed in Saccharomyces cerevisiae using the yeast acid phosphatase PHO5 promoter . The product encoded by the PSTI-coding cDNA was correctly processed in yeast cells, and the PSTI molecules were efficiently secreted into the medium . The amino acid composition and the N-terminal amino acid sequence of the secreted PSTI molecules were identical to those of the authentic PSTI polypeptides from human pancreas, and the product exhibited trypsin-inhibitory activity.

Gene, 1987, 58(1), 137 - 48
Structural characteristics of the PHO8 gene encoding repressible alkaline phosphatase in Saccharomyces cerevisiae; Kaneko Y et al.; The nucleotide sequence of a 3694-bp DNA fragment bearing the PHO8 gene encoding nonspecific repressible alkaline phosphatase (rALPase; EC 3.1.3.1) of Saccharomyces cerevisiae was determined . The sequence contains a 1698 bp open reading frame (ORF), and the major PHO8 transcription start point at 32 bp upstream from the ATG codon; several minor transcription start points are located between the major start point and ATG . The major start point is most responsive to the phosphate signals . The amino acid (aa) sequence deduced from the ORF contains several homologous regions in common with alkaline phosphatases of Escherichia coli and human placenta . A PHO8 DNA fragment previously isolated {Kaneko et al., Mol . Cell . Biol . 5 (1985) 248-252} was found to be truncated for the region encoding the 22 aa residues at the C terminus of the enzyme, which were replaced with 17 aa encoded by a pBR322 DNA . The modified gene could produce significant rALPase activity without the function of proteinase A which is required for the maturation of rALPase from its precursor.

Annu Rev Microbiol, 1987, 41, 595 - 616
High-resolution NMR studies of Saccharomyces cerevisiae; Campbell-Burk SL et al.; High-resolution NMR studies of yeast cells have contributed to our understanding of metabolism and energetics . The above studies of glycolytic control, enzyme kinetics, and metabolism during dormancy have shown how the strengths of NMR investigations can build upon existing knowledge to create a qualitatively different understanding of the processes in yeast.

Microbios, 1987, 51(208-209), 183 - 90
The isolation and characterization of Ni2+ resistant mutants of Saccharomyces cerevisiae; Joho M et al.; Four Ni2+ resistant mutants of Saccharomyces cerevisiae have been isolated and characterized . In the accumulation of Ni2+, there was no significant difference between the wild type strain and the Ni2+ resistant mutants . The synthesis of RNA in the yeast was severely inhibited by Ni2+ while inhibition of protein synthesis was less pronounced . The inhibitory effect of Ni2+ on both RNA and protein synthesis in Ni2+ resistant mutants of yeast was less pronounced than in the wild type strain, while four Ni2+ resistant mutants were more sensitive to Cd2+ and Cu2+ than was the wild type strain . Ni2+ resistant mechanism(s) appeared to exist within the cells and differed from those of Cd2+ or Cu2+.

Gene, 1987, 55(2-3), 353 - 6
Synthesis and secretion of wheat alpha-amylase in Saccharomyces cerevisiae; Rothstein SJ et al.; A wheat alpha-amylase cDNA clone has been fused to the phosphoglycerate kinase initiator methionine to enable synthesis in the yeast Saccharomyces cerevisiae of an alpha-amylase enzyme that is identical in size to the wild-type alpha-amylase . The alpha-amylase is synthesized with an N-terminal plant signal peptide which is recognized in the yeast host, leading to efficient processing and secretion into the medium . The secretion of alpha-amylase into the medium is quite efficient in rich medium, but barely detectable in a minimal medium.

Gene, 1987, 55(2-3), 277 - 85
Regulation of arginine metabolism in Saccharomyces cerevisiae: expression of the three ARGR regulatory genes and cellular localization of their products; Bercy J et al.; Three regulatory proteins are involved in the control of arginine metabolism in yeast: ARGRI, ARGRII and ARGRIII . The control region and part of the coding sequence of the ARGR genes were fused to the Escherichia coli lacZ gene . These chimeras were used to study the expression of the regulatory genes as well as the cellular compartmentalization of the regulatory products . Our results show that the three ARGR proteins are localized in the nucleus and that their synthesis is not regulated by arginine nor by any of the other ARGR products . However, some data suggest that the ARGRIII protein could control ARGRI activity.

Gene, 1987, 55(2-3), 265 - 75
Characterization of two new genes essential for vegetative growth in Saccharomyces cerevisiae: nucleotide sequence determination and chromosome mapping; Dubois E et al.; Based on nucleotide sequence determination, we have identified two new yeast genes FUN80 and FUN81 located on chromosome XIII . They are both essential for cellular growth but their function is still unknown . FUN80 is closely linked to the ARGRI (or ARG80) gene while FUN81 is located next to the ARGRII (or ARG81) gene . Interestingly, the proteins encoded by these two genes have a long stretch of acidic amino acids within their C-terminal portions.

J Gen Microbiol, 1987 Jan, 133 ( Pt 1), 9 - 14
Glutamine degradation through the omega-amidase pathway in Saccharomyces cerevisiae; Soberon M et al.; A glutamine transaminase activity has been identified in Saccharomyces cerevisiae, and the existence of the omega-amidase activity previously described in this yeast has been confirmed . The glutamine transaminase utilizes different 2-oxo acids as substrates, including pyruvate and glyoxylate, and is regulated by the available nitrogen source . The glutamine transaminase activity decreases when lysine or glycine is added to the medium; the inhibition by lysine diminishes under microaerophilic culture conditions.

J Gen Microbiol, 1987 Jan, 133 ( Pt 1), 135 - 40
The cdc30 mutation in Saccharomyces cerevisiae results in a temperature-sensitive isoenzyme of phosphoglucose isomerase; Dickinson JR et al.; The cdc30 mutation in the yeast Saccharomyces cerevisiae causes cell cycle arrest late in nuclear division when cells are shifted from the permissive temperature of 25 degrees C to the restrictive temperature of 36.5 degrees C . Cell cycle arrest at 36.5 degrees C is dependent upon the carbon source used: a shift-up in glucose containing media results in cell cycle blockade, whereas a shift-up in ethanol, fructose, glycerol, glycerol plus ethanol, or mannose does not . Metabolite analyses showed accumulation of glucose 6-phosphate in a cdc30-bearing strain after a temperature shift-up in glucose-containing medium . Thermal denaturation studies and kinetic measurements indicate the existence of two isoenzymes of phosphoglucose isomerase (EC 5.3.1.9); one of which is apparently altered in the temperature-sensitive cell cycle mutant . We propose that the gene products of both the CDC30 and PG11 genes are required for cell cycle progression in glucose media and that the PGI1 gene product has a regulatory function over the CDC30 gene product.

J Gen Microbiol, 1987 Jan, 133 ( Pt 1), 1 - 8
Physiological role of glutaminase activity in Saccharomyces cerevisiae; Soberon M et al.; The participation of glutaminase activity in glutamine degradation was studied in a wild-type strain (S288C) of Saccharomyces cerevisiae . Evidence is presented that this strain has two glutaminase activities, a readily extractable form (glutaminase B) and a membrane-bound enzyme (glutaminase A) . Glutaminase A and B activities could also be distinguished by their thermostability, pyruvate sensitivity and pH optimum . Glutaminase B activity was negatively modulated by some 2-oxo acids, and in vivo pyruvate accumulation inhibited this activity . A mutant strain (CN10) with an altered glutaminase B activity was isolated and partially characterized . Its glutaminase B activity was more sensitive to inhibition by pyruvate and 2-oxoglutarate than the wild type, thus resulting in inactivation of this enzyme in vivo . The physiological role of glutaminase activity is discussed with regard to the phenotype shown by the mutant strain.

J Cell Sci Suppl, 1987, 6, 25 - 38
Excision repair in the yeast, Saccharomyces cerevisiae; McCready SJ et al.; cdc9 mutants of yeast lack detectable DNA ligase activity at restrictive temperatures . They also appear to be more sensitive than wild-type cells to ultraviolet (u.v.) radiation and it has been assumed that this is because the CDC9 ligase is needed for the final ligation step in excision repair . The fact that single-strand breaks have been demonstrated in u.v.-irradiated cdc9 mutants has been regarded as evidence for this interpretation . However, the kinetics of appearance of nicks in the DNA do not support this since maximal levels of strand breaks appear almost immediately after exposure to u.v . light and not progressively as repair events are initiated . We believe, therefore, that these strand breaks are connected with a u.v.-dependent preincision event, possibly connected with reorganization of chromatin.

Gene, 1987, 54(1), 125 - 32
Nucleotide sequence of the CLS4 (CDC24) gene of Saccharomyces cerevisiae; Miyamoto S et al.; The nucleotide sequence of the CLS4 gene controlling Ca2+ regulatory process of bud emergence, which was cloned previously {Ohya et al., J . Bacteriol . 165 (1986) 28-33}, was determined . The CLS4 (CDC24) locus encodes a protein consisting of 736 amino acid (aa) residues with an Mr of 83,970 . By primer extension mapping, the mRNA start point was located 139 bp upstream from the translation start codon . The predicted CLS4 protein was hydrophilic with two serine + threonine-rich domains in the middle and C-terminal regions . It has two putative Ca2+-binding regions, one being partly homologous to the Ca2+-binding domain of the S-100a protein and the other that of alpha-lactalbumin.

Microbios, 1987, 50(203), 99 - 108
Study of an artificial endoassociation between Saccharomyces cerevisiae and Escherichia coli; Gonzalez MT et al.; The establishment of an artificial endoassociation between Escherichia coli (JC 5466, trp, his, recA 56, lac delta X 74, SpcR, harbouring the plasmid pRD1 which confers on it the capacity to produce penicillinase), and Saccharomyces cerevisiae (3.2, a, ade, ura, lys) was carried out in order to study its behaviour and stability . The pattern of protoplast reversion to whole cells, the penicillinase production capacity, the stability without selective pressure and the bacterial localization in the yeast cells, is described and discussed.

Gene, 1987, 61(2), 207 - 15
Construction of expression plasmids for Saccharomyces cerevisiae: application for synthesis of poliovirus protein VP2; Verbakel JM et al.; A series of expression plasmids was constructed to compare the usefulness of various promoters for the synthesis of a given protein in the Saccharomyces cerevisiae . The plasmids pMBL212, -213, -214, -215 and -216 can be used to synthesize the protein of interest directly as a non-fused protein or, if the protein is difficult to detect, indirectly as an enzymatically active beta-galactosidase fusion protein . The plasmids were employed to identify which yeast promoter and strain are suitable for the synthesis of poliovirus protein VP2 . It was concluded that the GAL7 and PGK promoters in combination with strain X904 can be used for efficient synthesis of a VP2 in the form of a N-terminally fused VP2-beta-galactosidase protein.

J Bacteriol, 1987 Jan, 169(1), 416 - 8
Purification and properties of saccharopine dehydrogenase (glutamate forming) in the Saccharomyces cerevisiae lysine biosynthetic pathway; Storts DR et al.; Saccharopine dehydrogenase (glutamate forming) of the biosynthetic pathway of lysine in Saccharomyces cerevisiae was purified 1,122-fold by using acid precipitation, ammonium sulfate precipitation, DEAE-Sepharose, gel filtration, and Reactive Red-120 agarose chromatography . The enzyme exhibited a native molecular size of 69,000 daltons by gel filtration and consisted of a single 50,000-dalton polypeptide based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme was readily denatured by exposures to temperatures exceeding 46 degrees C . The pH optimum for the reverse reaction was 9.5 . The apparent Kms for L-saccharopine and NAD+ were 2.32 and 0.054 mM, respectively . The enzyme was inhibited by mercuric chloride but not by carbonyl or metal complexing agents.

Gene, 1987, 52(1), 59 - 70
Sequence and expression characteristics of a shuttle chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli; Hadfield C et al.; An efficiently transforming chloramphenicol-resistance (CmR) shuttle marker for Saccharomyces cerevisiae and Escherichia coli has been characterized in terms of its primary structure and expression characteristics . The complete nucleotide (nt) sequence of the CmR marker is given, with details on restriction sites, apparent expression signals for both organisms, and translation of the Cm acetyltransferase (CAT)-coding sequence . SDS-polyacrylamide gel electrophoresis and Western blotting have confirmed that the marker produced an identical CAT protein in yeast and E . coli . Each copy of the marker, whether present in multiple copies or as a single copy, gave rise to approx . 0.1% of the total soluble protein as CAT in haploid yeast cells . When compared with homologous expression of alcohol dehydrogenase (ADH-I) by the same ADC1 promoter, this represents a 27-fold reduction for CAT expression, which is typical of heterologous gene expression in yeast . When the marker was on a multicopy plasmid in yeast, up to 2.1% of the total soluble cell protein was produced as CAT, but this did not adversely affect the growth of host cells . Increase of the Cm concentration in the medium did not result in an increase in the number of plasmids nor the amount of CAT protein produced, showing that plasmid copy number and marker expression are regulated independently of the selection pressure . In E . coli, the ADC1 yeast-promoter DNA was found to contain both forwards and backwards promoter activity . The level of expression provided by these promoters was equivalent to that of an average E . coli gene.

EMBO J, 1987 Jan, 6(1), 235 - 41
Identification and sequence of the gene encoding cytochrome c heme lyase in the yeast Saccharomyces cerevisiae; Dumont ME et al.; Mitochondrial cytochrome c contains a heme group covalently attached through thioether linkages to two cysteinyl residues of the protein . We demonstrate here that the nuclear gene, CYC3, in the yeast Saccharomyces cerevisiae, encodes cytochrome c heme lyase (CCHL), the enzyme catalyzing the attachment of heme to apocytochrome c . Mitochondrial extracts from cyc3- mutants are deficient in CCHL activity compared with extracts from normal strains, whereas strains carrying multiple copies of the CYC3 gene exhibit high levels of the activity . The CYC3 gene was cloned by functional complementation of a cyc3- mutant using a previously isolated plasmid containing the gene PYK1, which is tightly linked to CYC3 . An open reading frame encoding a protein of 269 amino acids was identified from the DNA sequence of a fragment encompassing the CYC3 gene, and the corresponding transcript shown to be approximately 0.9 kb in length . CCHL appears to be a single polypeptide chain which acts specifically on the two forms of cytochrome c, but not on cytochrome c1.

Mol Cell Biol, 1987 Jan, 7(1), 244 - 50
Heat shock response of Saccharomyces cerevisiae mutants altered in cyclic AMP-dependent protein phosphorylation; Shin DY et al.; When Saccharomyces cerevisiae cells grown at 23 degrees C were transferred to 36 degrees C, they initiated synthesis of heat shock proteins, acquired thermotolerance to a lethal heat treatment given after the temperature shift, and arrested their growth transiently at the G1 phase of the cell division cycle . The bcy1 mutant which resulted in production of cyclic AMP (cAMP)-independent protein kinase did not synthesize the three heat shock proteins hsp72A, hsp72B, and hsp41 after the temperature shift . The bcy1 cells failed to acquire thermotolerance to the lethal heat treatment and were not arrested at the G1 phase after the temperature shift . In contrast, the cyr1-2 mutant, which produced a low level of cAMP, constitutively produced three heat shock proteins and four other proteins without the temperature shift and was resistant to the lethal heat treatment . The results suggest that a decrease in the level of cAMP-dependent protein phosphorylation results in the heat shock response, including elevated synthesis of three heat shock proteins, acquisition of thermotolerance, and transient arrest of the cell cycle.

Mol Cell Biol, 1987 Jan, 7(1), 177 - 84
Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae; Dihanich ME et al.; The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N6-(delta 2-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to less than 1.5% of wild-type levels . The tRNA modification enzyme, delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells . A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation . To that end we have cloned the MOD5 gene and shown that it restores delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase activity and N6-(delta 2-isopentenyl)adenosine to tRNA in both the mitochondria and the nucleus/cytoplasm compartments of mod5-1 yeast cells . That MOD5 sequences are expressed in Escherichia coli and can complement an N6-(delta 2-isopentenyl)-2-methylthioadenosine-deficient E . coli mutant leads us to conclude that MOD5 is the structural gene for delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase.

Mol Cell Biol, 1987 Jan, 7(1), 104 - 10
Two related regulatory sequences are required for maximal induction of Saccharomyces cerevisiae his3 transcription; Struhl K et al.; In Saccharomyces cerevisiae, the coordinate induction of his3 and other amino acid biosynthesis genes is mediated by the binding of GCN4 activator protein to specific promoter sequences . The his3 regulatory region contains the sequence TGACTC, which with some variation is repeated six times upstream of the mRNA initiation site . The requirements for maximal his3 induction were examined with a series of sequential 5' deletion mutations as well as a set of small internal deletions . Deletions encroaching as far downstream as position -142 behave indistinguishably from the wild-type gene, thus indicating that the two proximal copies of the regulatory sequence are sufficient for maximal induction . Deletions with breakpoints between -137 and -99 confer inducibility, but not to the normal wild-type level . A deletion ending immediately upstream of the proximal TGACTC sequence (position -99) shows some constitutive expression that is independent of the gcn4 gene product . Deletions extending to -94 or beyond do not produce detectable levels of his3 mRNA . Small internal deletions that only remove the proximal regulatory sequence and a 1-base-pair deletion of the thymine residue at -99 abolish induction, but do not affect the basal level of transcription . These results indicate that the proximal copy between -99 and -94 is absolutely required for his3 induction, whereas the copy between -142 and -137 is required only for the maximal level of induction and is inactive by itself . From these and other observations, we suggest the possibility that these related regulatory sequences may be targets for two distinct proteins.

Biochimie, 1987 Jan, 69(1), 25 - 36
Effects of a missense exonic mutation in cytochrome b gene, observed on isolated mitochondrial complex III of Saccharomyces cerevisiae: consequence for the antimycin binding site; Chevillotte-Brivet P et al.; The mitochondrial complex III was isolated from a wild type strain of Saccharomyces cerevisiae PS409 and from two mutants, PS490 and PS493, carrying a missense mutation in the structural gene of cytochrome b (in exons B1 and B4 respectively) . These mutants synthesize cytochrome b in variable proportions, but they are unable to grow on a respirable substrate . Strain PS493 does not bind antimycin, whereas strain PS490 contains less cytochromes b and c1 but shows a strong binding to the inhibitor . The complex isolated from the wild type strain or mutant PS493 exhibited a specific cytochrome b and cytochrome c1 heme content of approximately 8 and 4 nmol/mg of protein respectively . This content was about 3 and 2 nmol/mg with PS490, which leads to a molar stoichiometry of 1.3 : 1 for cytochromes b and c1, instead of an 'ideal' ratio of 2 : 1 expected with b-c1 complex, and obtained with the two other strains . This implies that the association (or presence) of b and c1 cytochromes is not pre-requisite for complex III assembly . The wild type complex III isolated from PS409 was found to have a high level of CoQ2H2 activity, using a synthetic coenzyme Q analog as substrate (440 s-1 mol of cytochrome c reduced/mol of cytochrome c1) . This activity is fully inhibited by antimycin . The complexes isolated from the two box mutants exhibited no such activity . Analysis of the subunit composition of the three isolated complexes on sodium dodecyl sulfate-gel electrophoresis showed that all the bands belonging to the b-c1 complex were synthesized in both mutants as well as in the wild type strain . Some of them appeared to have slightly diminished, but no specific decrease of a band has been observed in mutant PS493 that does not bind antimycin, with respect to mutant PS490 which binds strongly to the inhibitor . It should be noted that the subunit of about 12-13 kDa, qualified as the antimycin binding protein, is equally present in the three complexes . The results suggest that the loss of antimycin binding in mutant PS493 might be due to conformational perturbations in the modified complex rather than to the disappearance or significant modification of some protein support.

J Basic Microbiol, 1987, 27(4), 185 - 90
Chitin synthase activity and the rate of chitin formation in cell-division cycle mutant Saccharomyces cerevisiae cdc 24; Dankova R et al.; At the nonpermissive temperature (37 degrees C) the cells of the temperature-sensitive mutant Saccharomyces cerevisiae cdc 24 accumulated chitin 10 times faster than at 22 degrees C . In situ determinations of the activity of chitin synthase revealed that in the cells grown at 37 degrees C more than 37% of the total chitin synthase were in the active state whereas in cells grown at 22 degrees C only 7% of the potential enzyme activity were expressed . When the enzyme activity was calculated per cell number unit, there was 10 times more of the active chitin synthase per cell in the cells grown at 37 degrees C than in the cells grown at 22 degrees C, a value which correlated well with the observed difference in the rates of chitin accumulation at different temperatures.

Curr Genet, 1987, 12(8), 577 - 82
Control of the G1-G0 transition and G0 protein synthesis by cyclic AMP in Saccharomyces cerevisiae; Shin DY et al.; When the cyr1-1 cells of Saccharomyces cerevisiae, which require cyclic AMP (cAMP) for growth, were starved for cAMP, cell division was arrested at the G1 state of the mitotic cell cycle and the cells entered the resting state (G0) also observed in wild-type cells transferred to sulfur-free medium . The level of cAMP in wild-type cells decreased rapidly when the cells were starved for sulfur and subsequently increased following its addition . The cyr1-1 cells starved for cAMP preferentially synthesized nine G0 proteins . The synthesis of these G0 proteins in the sulfur-starved cells was repressed by the addition of cAMP . The RAS2val9 or bcy1 cells, which produced an elevated level of cAMP or cAMP-independent protein kinase, did not synthesize the G0 proteins under the sulfur-starved condition . The results suggest that cAMP plays a role in the transition between the proliferating state and G0 state.

Curr Genet, 1987, 12(2), 111 - 7
Missense exonic mitochondrial mutation in cytochrome b gene of Saccharomyces cerevisiae, resulting in core protein deficiency in complex III of the respiratory chain; Chevillotte-Brivet P et al.; The level of core protein I and subunit VI of mitochondrial complex III (which are coded by the nuclear genome) was found to be greatly diminished in a yeast strain carrying a mutation (W7) in the mitochondrial gene coding for cytochrome b . This suggests that intricate interactions occur in complex III biogenesis between proteins of cytoplasmic and mitochondrial origin . This mutant was characterized by a low cytochrome b level and a loss of activity in the b-c1 segment of the respiratory chain . It was compared to another mutant showing similar biochemical characteristics, but which had integrated core protein I, as shown by antibody binding experiments . In mutant devoid of core protein I, cytochrome b was found to be reducible by NADH but not by succinate, suggesting two different electron transfer pathways inside comples III from each substrate to cytochrome b heme(s).

Curr Genet, 1987, 12(5), 311 - 22
Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae: multiple trans-acting nuclear genes exert specific effects on expression of each of the cytochrome c oxidase subunits encoded on mitochondrial DNA; Kloeckener-Gruissem B et al.; Fourteen nuclear complementation groups of mutants that specifically affect the three mitochondrially-encoded subunits of yeast cytochrome c oxidase have been characterized . Genes represented by these complementation groups are not required for mitochondrial transcription, transcript processing, or translation per se but are required for the expression of one of the three genes--COX1, COX2, or COX3--which encode the cytochrome c oxicase subunits I, II, or III, respectively . Five of these genes affect the biogenesis of cytochrome c oxidase subunit I, 3 affect the biogenesis of subunit II, 3 affect the biogenesis of subunit III and 3 affect the biogenesis of both cytochrome c oxidase subunit I and cytochrome b, the product of COB . Among the 5 complementation groups of mutants that affect the expression of COX1, 2 lack COX1 transcripts, 1 produces incompletely processed COX1 transcripts, and 2 contain normal levels of normal-sized COX1 transcripts . In contrast, all 3 complementation groups which affect the expression of COX2 and all 3 complementation groups which affect the expression of COX3 exhibit no, or little, detectable difference with respect to the wild type pattern of transcripts . The 3 complementation groups which affect the expression of both COX1 and COB all have aberrant COX1 and COB transcript patterns . These findings indicate that multiple trans-acting nuclear genes are required for specific expression of each COX gene encoded on mitochondrial DNA and suggest that their products act at different steps in the expression of these mitochondrial genes.

J Cell Biol, 1987 Jan, 104(1), 67 - 75
Transport of a fluorescent macromolecule via endosomes to the vacuole in Saccharomyces cerevisiae; Makarow M et al.; Fluorescein isothiocyanate-conjugated dextran (FITC-dextran) is internalized by endocytosis into the lysosome-like vacuoles of Saccharomyces cerevisiae (Makarow, M., 1985, EMBO (Eur . Mol . Biol . Organ.) J . 4:1861-1866) . Here we show that under energy depletion conditions FITC-dextran accumulated in a cytoplasmic compartment, from which it could be chased to the vacuole when the energy block was removed . The internal pH of the intermediate compartment under energy depletion was determined by fluorometry to be 5.8 . The pH could be raised by the lysosomotropic agent ammonium chloride, the protonophore carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (CCCP) and the ATPase inhibitors dicyclohexylcarbodiimide (DCCD) and sodium vanadate . The pH of the vacuole was found to be 6.5 . It was raised by ammonium chloride, CCCP, and DCCD, but not with sodium vanadate . Efrapeptin had no effect on the internal pH of either compartment . By dissecting the endocytic pathway, two portions of the route leading to the vacuole could be studied separately . The internalization of FITC-dextran from the extracellular fluid to the intermediate compartment followed linear kinetics, was independent of energy, and occurred at temperatures of between 15 degrees and 37 degrees C . Transfer of the marker from the intermediate compartment to the vacuole required energy, took place at temperatures between 19 degrees and 37 degrees C, and had a half-time of 7 min at 37 degrees C . Transport of the marker from the exterior of the cell to the vacuole did not require acidic pH values in the intermediate compartment or the vacuole . We suggest that the cytoplasmic compartment revealed by FITC-dextran, under energy depletion, represents the equivalent of the endosomes of mammalian cells.

J Bacteriol, 1987 Jan, 169(1), 180 - 3
Apurinic endonucleases from Saccharomyces cerevisiae also recognize urea residues in oxidized DNA; Chang CC et al.; Saccharomyces cerevisiae apurinic endonucleases E cochromatographed with activity against a DNA substrate containing urea residues . The urea-recognizing activity of endonuclease E was competitively inhibited by apurinic DNA, and the heat labilities of both activities were the same . The apparent VmaxS of endonuclease E for both substrates were about the same, while the apparent Km for urea-containing DNA was about threefold greater than that for apurinic DNA . These results were similar to those obtained previously with Escherichia coli exonuclease III (Y . Kow and S . Wallace, Proc . Natl . Acad . Sci . USA 82:8354-8358, 1985) and suggest that the ability to recognize urea residues may be a general property of apurinic endonucleases.

Nucleic Acids Res, 1986 Dec 22, 14(24), 9561 - 78
Sequence and expression of four mutant aspartic acid tRNA genes from the mitochondria of Saccharomyces cerevisiae; Najarian D et al.; Expression of the mitochondrial tRNAAsp gene of Saccharomyces cerevisiae has been examined in five syn- mutants known to affect tRNAAsp function, and in a rho- mutant which accumulates precursor tRNAs . By comparison of wild-type versus mutant DNA sequence, the lesion in each syn- mutant has been identified as a single base change within the mitochondrial tRNAAsp structural gene . The mutant tRNAAsp genes are transcribed, and the transcripts can be processed to mature 4S-size tRNAAsp . The steady-state level of each mutant tRNAAsp is lower than that of wild-type tRNAAsp . The RNA from two of the syn- mutants contained a second, slow-migrating form of mitochondrial tRNAAsp which is correctly processed at the 5' end . We conclude that the lesions in the syn- mitochondrial tRNAAsp genes block neither transcription of these genes, nor 5'-end processing of the transcripts . The effect of each point mutation must be manifested at the level of 3'-end processing, or at a functional level.

Nucleic Acids Res, 1986 Dec 22, 14(24), 9631 - 51
The ILV5 gene of Saccharomyces cerevisiae is highly expressed; Petersen JG et al.; The nucleotide sequence of the yeast ILV5 gene, which codes for the branched-chain amino acid biosynthesis enzyme acetohydroxyacid reductoisomerase, has been determined . The ILV5 coding region is 1,185 nucleotides, corresponding to a polypeptide with a molecular weight of 44,280 . Transcription of the ILV5 mRNA initiates at position -81 upstream from the ATG translation start codon and terminates between 218 and 222 bases downstream from the stop codon . Consensus sequences have been identified for initiation and termination of transcription, and for general control of amino acid biosynthesis, as well as repression by leucine . The ILV5 gene is regulated slightly by general amino acid control . Codon usage of the ILV5 gene has the strong bias observed in yeast genes that are highly expressed . In agreement with this, the reductoisomerase monomer, with an apparent molecular weight of 40,000, has been identified in an SDS polyacrylamide gel pattern of total soluble yeast proteins as a gene dosage dependent band.

Eur J Biochem, 1986 Dec 15, 161(3), 565 - 9
Hexokinase PII from Saccharomyces cerevisiae is regulated by changes in the cytosolic Mg2+-free ATP concentration; Moreno F et al.; Hexokinase PII is not inhibited by high Mg-ATP concentrations if the Mg2+-free ATP is kept at low levels (0.01 mM) in the assay mixture . Hexokinase PI activity is not affected either by Mg2+-free ATP nor by free Mg2+ in the assay mixture . Thus, hexokinase PI and PII activities appear not to be regulated by substrate inhibition as proposed previously {Kopetzki, E . & Entian, K . D . (1985) Eur . J . Biochem . 146, 657-662} . However, the level of Mg2+-free ATP in the hexokinase PII assay mixture strongly affects the enzyme activity by decreasing the Vmax and increasing the Km value for Mg-ATP from 0.15 mM to 5.0 mM . The physiological role of this inhibition, which has not been described previously, was investigated by determining the cytosolic ATP and Mg2+ concentrations in yeast cells grown under derepressing and repressing conditions . Derepression is accompanied by an important loss of Mg2+ from the cells, maintaining the ATP concentration constant . This produces an increase of Mg2+-free ATP in the cytosol from 0.01 mM to 0.1 mM . This free ATP concentration would lead to a maximal inhibition of hexokinase PII.

EMBO J, 1986 Dec 1, 5(12), 3381 - 9
Mapping of functional domains within the Saccharomyces cerevisiae type 1 killer preprotoxin; Sturley SL et al.; Strains of Saccharomyces cerevisiae harboring M1-dsRNA, the determinant of type 1 killer and immunity phenotypes, secrete a dimeric 19-kd toxin that kills sensitive yeast cells by the production of cation-permeable pores in the cytoplasmic membrane . The preprotoxin, an intracellular precursor to toxin, has the domain sequence delta-alpha-gamma-beta where alpha and beta are the 9.5-and 9.0-kd subunits of secreted toxin . Plasmids containing a partial cDNA copy of M1, in which alpha, gamma, and beta are fused to the PH05 promoter and signal peptide, have previously been shown to express phosphate-repressible toxin production and immunity . Here the construction of a complete DNA copy of the preprotoxin gene and its mutagenesis are described . Analysis of the expression of these mutants from the PH05 promoter elucidates the functions of the preprotoxin domains . delta acts as a leader peptide and efficiently mediates the secretion, glycosylation and maturation of killer toxin . Mutations within the beta subunit indicate it to be essential for binding of toxin to and killing of whole cells but unnecessary for the killing of spheroplasts . Mutations within the putative active site of alpha prevent killing of both cells and spheroplasts . The probable role of beta is therefore recognition and binding to the cell wall receptor whereas alpha is the active ionophore . Mutations within alpha causing loss of toxicity also cause loss of immunity, while the mutants described within gamma and beta retain partial or complete immunity . Expression of gamma without alpha or beta confers no phenotype . The immunity determinant may minimally consist of the alpha domain and the N-terminal portion of gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1986 Dec, 6(12), 4509 - 15
Mitochondrial and nonmitochondrial citrate synthases in Saccharomyces cerevisiae are encoded by distinct homologous genes; Rosenkrantz M et al.; Saccharomyces cerevisiae contains two genes, CIT1 and CIT2, encoding functional citrate synthase (K.-S . Kim, M . S . Rosenkrantz, and L . Guarente, Mol . Cell . Biol . 6:1936-1942, 1986) . We show here that CIT2 encodes a nonmitochondrial form of citrate synthase . The DNA sequence of CIT2 presented provides a possible explanation for why the CIT2 product, unlike the CIT1 product, fails to be imported into mitochondria . While the products of these two genes are highly homologous, they diverge strikingly at their amino termini . The amino terminus of the CIT1 primary translation product extends 39 residues beyond the amino termini of Escherichia coli and porcine citrate synthases . This extension consists of a typical mitochondrial targeting motif . The amino terminus of the CIT2 primary translation product extends 20 residues beyond the amino termini of the E . coli and porcine enzymes . The CIT2-encoded extension is not homologous to that of CIT1, resulting in a nonmitochondrial localization of the product . The CIT2-encoded extension, however, does bear certain similarities to mitochondrial targeting sequences . The possible role of this sequence in targeting this CIT2 product to a nonmitochondrial organelle is discussed.

Mol Cell Biol, 1986 Dec, 6(12), 4478 - 85
The SPS4 gene of Saccharomyces cerevisiae encodes a major sporulation-specific mRNA; Garber AT et al.; The SPS4 gene of Saccharomyces cerevisiae, a sporulation-specific gene identified previously in a differential hybridization screen of a genomic yeast DNA library, has been characterized further . The protein encoded by this gene was inferred from its nucleotide sequence to be 38,600 daltons with an isoelectric pH of 8.2 . Consistent with this, two-dimensional polyacrylamide gel electrophoresis of the in vitro translation products of RNA purified by hybridization with the cloned SPS4 DNA indicated that the SPS4 gene product is a 39-kilodalton, basic protein . This protein was found to be identical in size and charge to a major, sporulation-specific protein identified in a two-dimensional polyacrylamide gel electrophoretic comparison of the in vitro translation products of total RNA from sporulating MATa/MAT alpha cells and asporogenous MAT alpha/MAT alpha cells . A MATa/MAT alpha strain homozygous for a partial deletion of the SPS4 gene appeared, however, to be unaffected in its ability to form viable ascospores.

Mol Cell Biol, 1986 Dec, 6(12), 4335 - 43
Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences; Ogden JE et al.; The Saccharomyces cerevisiae PGK (phosphoglycerate kinase) gene encodes one of the most abundant mRNA and protein species in the cell . To identify the promoter sequences required for the efficient expression of PGK, we undertook a detailed internal deletion analysis of the 5' noncoding region of the gene . Our analysis revealed that PGK has an upstream activation sequence (UASPGK) located between 402 and 479 nucleotides upstream from the initiating ATG sequence which is required for full transcriptional activity . Deletion of this sequence caused a marked reduction in the levels of PGK transcription . We showed that PGK has no requirement for TATA sequences; deletion of one or both potential TATA sequences had no effect on either the levels of PGK expression or the accuracy of transcription initiation . We also showed that the UASPGK functions as efficiently when in the inverted orientation and that it can enhance transcription when placed upstream of a TRP1-IFN fusion gene comprising the promoter of TRP1 fused to the coding region of human interferon alpha-2.

Mol Cell Biol, 1986 Dec, 6(12), 4274 - 80
In vivo evidence for posttranslational translocation and signal cleavage of the killer preprotoxin of Saccharomyces cerevisiae; Lolle SJ et al.; A full-length cDNA of the M1 double-stranded RNA killer preprotoxin coding region successfully directed the synthesis of secreted K1 toxin when expressed in Saccharomyces cerevisiae from a plasmid vector . Three protein species immunoreactive with antitoxin antiserum were detected intracellularly in transformants harboring this killer cDNA plasmid . These toxin precursor species were characterized by using secretory-defective hosts, by comparative electrophoretic mobilities, and by tunicamycin susceptibility . Such studies indicate that these three protein species represent intermediates generated by signal cleavage of the preprotoxin and its subsequent glycosylation and provide evidence that these events occur posttranslationally.

Mol Cell Biol, 1986 Dec, 6(12), 4145 - 8
Negative regulation of the Saccharomyces cerevisiae ANB1 gene by heme, as mediated by the ROX1 gene product; Lowry CV et al.; In Saccharomyces cerevisiae the anaerobic (oxygen-repressed) ANB1 gene and a group of aerobic (oxygen-induced) genes are coordinately regulated by the ROX1 gene . We report here that heme, known as an inducer of aerobic genes, also causes inhibition of ANB1 expression . Thus, in combination with the ROX1 gene product heme has an opposite effect on the expression of anaerobic and aerobic genes . Accumulation of ANB1 mRNA was sharply decreased in anaerobic cells grown in the presence of heme . This effect must operate at the level of transcription since heme also inhibited accumulation of CYC1 mRNA from an ANB1-CYC1 fusion . Heme precursors did not appear to function either as inhibitors or as activators . Oxygen itself also had no effect on transcription of ANB1 . Repression by heme cannot be attributed to the respiratory competence conferred by heme since both ANB1 and the aerobic genes tr-1 and CYC1 were regulated normally in {rho 0} mutants . The results are consistent with a classical allosteric coeffector function for heme, although more indirect explanations are tenable . A role for the ROX1 gene product in transcriptional regulation can be inferred from the observation that there was no inhibition of ANB1 expression by heme in rox1 mutants . Judging from this epistasis the rox1 phenotype is not due to a defect in heme production; this would indicate that the ROX1 factor functions by mediating the effect of heme on transcription.

J Bacteriol, 1986 Dec, 168(3), 1472 - 5
Agglutination and mating activity of the MF alpha 2-encoded alpha-factor analog in Saccharomyces cerevisiae; Kurjan J et al.; The MF alpha 2-encoded Asn-5,Arg-7 alpha-factor-like peptide has been shown shown to have similar activity to Gln-5,Lys-7 alpha-factor in morphogenesis and growth arrest studies (S . Raths, P . Shenbagamurthi, F . Naider, and J . M . Becker, J . Bacteriol . 168:1468-1471, 1986) . We tested the Asn-5,Arg-7 peptide in agglutination and mating assays and found that its activity was similar to or slightly less than that of the Gln-5,Lys-7 alpha-factor . The Asn-5,Arg-7 alpha-factor-like peptide is thus the most active analog of the Gln-5,Lys-7 alpha-factor known.

J Bacteriol, 1986 Dec, 168(3), 1352 - 7
Fluphenazine-resistant Saccharomyces cerevisiae mutants defective in the cell division cycle; Matsumoto K et al.; An fls1 mutant of Saccharomyces cerevisiae, which did not grow in the presence of 30 micrograms of fluphenazine per ml, was isolated . Mutants that were resistant to 90 micrograms of fluphenazine per ml and temperature sensitive for growth were obtained from the fls1 mutant . One fluphenazine-resistance mutation, fsr1, was located near the his7 locus on chromosome II . Growth of the fsr1 mutants at 35 degrees C was arrested after nuclear division . The other group of fluphenazine-resistant mutants, carrying fsr2 mutations, showed Ca2+-dependent growth at 35 degrees C . Growth of the fsr2 mutants at 35 degrees C was arrested at the G2 stage of the cell cycle in Ca2+-poor medium.

J Bacteriol, 1986 Dec, 168(3), 1336 - 42
Growth-rate-dependent regulation of the expression and inactivation of thymidylate synthase in Saccharomyces cerevisiae; Greenwood MT et al.; Thymidylate synthase activity fluctuated dramatically as cultures of Saccharomyces cerevisiae progressed through the different stages of batch culture growth . During logarithmic growth these yeast cultures each contained about 40 microU (1 microU is 1 pmol of 3H released per min) of thymidylate synthase activity per 10(8) haploid cells, but as cultures entered the stationary phase and during the stationary phase, activity dropped dramatically, eventually reaching undetectable levels . Stimulation of stationary-phase cells with fresh medium resulted in rapid reestablishment of log phase levels . Two mechanisms, the regulation of thymidylate synthase-specific transcripts and the irreversible inactivation of thymidylate synthase activity, acted in concert to regulate activity levels . These results suggested that thymidylate synthase represents a special subset of yeast proteins whose levels per cell vary quickly and dramatically in response to changes in proliferation rates.

J Bacteriol, 1986 Dec, 168(3), 1250 - 3
Germination conditions that require mitochondrial function in Saccharomyces cerevisiae: utilization of acetate and galactose; Donnini C et al.; Ascospores of Saccharomyces cerevisiae inherited at least one functioning mitochondrion as shown by their ability to germinate on nonfermentable carbon sources . After transfer to germination medium, the optical density of the culture at 600 nm decreased (phase-dark), reaching a minimum within 60 min in the presence of glucose and within 180 min after transfer to acetate medium; thereafter, the optical density increased . Budding cells first appeared 90 min after transfer to glucose and 150 min after transfer to acetate . Augmentation of respiratory components, respiratory activity, and macromolecular synthesis (except for DNA synthesis) started at about the same time on glucose and on acetate, although the highest values for all these processes were reached in the presence of glucose . Mitochondrial inhibitors which affected germination on acetate did not arrest germination on glucose . However, mitochondrial activity was required for germination on galactose in a strain carrying the mutated allele imp1 of the nucleomitochondrion-connecting gene IMP1.

J Gen Microbiol, 1986 Dec, 132 ( Pt 12), 3467 - 72
Mechanism of inactivation of hexokinase PII of Saccharomyces cerevisiae by D-xylose; Fernandez R et al.; The mechanism of inactivation of hexokinase PII of Saccharomyces cerevisiae by D-xylose was characterized . Inactivation was dependent on the presence of MgATP and was irreversible . Inactivation involved phosphorylation of the protein . Observation of the carbon catabolite repression of selected enzymes showed that invertase and maltase synthesis were not repressed when hexokinase PII was phosphorylated.

J Gen Microbiol, 1986 Dec, 132 ( Pt 12), 3309 - 13
Catabolite repressive effects of 5-thio-D-glucose on Saccharomyces cerevisiae; Egilsson V et al.; The effect of the glucose analogue 5-thio-D-glucose (5TG) on the yeast Saccharomyces cerevisiae was studied . Derepression of mitochondrial respiratory chain cytochromes, alcohol dehydrogenase (isoenzyme II), NADH dehydrogenase and maltase was inhibited by 0.5-2 mM-5TG . Growth rate was only slightly affected . Ethanol was efficiently produced with 2 mM-5TG in medium initially containing 0.25% glucose . Mutants resistant to the growth inhibitory effects of 5TG on glycerol medium showed resistance to the catabolite repressing effects of glucose . Other mutants, known to be catabolite repression resistant, showed resistance to 5TG . The analogue seems to inhibit derepression of glucose repressible enzymes with greater potency than glucose itself.

Mutat Res, 1986 Dec, 175(4), 223 - 9
Endogenous promutagen activation in the yeast Saccharomyces cerevisiae: factors influencing aflatoxin B1 mutagenicity; Niggli B et al.; The formation of convertants, revertants and other types of mitotic segregants was induced in Saccharomyces cerevisiae D7 upon incubation with aflatoxin B1 (AFB1) . The most distinct effects were observed for gene conversion to tryptophan prototrophy . The fact that different cytochrome P-450 inhibitors (ellipticine, penconazole and propiconazole as yeast-specific P-450 inhibitors) abolished the AFB1-induced mutagenicity indicates that activation of the promutagen AFB1 depends on the cytochrome P-450-catalyzed electron-transfer reactions . This hypothesis is further supported by the observation that the cytochrome P-450 content of yeast cells harvested at different phases during growth is directly correlated with their sensitivity for AFB1-induced tryptophan conversion.

DNA, 1986 Dec, 5(6), 483 - 91
Improved secretion of heterologous proteins by Saccharomyces cerevisiae: effects of promoter substitution in alpha-factor fusions; Ernst JF; The effects of promoter strength on secretion of a heterologous protein, somatomedin-C (SMC), by the yeast Saccharomyces cerevisiae were studied by using the promoters of the MF alpha 1, ACT, and CYC1 genes to control expression of alpha-factor/SMC gene fusions . When a low-copy centromere vector was used to carry the gene fusions in yeast transformants, the greatest secretion was obtained with the MF alpha 1 promoter construction and the least with the CYC1 promoter construction . Unexpectedly, using two types of multicopy vectors, the greatest secretion was obtained with the CYC1 promoter construction and the least with the MF alpha 1 promoter construction . The decrease in secretion by the strongest promoter construction (MF alpha 1 promoter) on multicopy vectors was associated with a decrease in SMC mRNA during growth, a decrease in vector copy number, a decrease in vector stability, and a decrease in transformation frequency . The results demonstrate that, unlike in intracellular expression, promoter strength is not simply related to secretion expression levels . Selection against oversecreting cells during growth may explain the reduced secretion efficiency of strong promoter constructions.

Proc Natl Acad Sci U S A, 1986 Dec, 83(24), 9303 - 7
Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae; Sass P et al.; A gene, PDE2, has been cloned from the yeast Saccharomyces cerevisiae that, when present in high copy, reverses the phenotypic effects of RAS2Val19, a mutant form of the RAS2 gene that renders yeast cells sensitive to heat shock and starvation . It has previously been shown that the RAS proteins are potent activators of yeast adenylate cyclase . We report here that PDE2 encodes a high-affinity cAMP phosphodiesterase that shares sequence homology with animal cell phosphodiesterases . These results therefore imply that the effects of RAS2Val19 are mediated through its changes in cAMP concentration.

Mol Cell Biol, 1986 Dec, 6(12), 4690 - 6
A single Saccharomyces cerevisiae upstream activation site (UAS1) has two distinct regions essential for its activity; Lalonde B et al.; Several site-directed mutagenesis regimens were used to generate single- and multiple-base substitutions in the upstream activation site UAS1 of the Saccharomyces cerevisiae CYC1 gene . Mutations resulting in large reductions in activity of the site lie in two distinct regions . Six single-base changes in a region A, between -288 and -285, all resulted in a 15-fold reduction in activity . Synthetic sites built up solely of multimers of the -289 to -285 sequence ACCGA behaved as carbon catabolite-sensitive UASs . In addition, substitution mutations in a second region, at nucleotides -266 and -265, virtually eliminated UAS1 activity . These mutations abolished the binding of a heme-dependent protein factor in vitro . Thus, UAS1 contains two essential regions both of which are required for its activity.

Mol Cell Biol, 1986 Dec, 6(12), 4419 - 24
Isolation and characterization of PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae; Keierleber C et al.; We isolated a cloned DNA fragment containing PRT1, a gene required for the initiation of protein biosynthesis in Saccharomyces cerevisiae, by complementation of the temperature-sensitive prtl-1 mutation . The entire PRT1 gene is contained within a 3.2-kilobase-pair segment of the cloned DNA in YEp13 H1.2 . Southern blot analysis demonstrated that PRT1 is a single copy gene which is transcribed into a 2.3-kilobase RNA . We determined the direction of transcription and mapped the 5' and 3' ends of the gene.

Mol Cell Biol, 1986 Dec, 6(12), 4251 - 8
Multiple control elements in the TRP1 promoter of Saccharomyces cerevisiae; Kim S et al.; The TRP1 promoter generates two groups of mRNAs, transcript I and transcript II . The difference in size between the largest and smallest mRNAs is about 200 base pairs . A series of one-sided and internal deletions were constructed in vitro throughout the TRP1 promoter, and the effect of each deletion on transcription was assessed by Northern blotting . We showed that 395 base pairs of the TRP1 promoter were sufficient for the normal transcription of all RNAs and that the promoter contained two control domains . The control domain for transcript I consisted of one positive element and one negative element, while the control domain for transcript II contained two positive elements . The negative element, mapped between -293 and -318, expression of transcript I . Two regions of transcript I . Two regions (-280 to -236 and -235 to -209) were required for accurate initiation of transcript I . Each region contained sequences homologous to known consensus sequences of the TATA box.

Biochem J, 1986 Dec 1, 240(2), 541 - 7
Regulation of early enzymes of ergosterol biosynthesis in Saccharomyces cerevisiae; Servouse M et al.; In order to determine the regulation mechanisms of ergosterol biosynthesis in yeast, we developed growth conditions leading to high or limiting ergosterol levels in wild type and sterol-auxotrophic mutant strains . An excess of sterol is obtained in anaerobic sterol-supplemented cultures of mutant and wild type strains . A low sterol level is obtained in aerobic growth conditions in mutant strains cultured with optimal sterol supplementation and in wild type strain deprived of pantothenic acid, as well as in anaerobic cultures without sterol supplementation . Measurements of the specific activities of acetoacetyl-CoA thiolase, HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase and HMG-CoA reductase (the first three enzymes of the pathway), show that in cells deprived of ergosterol, acetoacetyl-CoA thiolase and HMG-CoA synthase are generally increased . In an excess of ergosterol, in anaerobiosis, the same enzymes are strongly decreased . A 5-10-fold decrease is observed for acetoacetyl-CoA thiolase and HMG-CoA synthase . In contrast, HMG-CoA reductase is only slightly affected by these conditions . These results show that ergosterol could regulate its own synthesis, at least partially, by repression of the first two enzymes of the pathway . Our results also show that exogenous sterols, even if strongly incorporated by auxotrophic mutant cells, cannot suppress enzyme activities in aerobic growth conditions . Measurement of specific enzyme activities in mutant cells also revealed that farnesyl pyrophosphate thwarts the enhancement of the activities of the two first enzymes.

J Bacteriol, 1986 Dec, 168(3), 1254 - 7
The Saccharomyces cerevisiae start mutant carrying the cdc25 mutation is defective in activation of plasma membrane ATPase by glucose; Portillo F et al.; Activation of plasma membrane ATPase by the addition of glucose was examined in several cell division cycle mutants of Saccharomyces cerevisiae . The start mutant carrying the cdc25 mutation was shown to be defective in ATPase activation at the restrictive temperature . Genetic analysis showed that lack of growth and defective activation of ATPase at the restrictive temperature were caused by the same mutation . It was also found that CDC25 does not map at the same locus as the structural gene of plasma membrane ATPase (PMA1) . We conclude that the product of CDC25 controls the activation of ATPase.

FEBS Lett, 1986 Nov 24, 208(2), 208 - 10
DNA sequence analysis of diuron-resistant mutations in the mitochondrial cytochrome b gene of Saccharomyces cerevisiae; di Rago JP et al.; Diuron (3-{3,4-dichlorophenyl}-1,1-dimethylurea), an inhibitor of mitochondrial respiration, blocks the yeast respiratory chain between cytochrome b and c1 . Diuron-resistant mutants of Saccharomyces cerevisiae have been selected and several mutations localized to the mitochondrial cytochrome b gene . The present paper identifies specific DNA base changes within the cytochrome b gene conferring diuron-resistance . DNA sequence analysis was done utilizing primer extension of crude mitochondrial RNA preparations in the presence of reverse transcriptase . Five independent diuron-resistant mutations have been sequenced.

Biochim Biophys Acta, 1986 Nov 17, 862(2), 407 - 12
Possible role of histidine in the L-proline transport system of Saccharomyces cerevisiae; Horak J; The L-proline transport system of Saccharomyces cerevisiae is shown to be specifically inactivated upon incubation of intact yeast cells with the histidine modifier diethylpyrocarbonate . The extent of inactivation is half-maximum at 0.5 mM diethylpyrocarbonate for an incubation of 2 min at 30 degrees C and pH 6.0 . Under the same conditions, the time dependence of inactivation is monophasic with the second-order rate constant of 5.5 M-1 X s-1 and the maximum rate Jmax of L-proline transport is lowered by about 50%, while the KT value remains unchanged . Moreover, L-proline afforded significant protection against diethylpyrocarbonate inactivation . The complete reactivation of a partially inactivated L-proline transport system by neutral hydroxylamine and the elimination of the possibility that the modification of other amino acid residues are responsible for the inactivation, suggested that the transport protein inactivation occurs solely by a modification of histidine residues.

Eur J Biochem, 1986 Nov 17, 161(1), 1 - 6
Protein synthesis in yeast Saccharomyces cerevisiae . Purification of Co-eIF-2A and 'mRNA-binding factor(s)' and studies of their roles in Met-tRNAf.40S.mRNA complex formation; Nasrin N et al.; Antibodies prepared against a homogeneous preparation of Co-eIF-2A20 {Ahmad et al . (1985) J . Biol . Chem . 260, 6955-6959} reacted with several polypeptides including an 80-kDa polypeptide present in a crude yeast ribosomal salt wash . This 80-kDa polypeptide, containing Co-eIF-2A (Co-eIF-2A80) activity, has been extensively purified using a two-step purification procedure involving an immunoaffinity column chromatograph prepared using antibodies against Co-eIF-2A20 (fraction II) and hydroxyapatite chromatography (fraction III) . The factors, eIF-2 + homogeneous Co-eIF-2A80 (fraction III) promoted Met-tRNAf.40S complex formation with an AUG codon but not with a physiological mRNA or a polyribonucleotide messenger poly(U,G) whereas eIF-2 + a partially purified Co-eIF-2A80 preparation (fraction II) promoted Met-tRNAf.40S complex formation with an AUG codon as well as with globin mRNA and poly(U,G) messenger . This factor-promoted Met-tRNAf binding to 40S ribosomes depends absolutely on the presence of a polyribonucleotide messenger containing an initiation codon (such as AUG or GUG) . Other polyribonucleotide messengers tested, such as poly(U), poly(A) and poly(A,C) were completely ineffective in this binding reaction . This result indicates that the Met-tRNAf.40S.mRNA complex is formed by a direct interaction between Met-tRNAf, 40S ribosomes and the initiation site in mRNA . A mechanism has been proposed for Met-tRNAf.40S.mRNA complex formation in yeast.

Arch Biochem Biophys, 1986 Nov 15, 251(1), 205 - 14
Partial purification and characterization of the interconvertible forms of trehalase from Saccharomyces cerevisiae; Dellamora-Ortiz GM et al.; Cryptic trehalase from Saccharomyces cerevisiae was purified about 3000-fold . The recovery of 970% of the original "activity" indicated the removal of an inhibitor of the enzyme . Active trehalase, obtained through phosphorylation of cryptic trehalase by cAMP-dependent protein kinase, was isolated by chromatography on DEAE-cellulose . A major phosphorylated protein, with an apparent Mr of 86,000, was detected after SDS-polyacrylamide gel electrophoresis . This protein band correlated exactly with the elution profile of trehalase activity and 32Pi incorporation into the enzyme on DEAE-cellulose chromatography . Partially purified active trehalase showed absolute specificity towards trehalose with an apparent Km of 4.79 X 10(-3) M . Both forms of the enzyme showed an apparent molecular weight of 160,000, by gel filtration . Centrifugation on a glycerol density gradient indicated multiple forms of trehalase-c, with Mr of 320,000, 160,000, and 80,000 . After activation of each of these forms by protein kinase, a single form of trehalase-a was observed, with a Mr of 160,000 . Trehalase-c appears to be a totally inactive form of the enzyme . The only mechanism of activation seems to be phosphorylation by cAMP-dependent protein kinase . When the protein kinase concentration was varied, at a fixed trehalase-c concentration, a sigmoidal activation plot was obtained . This result suggests the occurrence of multiple forms of cryptic trehalase.

J Biol Chem, 1986 Nov 15, 261(32), 15147 - 52
Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae; Sburlati A et al.; Strains containing a disrupted structural gene for chitin synthetase (chs1::URA3) are defective in chitin synthetase 1 (Chs1) activity but contain normal amounts of chitin (Bulawa, C.E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, L., and Robbins, P . W . (1986) Cell 46, 213-225) . We have now detected in such strains a new chitin synthetase activity (Chs2), at levels about 5% of those of Chs1 in wild-type cells . Thus, Chs2 is presumably the physiological agent for chitin deposition in strains with a disrupted CHS1 gene and probably also in wild-type strains . Chs1 and Chs2 share certain properties, such as stimulation by N-acetylglucosamine and by partial proteolysis . They differ sharply, however, in divalent cation specificity and in pH optimum . Chs2 also shows less sensitivity than Chs1 to inhibition by polyoxin D or sodium chloride, a property that was used to demonstrate the presence of Chs2 in wild-type extracts . As in the case of Chs1, most of the Chs2 activity was found to be associated with the plasma membranes . This finding, together with the apparent zymogenic nature of Chs2, is consistent with the hypothesis, previously put forward for Chs1, that localized deposition of chitin is attained by activation of the zymogen form at a specific time and place . Function and significance of the two chitin synthetases are discussed in connection with fungal morphogenesis and evolution.

Eur J Biochem, 1986 Nov 3, 160(3), 487 - 90
Nucleotide sequence of the Saccharomyces cerevisiae CTT1 gene and deduced amino-acid sequence of yeast catalase T; Hartig A et al.; A 2642-base-pair DNA fragment containing the catalase T (CTT1) structural gene of the yeast Saccharomyces cerevisiae and its flanking regions has been sequenced . The gene codes for a protein of 562 amino acids (relative molecular mass 64,449) and appears to contain no intron . The amino acid sequence of catalase T derived from the DNA sequence shows 40.7% homology (52.2% including conservative replacements) to that of bovine liver catalase . All amino acids previously postulated to participate directly in catalysis by liver catalase and most of the amino acids of the immediate environment of hemin, the prosthetic group of catalase, are conserved in catalase T . The data obtained indicate that the folding of polypeptide chains of the two catalases compared has been conserved within a central region consisting mainly of the beta-barrel domain, which bears the prosthetic group, and a major part of the "wrapping domain" . N- and C-terminal regions involved in subunit interactions are less well conserved . It is suggested that their structure is more similar to that of the corresponding regions of Penicillium vitale catalase . However, catalase T lacks the C-terminal flavodoxin-like domain present in this protein.

Mol Cell Biol, 1986 Nov, 6(11), 4099 - 103
Sequence analysis of temperature-sensitive mutations in the Saccharomyces cerevisiae gene CDC28; Lorincz AT et al.; Eleven independently isolated temperature-sensitive mutations in the cell division cycle gene CDC28 were mapped with respect to the DNA sequence of the wild-type gene and then sequenced to determine the precise nature of each mutation . The set yielded six different point mutations, each of which predicts a single amino acid substitution in the CDC28 product . The positions of the mutations did not correlate in any obvious way with observable biological characteristics of the mutant alleles . When the positions of substitutions were collated with a predicted secondary structural analysis of the CDC28 protein kinase, they were found to correlate strongly with probable regions of structural transition.

Mol Cell Biol, 1986 Nov, 6(11), 3990 - 8
Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae; Harashima S et al.; GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae . Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression . We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13 . All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles . By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell . Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion . Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions . Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions . In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype . This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

Mol Cell Biol, 1986 Nov, 6(11), 3685 - 93
Mitotic gene conversion lengths, coconversion patterns, and the incidence of reciprocal recombination in a Saccharomyces cerevisiae plasmid system; Ahn BY et al.; Plasmids capable of undergoing genetic exchange in mitotically dividing Saccharomyces cerevisiae cells were used to measure the length of gene conversion events, to determine patterns of coconversion when multiple markers were present, and to correlate the incidence of reciprocal recombination with the length of conversion tracts . To construct such plasmids, restriction site linkers were inserted both within the HIS3 gene and in the flanking sequences, and two different his3- alleles were placed in a vector . Characterization of the genetic exchanges in these plasmids showed that most occur with the conversion of one his3- allele . Many of these events included coconversions in which more than one marker along the allelic sequence was replaced . The frequency of coconversion decreased with the distance between two markers such that markers further than 1 kilobase apart were infrequently coconverted . From these results the average length of conversion was determined to be approximately 0.5 kilobase . Examination of coconversions involving three or more markers revealed an almost obligatory, simultaneous coconversion pattern of all markers . Thus, when two markers which flank an intervening marker are converted, the intervening marker is 20 times more likely to be converted than to remain unchanged . The results of these studies also showed that the incidence of reciprocal recombination, which accompanies more than 20% of the conversion events, is more frequent when the conversion tract is longer than average.

Mol Cell Biol, 1986 Nov, 6(11), 3643 - 51
Molecular analysis of SNF2 and SNF5, genes required for expression of glucose-repressible genes in Saccharomyces cerevisiae; Abrams E et al.; The SNF2 and SNF5 genes are required for derepression of SUC2 and other glucose-repressible genes of Saccharomyces cerevisiae in response to glucose deprivation . Previous genetic evidence suggested that SNF2 and SNF5 have functionally related roles . We cloned both genes by complementation and showed that the cloned DNA was tightly linked to the corresponding chromosomal locus . Both genes in multiple copy complemented only the cognate snf mutation . The SNF2 gene encodes a 5.7-kilobase RNA, and the SNF5 gene encodes a 3-kilobase RNA . Both RNAs contained poly(A) and were present in low abundance . Neither was regulated by glucose repression, and the level of SNF2 RNA was not dependent on SNF5 function or vice versa . Disruption of either gene at its chromosomal locus still allowed low-level derepression of secreted invertase activity, suggesting that these genes are required for high-level expression but are not directly involved in regulation . Further evidence was the finding that snf2 and snf5 mutants failed to derepress acid phosphatase, which is not regulated by glucose repression . The SNF2 and SNF5 functions were required for derepression of SUC2 mRNA.

Mol Cell Biol, 1986 Nov, 6(11), 3621 - 5
Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome; Boylan MT et al.; We constructed Aspergillus nidulans transformation plasmids containing the A . nidulans argB+ gene and either containing or lacking centromeric DNA from Saccharomyces cerevisiae chromosome XI (CEN11) . The plasmids transformed an argB Aspergillus strain to arginine independence at indistinguishable frequencies . Stable haploid transformants were obtained with both plasmids, and strains were identified in which the plasmids had integrated into chromosome III by homologous recombination at the argB locus . Plasmid DNA was recovered from a transformant containing CEN11, and the sequence of the essential portion of CEN11 was determined to be unaltered . The transformants were further characterized by using them to construct heterozygous diploids and then testing the diploids for preferential loss of the plasmid-containing chromosomes . The CEN11 sequence had little or no effect on chromosome stability . Thus, CEN11 does not prevent chromosomal integration of plasmid DNA and probably lacks centromere activity in Aspergillus spp.

Mol Cell Biol, 1986 Nov, 6(11), 3569 - 74
Null mutations in the SNF3 gene of Saccharomyces cerevisiae cause a different phenotype than do previously isolated missense mutations; Neigeborn L et al.; Missense mutations in the SNF3 gene of Saccharomyces cerevisiae were previously found to cause defects in both glucose repression and derepression of the SUC2 (invertase) gene . In addition, the growth properties of snf3 mutants suggested that they were defective in uptake of glucose and fructose . We have cloned the SNF3 gene by complementation and demonstrated linkage of the cloned DNA to the chromosomal SNF3 locus . The gene encodes a 3-kilobase poly(A)-containing RNA, which was fivefold more abundant in cells deprived of glucose . The SNF3 gene was disrupted at its chromosomal locus by several methods to create null mutations . Disruption resulted in growth phenotypes consistent with a defect in glucose uptake . Surprisingly, gene disruption did not cause aberrant regulation of SUC2 expression . We discuss possible mechanisms by which abnormal SNF3 gene products encoded by missense alleles could perturb regulatory functions.

Genetics, 1986 Nov, 114(3), 769 - 89
Repression of meiotic crossing over by a centromere (CEN3) in Saccharomyces cerevisiae; Lambie EJ et al.; The location of the centromere of chromosome III (CEN3) of Saccharomyces cerevisiae has been altered by means of transformation . The frequency of meiotic crossing over in the CEN3-PGK1 and LEU2-CEN3 intervals increases approximately 1.5- and fourfold, respectively, when CEN3 is repositioned at HIS4 . The centromere-distal HIS4-LEU2 region experiences a three- to fivefold decrease in the frequency of meiotic exchange when CEN3 is repositioned at HIS4 . The inhibition of meiotic crossing over is conferred by a 627-base-pair fragment of CEN3 DNA and is not dependent on the orientation of CEN3 relative to the rest of chromosome III.

Arch Biochem Biophys, 1986 Nov 1, 250(2), 382 - 9
Effect of fatty acid supplementation on thermotropic behavior of membrane lipids and leucine transport in Saccharomyces cerevisiae; Basu J et al.; An unsaturated fatty acid-requiring mutant (KD 115) of Saccharomyces cerevisiae shows altered phospholipid composition, transport behavior, and physical properties of membrane lipids when grown in the presence of different cis- and trans-unsaturated fatty acids . There is an increase in phosphatidyl ethanolamine content and a concomitant decrease in phosphatidyl choline content in the cells supplemented with trans-unsaturated fatty acids . The affinity for uptake of L-leucine is higher in the cis-unsaturated fatty acid-supplemented cells compared with the trans-unsaturated fatty acid-supplemented cells . The temperature-dependence of L-leucine uptake bears a reasonably good correlation with the thermotropic behavior of the membrane lipids as studied by the steady-state fluorescence polarization technique . The present findings are discussed in light of the importance of the lipid environment in modulating membrane-associated functions.

Arch Biochem Biophys, 1986 Nov 1, 250(2), 373 - 81
Electron transfer between reduced methyl viologen and oxidized glutathione: a new assay of Saccharomyces cerevisiae glutathione reductase; Llobell A et al.; Pure glutathione reductase from Saccharomyces cerevisiae catalyzed under anaerobic conditions the enzymatic reduction of GSSG using electrochemically reduced methyl viologen as electron donor . The new assay was completely dependent on the amount of active enzyme present, and involved the formation of 1 mol GSH per mole of reduced methyl viologen consumed . The enzyme followed a standard Michaelis-Menten kinetics; a Km = 230 microM for reduced methyl viologen and a turnover number of 969 mumol GSSG reduced per minute per micromole enzyme were determined . The enzymatic activity seemed to depend on the redox potential, showing half-maximal activity at -0.407 V . The enzyme was quite specific: the activity using reduced benzyl viologen as electron donor was just 1.5% of that obtained with reduced methyl viologen at the same concentration and potential . Glutathione reductase was totally inactivated after a brief anaerobic exposure with reduced methyl viologen in the absence of GSSG; a partial reactivation was observed following addition of glutathione disulfide . No inhibition of the methyl viologen-dependent activity was observed in the presence of 2',5'-ADP or 2'-P-5'-ADP-ribose, two NADP(H) analogs, at concentrations which drastically inhibited the NADPH-dependent activity, thus suggesting that the reduced viologen does not interact with the pyridine nucleotide-binding site.

Proc Natl Acad Sci U S A, 1986 Nov, 83(21), 8097 - 101
Small nuclear RNAs from Saccharomyces cerevisiae: unexpected diversity in abundance, size, and molecular complexity; Riedel N et al.; Previous work showed that the simple eukaryote Saccharomyces cerevisiae contains a group of RNAs with the general structural properties predicted for small nuclear RNAs (snRNAs), including possession of the characteristic trimethylguanosine 5'-terminal cap . It was also demonstrated that, unlike their metazoan counterparts, the yeast snRNAs are present in low abundance (200-500 molecules per haploid cell) . We have now used antibody directed against the 5' cap to investigate the total set size of snRNAs in this organism . We present evidence that the number of distinct yeast snRNAs is on the order of several dozen, that the length of the capped RNAs can exceed 1000 nucleotides, and that the relative abundance of a subset of these RNAs is 1/5th to 1/20th that of the class of snRNAs described previously . These findings suggest that the six highly abundant species of snRNAs (U1-U6) typically reported in metazoans may represent a serious underestimation of the total diversity of snRNAs in eukaryotes.

Mol Gen Genet, 1986 Nov, 205(2), 276 - 84
Transcriptional regulation of DNA damage responsive (DDR) genes in different rad mutant strains of Saccharomyces cerevisiae; Maga JA et al.; The roles of the RAD genes of Saccharomyces cerevisiae in the regulation of transcription of two DNA damage responsive (DDR) genes were investigated by examining the levels of the DDRA2 and DDR48 transcripts in different rad mutants after exposure to two different DNA damaging agents . Strains carrying mutations in either the RAD3, RAD6 or RAD52 genes were treated with increasing concentrations of 4-nitroquinoline-1-oxide (NQO) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the DDR transcript levels were determined by Northern hybridization analysis . Our results indicate that the RAD3 gene is required for DDRA2 transcript production following NQO or MNNG treatments . Strains carrying mutations in either the RAD6 or RAD52 genes show an increased level of DDRA2 transcript in undamaged cells . However, the rad6 and rad52 mutants show a normal dose-dependent increase in DDRA2 transcript levels after NQO or MNNG exposure . The DDR48 gene appears to be regulated differently from DDRA2 in that this gene is induced in rad3 cells after damaging treatment but transcript induction is severely reduced in both rad6 and rad52 mutant strains . Although the rad mutations influence the kinetics of transcript accumulation, these effects do not account for the altered dose responses of the DDRA2 and DDR48 genes . Our results also demonstrate that the regulation of DDRA2 and DDR48 transcript levels by heat shock treatment is affected less severely in the different rad strains, a result which suggests that the RAD genes play an indirect role in DDR gene control.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1986 Nov, 6(11), 3694 - 703
Product of Saccharomyces cerevisiae nuclear gene PET494 activates translation of a specific mitochondrial mRNA; Costanzo MC et al.; The product of Saccharomyces cerevisiae nuclear gene PET494 is known to be required for a posttranscriptional step in the accumulation of one mitochondrial gene product, subunit III of cytochrome c oxidase (coxIII) . Here we show that the PET494 protein probably acts in mitochondria by demonstrating that both a PET494-beta-galactosidase fusion protein and unmodified PET494 are specifically associated with mitochondria . To define the PET494 site of action, we isolated mutations that suppress a pet494 deletion . These mutations were rearrangements of the mitochondrial gene oxi2 that encodes coxIII . The suppressor oxi2 genes had acquired the 5'-flanking sequences of other mitochondrial genes and gave rise to oxi2 transcripts carrying the 5'-untranslated leaders of their mRNAs . These results demonstrate that in wild-type cells PET494 specifically promotes coxIII translation, probably by interacting with the 5'-untranslated leader of the oxi2 mRNA.

Mol Gen Genet, 1986 Nov, 205(2), 305 - 11
Mutations in the right boundary of Saccharomyces cerevisiae centromere 6 lead to nonfunctional or partially functional centromeres; Hegemann JH et al.; Centromeres most likely consist of DNA (CEN DNA) interacting with specific proteins . In Saccharomyces cerevisiae a clear picture has emerged of a 120 bp sequence that is characteristic of CEN DNA . We have investigated the 25 bp centromere DNA element (CDEIII) that represents the right part of a CEN DNA . We showed using a series of mutants generated in vitro that the right most triple A of the consensus sequence TGT.T.TG. . TTCCGAA.....AAA participates in the assembly of a functional centromere and that no further sequences to the right are needed . Distance changes between the centre dyad TTCCGAA and the triple A have two effects: Addition of one base pair leads to a reduction, and addition of two or four base pairs to a loss of centromere function implying a participation of the centre dyad and the triple A region in protein binding . Indeed, a synthetic oligonucleotide of 39 bp containing CDEIII shows specific protein binding.

Mol Cell Biol, 1986 Nov, 6(11), 4053 - 9
A nuclear gene of Saccharomyces cerevisiae needed for stable maintenance of plasmids; Kikuchi Y et al.; We have isolated host mutants of Saccharomyces cerevisiae in which the 2 microns plasmid is poorly maintained . All the mutants tested constituted one complementation group, which was designated map1 (maintenance of plasmid) . Minichromosomes carrying a chromosomal replication origin and a centromere were affected in the mutants . Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2 microns inverted repeat sequences . These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication.

Mol Cell Biol, 1986 Nov, 6(11), 3954 - 64
Induction and repression of the urea amidolyase gene in Saccharomyces cerevisiae; Genbauffe FS et al.; The DUR1,2 gene from Saccharomyces cerevisiae has been isolated on recombinant plasmids along with all DNA between the DUR1,2 and MET8 loci . DUR1,2 was found to encode a 5.7-kilobase transcript, which is consistent with our earlier suggestion that the DUR1 and DUR2 loci are two domains of a single multifunctional gene . Steady-state levels of the DUR1,2 transcript responded to induction and nitrogen catabolite repression in the same way as urea amidolyase activity . dal81 mutants (grown with inducer) contained barely detectable amounts of DUR1,2 RNA, whereas dal80 mutants (grown without inducer) contained the same amount as a wild-type induced culture . These observations support our earlier hypothesis that DUR1,2 is transcriptionally regulated, with control being mediated by the DAL80 and DAL81 gene products . We cloned the DUR1,2-Oh mutation and found it to be a Ty insertion near sequences required for complementation of dur1,2 mutations . The ROAM phenotype of the DUR1,2-Oh mutation is sharply different from that of cis-dominant, DUR80 mutations, which enhance DUR1,2 expression but do not affect the normal control pattern of the gene . There is evidence that DUR80 mutations may also be Ty insertions, which generate phenotypes that are different from those in DUR1,2-Oh mutations.

Mol Cell Biol, 1986 Nov, 6(11), 3711 - 21
Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins; Schatz PJ et al.; Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology . Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species . Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences . Each gene had a single intervening sequence, located at an identical position in codon 9 . Cell fractionation studies showed that both gene products were present in yeast microtubules . These two genes, along with the TUB2 beta-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.

Mol Cell Biol, 1986 Nov, 6(11), 3575 - 81
Saccharomyces cerevisiae SPT3 gene is required for transposition and transpositional recombination of chromosomal Ty elements; Boeke JD et al.; Mutations in the Saccharomyces cerevisiae SPT3 gene have dramatic effects on the expression of Ty elements and genes adjacent to the element . The SPT3 gene is essential for Ty transposition, because transposition of chromosomal Ty elements ceased when the SPT3 gene was replaced with the frameshift mutation spt3-101 . Presumably, the elimination of transposition was due to the effect of the SPT3 gene product on Ty transcription; the transcripts of chromosomal Ty elements were largely abolished in the spt3-101 strain (F . Winston, K . J . Durbin, and G . R . Fink, Cell 39:675-682, 1984) . Ty transcription in an spt3-101 strain could be reestablished by introduction of the pGTyH3 plasmid, in which transcription of the Ty element TyH3 is under the control of the GAL1 promoter; these plasmid-derived Ty transcripts were SPT3-independent . Ty transposition resumed after galactose induction in spt3-101 strains containing the pGTyH3 plasmid . In spt3 mutants nearly all of the resulting transposition events derived from pGTyH3 plasmids and not from chromosomal elements.

Proc Natl Acad Sci U S A, 1986 Nov, 83(21), 8172 - 6
Possible involvement of RAS-encoded proteins in glucose-induced inositolphospholipid turnover in Saccharomyces cerevisiae; Kaibuchi K et al.; Incubation of yeast Saccharomyces cerevisiae at very low (0.02%) glucose levels led to arrest of the cell cycle at the G0/G1 phase . Readdition of glucose to these "starved" yeast resulted in cell proliferation . In glucose-starved yeast, glucose stimulated 32P incorporation into phosphatidic acid, phosphatidylinositol, phosphatidylinositol monophosphate, and phosphatidylinositol bisphosphate but not into phosphatidylethanolamine and phosphatidylcholine . Preincubation of yeast with {3H}inositol and subsequent exposure to glucose resulted in rapid formation of {3H}inositol monophosphate and {3H}inositol trisphosphate, presumably derived from phosphatidylinositol and phosphatidylinositol bisphosphate . Under similar conditions, glucose elicited both efflux and influx of Ca2+ in yeast . Glucose-induced 32P incorporation into inositolphospholipids and formation of {3H}inositol phosphates were more pronounced in RAS-related mutants such as ras1, ras1 ras2 bcy1, and RAS2Val19 than in the wild-type strain . These results strongly suggest that glucose stimulates inositolphospholipid turnover, Ca2+ mobilization, and subsequent cell proliferation in a manner similar to that of growth factors with mammalian cells, and that RAS-encoded proteins are involved in regulation of this glucose-induced inositolphospholipid turnover in yeast.

Proc Natl Acad Sci U S A, 1986 Nov, 83(21), 8117 - 21
Expression of the RNA genome of an animal virus in Saccharomyces cerevisiae; Makarow M et al.; The nucleocapsid of vesicular stomatitis virus (VSV) was introduced into the cytoplasm of Saccharomyces cerevisiae by low pH-dependent fusion of the viral envelope with the spheroplast plasma membrane . This led to de novo synthesis of the three major structural proteins of the virus--the G, N, and M proteins--as shown by immunoprecipitation of {35S}methionine-labeled spheroplast lysates . In NaDodSO4/polyacrylamide gel electrophoresis, M and N proteins comigrated with those of the virion, whereas the yeast-made G protein migrated as two bands with apparent molecular sizes of 60 and 70 kDa . Both polypeptides appeared to be N-glycosylated, since only one polypeptide with the apparent molecular mass of approximately equal to 55 kDa was produced in the presence of tunicamycin . Phase separation into Triton X-114 suggested that the unglycosylated G protein was membrane bound . According to immunofluorescent surface staining of live spheroplasts, at least part of the G protein was transported to the plasma membrane . Spheroplasts expressing the VSV genes could be fused together by low pH to form polykaryons, indicating that G protein synthetized by yeast was fusogenic--i.e., biologically active.

Mol Gen Genet, 1986 Nov, 205(2), 353 - 7
Cloning of the ARO3 gene of Saccharomyces cerevisiae and its regulation; Teshiba S et al.; Regulation of the two isozymes of 3-deoxy-D-arabino-heptulosonate-7phosphate synthase (DAHP synthase; EC 4.1.2.15) encoded by the genes ARO3 and ARO4 of Saccharomyces cerevisiae was studied . Both genes were shown to respond equally well to the general control of amino acid biosynthesis . Strains with mutations in these two genes were obtained by selecting first for a single aro3 mutation and afterwards for a double aro3 aro4 mutation . Gene ARO3, coding for the phenylalanine-dependent isozyme of DAHP synthase was cloned on the 2 micron multicopy vector pJDB207 by complementation of mutation aro3-1 in yeast . The ARO3 gene, carried originally on a 9.6 kb BamHI fragment (plasmid pME541A), was subcloned on a 1.9 kb HindIII-XbaI fragment (plasmid pME543) . A transcript of about 1.5 kb was shown to proceed from the HindIII towards the XbaI site . Expression from the 9.6 kb as well as from the 1.9 kb fragment was normal on a multicopy vector, since in both cases DAHP synthase levels of about 50-fold the wild-type level were observed.

FEBS Lett, 1986 Oct 27, 207(2), 217 - 21
Expression of cytochrome P-450d by Saccharomyces cerevisiae; Shimizu T et al.; Rat liver microsomal cytochrome P-450d was abundantly expressed in the yeast Saccharomyces cerevisiae by using a yeast-Escherichia coli shuttle vector consisting of rat liver P-450d cDNA and yeast acid phosphatase promoter . The expressed cytochrome P-450d was immunologically crossed with rat liver P-450d . The hydroxylase activity of estra-1,3,5(10)-triene-3, 17 beta-diol was 11 nmol/min per nmol P-450d, which is comparable to that reported previously for rat liver P-450d . The expressed P-450d content was nearlyt 1% of total yeast protein as estimated from immunoblotting, hydroxylase activity and optical absorpton of the reduced CO form.

J Biol Chem, 1986 Oct 25, 261(30), 14148 - 53
Protein topography of the 40 S ribosomal subunit from Saccharomyces cerevisiae as shown by chemical cross-linking; Yeh YC et al.; Protein-protein cross-linking was used to examine the spatial arrangement of proteins within the 40 S ribosomal subunits of Saccharomyces cerevisiae . Purified ribosomal subunits were treated with either 2-iminothiolane or dimethyl 3,3'-dithiobispropionimidate under conditions such that the ribosomal particle was intact and that formation of 40 S subunit dimers was minimized . Proteins were extracted from the treated subunits and fractionated on Sephadex G-150 or by acid-urea-polyacrylamide gel electrophoresis . Cross-linked proteins in these fractions were analyzed by two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Constituent members of cross-linked pairs were radiolabeled with 125I and identified by two-dimensional gel electrophoresis and comparison with nonradioactive ribosomal protein markers . Forty-two pairs involving 25 of the 32 40 S subunit proteins were identified . Many proteins were detected in several cross-linked dimers . These proteins with multiple cross-links form foci for the construction of a schematic model of the spatial arrangement of proteins within the 40 S subunit.

Nucleic Acids Res, 1986 Oct 24, 14(20), 7861 - 71
Nucleotide sequence of the Saccharomyces cerevisiae MET25 gene; Kerjan P et al.; To elucidate further the molecular basis of the specific regulatory mechanism modulating the expression of the genes implicated in methionine metabolism, we have cloned and characterized two genes, MET3 and MET25, and shown that the regulation of their expression is transcriptional . The sequence of the cloned yeast MET25 gene which encodes the O-acetyl homoserine - O-acetyl serine (OAH-OAS) sulfhydrylase is reported here along with its 5' and 3' flanking regions . The amino acid composition predicted from the DNA sequence is in good agreement with that determined by hydrolysis of the purified enzyme . In the 5' flanking region the signal for general amino acid control was not found, corroborating our previous finding that the synthesis of OAH-OAS sulfhydrylase is not submitted to general control . The transcription start points have been determined . The 5' and 3' flanking regions of the MET25 gene suggest initiation and termination signals similar to those associated with other yeast genes.

Biochim Biophys Acta, 1986 Oct 23, 861(3), 489 - 93
Effect of xylose incubation on the glucose transport system in Saccharomyces cerevisiae; Schuddemat J et al.; Incubation of Saccharomyces cerevisiae with xylose and ethanol for 16 hours leads to a decrease of hexokinase (and glucokinase) activity in the cells . It does not alter the levels of polyphosphate, orthophosphate and ATP . The transport of the glucose derivative 2-deoxy-D-glucose, a sugar that can be phosphorylated, is inhibited after this treatment, whereas transport of 6-deoxy-D-glucose, which has a blocked phosphorylation site, is not inhibited . Even though, both deoxyglucoses use the same transport system . The decrease in initial velocity of 2-deoxy-D-glucose transport is most pronounced under anaerobic conditions . Incubation of the cells with antimycin A, a treatment which has a similar effect as anaerobiosis, shows, that the inhibition of the transport of 2-deoxy-D-glucose is presumably the result of an increase in the Km of the carrier transport . Transport of glucose is probably regulated by kinase enzymes.

FEBS Lett, 1986 Oct 20, 207(1), 79 - 83
Amino acid substitutions in mitochondrial ATPase subunit 6 of Saccharomyces cerevisiae leading to oligomycin resistance; John UP et al.; The amino acid substitutions in subunit 6 of the mitochondrial ATPase complex have been determined for 4 oligomycin resistant mutants of Saccharomyces cerevisiae . The data were obtained for each mutant by nucleotide sequence analysis of the mitochondrial oli2 gene . Amino acid substitutions conferring oligomycin resistance in subunit 6 are located in two conserved regions that are thought to form domains which span the inner mitochondrial membrane . The disposition of these amino acid substitutions is consistent with the view that these two membrane-spanning domains interact structurally and functionally with the DCCD-binding proteolipid subunit 9 in the Fo-sector.

J Biol Chem, 1986 Oct 15, 261(29), 13401 - 3
A "CAT" family of repetitive DNA sequences in Saccharomyces cerevisiae; Wildeman AG et al.; An oligonucleotide probe was used to isolate yeast genomic clones containing DNA sequences with repetitive elements consisting primarily of a tandemly arranged trinucleotide, CAT . Hybridization analyses estimate that the yeast genome contains 40-50 CAT clusters, representing the first repetitive DNA sequence family found in yeast . Sequence analyses show short spacers between the CAT repeats consisting of closely related trinucleotides, primarily CGT . Some of the CAT clusters are located in longer repeating elements with lengths of 7 nucleotides or more . In one case a three-times-repeated 27-nucleotide sequence bears striking homology to the 21-base pair repeat region of the mammalian simian virus 40 promoter element . Hybridization studies further suggest that the "CAT" sequences may be widely dispersed in many diverse organisms including Escherichia coli, Drosophila, and man.

Mol Cell Biol, 1986 Oct, 6(10), 3490 - 7
Saccharomyces cerevisiae nuclear fusion requires prior activation by alpha factor; Rose MD et al.; We have developed a protocol for efficient fusion of spheroplasts of the same mating type . Nuclear fusion in this whole-cell system is also efficient and closely parallels nuclear fusion in heterosexual mating of intact cells . In the spheroplast fusion system, nuclear fusion is dependent on both the KAR1 gene and prior exposure to alpha factor . The major products of nuclear fusion in the spheroplast fusion assay were true diploids that were homozygous at the mating-type locus . An additional 10% of the products were cells of ploidy greater than diploid . The dependence of nuclear fusion on alpha factor treatment could not be replaced by synchronization in G1 by mutations in CDC28 and CDC35 or by prior arrest in stationary phase . These data suggest that nuclear fusion is not a constitutive function of the nucleus, but rather is specifically induced by mating hormone.

Mol Cell Biol, 1986 Oct, 6(10), 3357 - 67
Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle; McLeod M et al.; The FLP protein of the Saccharomyces cerevisiae plasmid 2 microns circle catalyzes site-specific recombination between two repeated segments present on the plasmid . In this paper we present results of experiments we performed to define more precisely the features of the FLP recognition target site, which we propose to designate FRT, and to determine the actual recombination crossover point in vivo . We found that essential sequences for the recombination event are limited to an 8-base-pair core sequence and two 13-base-pair repeated units immediately flanking it . This is the region identified as the FLP binding site in vitro and at which FLP protein promotes specific single-strand cleavages (B . J . Andrews, G . A . Proteau, L . G . Beatty, and P . D . Sadowski, Cell 40:795-803, 1985; J . F . Senecoff, R . C . Bruckner, and M . M . Cox, Proc . Natl . Acad . Sci . USA 82:7270-7274, 1985) . Mutations within the core domain can be suppressed by the presence of the identical mutation in the chromatid with which it recombines . However, mutations outside the core are not similarly suppressed . We found that strand exchange during FLP recombination occurs most of the time within the core region, proceeding through a heteroduplex intermediate . Finally, we found that most FLP-mediated events are reciprocal exchanges and that FLP-catalyzed gene conversions occur at low frequency . The low level of gene conversion associated with FLP recombination suggests that it proceeds by a breakage-joining reaction and that the two events are concerted.

Genetika, 1986 Oct, 22(10), 2416 - 22
{Genetic activity of carbamate herbicides in Escherichia coli and Saccharomyces cerevisiae}; Emnova EE et al.; DNA-damaging activity and herbicide-induction of gene point mutations, and intragenic mitotic recombination were studied in bacteria and the yeast tester strains . Herbicide (eptam, triallate, tillam, surpass) were not effective in DNA-damaging and mitotic recombination tests . Of the 4 chemicals, only eptam was strongly mutagenic . Dose response curves for eptam differed in bacteria and yeast; maximum mutagenic activity was registered in bacteria at 5 mg/l . Maximum yield of prototrophs was observed after 2 h incubation time . Triallate was moderate, tillam and surpass being weak mutagens for the strains used.

Genetics, 1986 Oct, 114(2), 363 - 74
Recessive nonsense suppressors in Saccharomyces cerevisiae: action spectra, complementation groups and map positions; Ono B et al.; Three genes SUP111, SUP112 and SUP113 of Saccharomyces cerevisiae have been identified that can mutate to give recessive omnipotent nonsense suppressors . Alleles of these loci can also act as allosuppressors; that is, different phenotypes, due apparently to different efficiencies of suppression, can result from different alleles at a given locus . The SUP111, SUP112 and SUP113 loci map to the right arms of chromosomes VIII, VII and XIII, respectively.

Proc Natl Acad Sci U S A, 1986 Oct, 83(20), 7618 - 22
Mismatch correction catalyzed by cell-free extracts of Saccharomyces cerevisiae; Muster-Nassal C et al.; Heteroduplex DNA substrates containing a 4- or 7-base-pair insertion/deletion mismatch or each of the eight possible single-base-pair mismatches were constructed . Extracts of mitotic Saccharomyces cerevisiae cells catalyzed the correction of mismatched nucleotides in a reaction that required Mg2+ and had a partial requirement for ATP and the four dNTPs . The insertion/deletion mismatches and the A X C and G X T mismatches were repaired efficiently, while the six other single-base-pair mismatches were repaired poorly or at undetectable rates . Mismatch correction was accompanied by the specific incorporation of less than 20 nucleotides at or near the site of the repaired mismatch.

Biochimie, 1986 Oct-Nov, 68(10-11), 1231 - 6
Accumulation of dinucleoside polyphosphates in Saccharomyces cerevisiae under stress conditions . High levels are associated with cell death; Baltzinger M et al.; Adenosine tetraphosphonucleosides (Ap4X) were measured in Saccharomyces cerevisiae by a coupled phosphodiesterase-luciferase assay . After exposure of the cells to cadmium or to hyperthermic treatment (46 degrees C) a marked increase of the cellular pool from 0.08 microM (base level) to 4 microM or higher was observed . The accumulation of Ap4X to high levels is associated with irreversible processes leading to cell death.

Mol Cell Biol, 1986 Oct, 6(10), 3555 - 8
Excision repair functions in Saccharomyces cerevisiae recognize and repair methylation of adenine by the Escherichia coli dam gene; Hoekstra MF et al.; Unlike the DNA of higher eucaryotes, the DNA of Saccharomyces cerevisiae (bakers' yeast) is not methylated . Introduction of the Escherichia coli dam gene into yeast cells results in methylation of the N-6 position of adenine . The UV excision repair system of yeast cells specifically responds to the methylation, suggesting that it is capable of recognizing modifications which do not lead to major helix distortion . The UV repair functions examined in this report are involved in the incision step of pyrimidine dimer repair . These observations may have relevance to the rearrangements and recombination events observed when yeast or higher eucaryotic cells are transformed or transfected with DNA grown in E . coli.

Mol Cell Biol, 1986 Oct, 6(10), 3320 - 8
Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis; Hirsch JP et al.; The INO1 gene of Saccharomyces cerevisiae encodes the regulated enzyme inositol-1-phosphate synthase, which catalyzes the first committed step in the synthesis of inositol-containing phospholipids . The expression of this gene was analyzed under conditions known to regulate phospholipid synthesis . RNA blot hybridization with a genomic clone for INO1 detected two RNA species of 1.8 and 0.6 kb . The abundance of the 1.8-kb RNA was greatly decreased when the cells were grown in the presence of the phospholipid precursor inositol, as was the enzyme activity of the synthase . Complementation analysis showed that this transcript encoded the INO1 gene product . The level of INO1 RNA was repressed 12-fold when the cells were grown in medium containing inositol, and it was repressed 33-fold when the cells were grown in the presence of inositol and choline together . The INO1 transcript was present at a very low level in cells containing mutations (ino2 and ino4) in regulatory genes unlinked to INO1 that result in inositol auxotrophy . The transcript was constitutively overproduced in cells containing a mutation (opi1) that causes constitutive expression of inositol-1-phosphate synthase and results in excretion of inositol . The expression of INO1 RNA was also examined in cells containing a mutation (cho2) affecting the synthesis of phosphatidylcholine . In contrast to what was observed in wild-type cells, growth of cho2 cells in medium containing inositol did not result in a significant decrease in INO1 RNA abundance . Inositol and choline together were required for repression of the INO1 transcript in these cells, providing evidence for a regulatory link between the synthesis of inositol- and choline-containing lipids . The level of the 0.6-kb RNA was affected, although to a lesser degree, by many of the same factors that influence INO1 expression.

Mol Gen Genet, 1986 Oct, 205(1), 74 - 81
Genetic characterization and isolation of the Saccharomyces cerevisiae gene coding for uridine monophosphokinase; Liljelund P et al.; We selected a 5-fluorouracil-resistant, thermosensitive mutant of the uridine monophosphokinase step in Saccharomyces cerevisiae . The mutant displays very weak thermolabile uridine monophosphokinase activity and wild-type uridine diphosphokinase activity . Growth of the mutant at the non-permissive temperature causes immediate reduction of pyrimidine triphosphate pools to 10% of the wild-type level as well as significantly lowering total RNA and protein synthesis . These conditions also provoke derepression of the first gene of the pathway, URA2, at both the levels of enzymatic activity and transcription . The mutation segregates independently of all known genes of the pyrimidine biosynthetic pathway . The corresponding gene has been isolated on a 4.8 kb fragment by complementation of the mutant phenotype . The new gene, named URA6, codes for a 2.2 kb polyadenylated messenger RNA, exists in a single copy per haploid genome, and was mapped to the centromere of chromosome XI.

Genetics, 1986 Oct, 114(2), 347 - 61
Frequency and directionality of gene conversion events involving the CYC7-H3 mutation in Saccharomyces cerevisiae; Pukkila PJ et al.; The CYC7-H3 mutation is a 5-kb deletion that causes overproduction of iso-2 cytochrome c . Unlike most mutations in yeast, the CYC7-H3 mutation is preferentially lost when it is involved in a gene conversion event . We have shown that cloned copies of CYC7-H3 DNA that are inserted into the yeast genome are associated with a high frequency of recombination and aberrant segregation events . Since parity in conversion frequency was observed when the extensive insertion/deletion heterozygosity at this locus was eliminated, we conclude that the CYC7-H3 sequences are inherently capable of acting as donors or recipients in gene conversion events, although they are unlikely to act as donors when they are located opposite a large heterology . DNA sequence comparisons revealed similarities between the CYC7-H3 junction region and the 2-micron circle DNA region that is involved in site-specific recombination.

FEBS Lett, 1986 Sep 29, 206(1), 147 - 50
Absence of structural homology between sup1 and sup2 genes of yeast Saccharomyces cerevisiae and identification of their transcripts; Surguchov AP et al.; The results of Southern blotting demonstrate that sup2 is a unique gene in Saccharomyces cerevisiae that does not possess homologous sequences in the yeast genome . The direct hybridization of DNA fragments, containing cloned sup1 and sup2 genes, did not reveal any structural homology between these two genes . By Northern blotting analysis the sizes of the transcripts were determined to be 1.6 kb for sup1 gene and 2.5 and 1.4kb for sup2 gene . Experiments with RNA isolated from yeast mutant with impaired splicing demonstrated that sup1 and sup2 genes do not contain introns.

J Biol Chem, 1986 Sep 25, 261(27), 12814 - 9
Regulation of arginine metabolism in Saccharomyces cerevisiae . Association of arginase and ornithine transcarbamoylase; Eisenstein E et al.; Association of arginase and ornithine transcarbamoylase (OTCase) has been proposed to play an essential role in the regulation of arginine metabolism in Saccharomyces cerevisiae (Wiame, J.-M . (1971) Curr . Top . Cell . Reg . 4, 1-39) . In this report multienzyme complex formation is directly demonstrated in the presence of the active-site ligands for OTCase and arginase . Using equilibrium sedimentation, a dissociation constant for complex formation was determined to be 2.3 X 10(-8) M in the presence of ornithine and agmatine, active-site ligands for OTCase and arginase, respectively . A molecular stoichiometry in the complex of one molecule of OTCase to one molecule of arginase was verified using transmission electron microscopy . The dimensions of the complex were determined by negative staining and rotary and unidirectional shadowing techniques to be 102 A wide by 81 A high . These dimensions are quantitively consistent with dimensions of the individual enzymes (Duong, L . T., Eisenstein, E., Green, S . M., Ornberg, R . L., and Hensley, P . (1986) J . Biol . Chem . 261, 12807-12813) . The enzymatic activity of OTCase is virtually completely inhibited when associated with arginase, reflecting the dramatic modulation of enzyme activity as a consequence of the acquisition of quaternary structure in this multienzyme complex.

J Biol Chem, 1986 Sep 25, 261(27), 12807 - 13
The quaternary structure of ornithine transcarbamoylase and arginase from Saccharomyces cerevisiae; Duong LT et al.; Electron microscopic studies employing negative staining, rotary shadowing, and unidirectional shadowing have revealed the subunit architecture of ornithine transcarbamoylase and arginase from Saccharomyces cerevisiae . These techniques have confirmed the quaternary structure of these enzymes, and have permitted an estimate of the shape and dimensions of each of the individual enzymes as well as those of the corresponding subunits to be determined . Both enzymes are trimers exhibiting 3-fold rotational symmetry with subunits which are oblate ellipsoids of revolution . The overall dimensions determined for ornithine transcarbamoylase are a height of 39 A and a width of 102 A . The consequent subunit dimensions are 59 X 59 X 39 A, yielding a subunit axial ratio of 1.51 . Arginase has a height of 42 A and a width of 97 A . The subunit dimensions are 56 X 56 X 42 A, yielding a subunit axial ratio of 1.33 . The hydrodynamic behavior of the two enzymes is fully consistent with this molecular architecture, suggesting that the structures proposed here are similar to those of the proteins in aqueous solution.

J Biol Chem, 1986 Sep 25, 261(27), 12599 - 603
Protein synthesis in yeast . Isolation of variant forms of elongation factor 1 from the yeast Saccharomyces cerevisiae; Saha SK et al.; Two species of the elongation factor 1 (EF-1) differing in molecular weight, subunit composition, and isoelectric point have been isolated from cell-free extracts of the yeast Saccharomyces cerevisiae . The ratio of these two forms of EF-1 activity (EF-1 alpha and EF-1H) seem to vary in different strains and upon the growth phase from which the cells have been isolated . The log phase cells of a protease negative yeast strain EJ101 show a distribution of EF-1 alpha and EF-1H in the ratio of 3:1 . Another laboratory yeast strain, D-587-4B, shows a distribution pattern of 4:1 . The two forms of EF-1 are completely separable by ion exchange, gel permeation, and hydrophobic and affinity chromatography . Yeast EF-1 alpha is a single polypeptide of molecular weight 50,000 and has an isoelectric point of 8.9 . The newly identified form of the yeast EF-1 (EF-1H) has a molecular weight of 200,000 . The isoelectric point of this protein is around 5.5 . Electrophoresis of the partially purified EF-1H in polyacrylamide gel containing sodium dodecyl sulfate indicates the presence of three nonidentical polypeptides having molecular weights of 50,000, 47,000, and 33,000 . The three polypeptides are present in the ratio of 2:1:1 . EF-1H is readily converted to EF-1 alpha and EF-1 beta gamma on anion exchange columns . The 50,000 dalton component of EF-1H immunologically cross-reacts with the antibody to EF-1 alpha . The other two polypeptides do not . On the basis of molecular weight, EF-1H is 2-3-fold more active than EF-1 alpha in poly(U)-dependent polyphenylalanine synthesis . EF-1H exchanges nucleotide (GDP----GTP) at a faster rate than EF-1 alpha . Both EF-1 alpha and EF-1H exhibit similar binding constants for GDP and GTP although the affinity of EF-1 alpha for guanine nucleotides is several-fold higher than that of EF-1H . The 33,000-dalton component of EF-1H appears to be functionally analogous to EF-1 beta (Ts) isolated from other eukaryotic sources . The function of EF-1 gamma is unknown.

J Biol Chem, 1986 Sep 25, 261(27), 12593 - 5
Protein synthesis in yeast . Identification of an altered elongation factor in thermolabile mutants of the yeast Saccharomyces cerevisiae; Kamath A et al.; Cell-free extracts from the wild type yeast strain (A364A) and from a group of noncomplementing mutants that are conditionally defective in translation were preincubated at a restrictive temperature prior to incubation at a permissive temperature for protein synthesis . Results of these experiments showed that upon exposure to the restrictive temperature (39 degrees C), all five of the noncomplementing mutants lost ability to incorporate amino acid into protein . The wild type parent strain retained better than 80% of the activity under identical conditions of heat treatment . Mutant extracts could be revived to incorporate amino acid by the addition of the purified yeast elongation factor 3 . Factors 1 and 2 had no effect . The heat-treated extract from one mutant did not supplement the activity of the other mutant . Although all five of the mutants were inactivated by preincubation at 39 degrees C, each showed a variable rate and extent of thermolability . Heat-treated mutant extracts were fully active in polyphenylalanine synthesis with liver ribosomes but not with the yeast ribosomes . Since liver ribosomes do not require factor 3, this assay then confirms that factor 3 is the thermolabile component in this group of noncomplementing mutants.

Nucleic Acids Res, 1986 Sep 25, 14(18), 7437 - 51
Biogenesis of mitochondria: DNA sequence analysis of mit- mutations in the mitochondrial oli2 gene coding for mitochondrial ATPase subunit 6 in Saccharomyces cerevisiae; John UP et al.; A series of yeast mitochondrial mit- mutants with defects in the oli2 gene, coding for subunit 6 of the mitochondrial ATPase complex, has been analyzed at the DNA sequence level . Fifteen of sixteen primary mit- mutants were shown to contain frameshift or nonsense mutations predicting truncated subunit 6 polypeptides, in various strains ranging from about 20% to 95% of the wild-type length of 259 amino acids . In only one strain could the defect in subunit 6 function be assigned to amino acid substitution in an otherwise full-length subunit 6 . Many mutants carried multiple base substitutions or insertions/deletions, presumably arising from the manganese chloride mutagenesis treatment . Revertants from three of the mit- mutants were analyzed: all contained full-length subunit 6 proteins with one or more amino acid substitutions . The preponderance of truncated proteins as opposed to substituted full-length proteins in oli2 mit- mutants is suggested to reflect the ability of subunit 6 to accommodate amino acid substitutions at many locations, with little or no change in its functional properties in the membrane FO-sector of the ATPase complex.

J Biol Chem, 1986 Sep 25, 261(27), 12596 - 8
Protein synthesis in yeast . Purification of elongation factor 3 from temperature-sensitive mutant 13-06 of the yeast Saccharomyces cerevisiae; Kamath A et al.; An altered form of the elongation factor 3 (EF-3) has been purified to near homogeneity from a thermolabile yeast mutant ts 13-06 . The isolation procedure involved chromatography on DEAE-Sephadex, CM-Sepharose, and hydroxylapatite columns . The final purification of this protein was obtained by affinity chromatography on an ATP-Sepharose column . Because of the extreme lability of the mutant protein, the yield was very poor . Silver stain analysis of the sodium dodecyl sulfate electrophoretograms indicated that the affinity-purified protein was better than 90% pure . From the studies of the physical and biochemical properties, the following characteristics of the purified wild type and the mutant protein have been established . The two proteins were indistinguishable by their molecular weight, amino acid composition, and isoelectric point . Purified mutant EF-3 was rapidly inactivated between 37 and 39 degrees C . Under this condition, wild type EF-3 was completely stable . Ribosome-dependent GTPase and ATPase activities of the mutant EF-3 were heat sensitive; GTPase activity was more labile than the ATPase activity . Mutant EF-3, after exposure to a nonpermissive temperature, failed to stimulate binding of the ternary complex of EF-1 X GTP X aminoacyl-tRNA to ribosome . The wild type protein was fully active under this condition . Other biochemical and physical properties of these two proteins are under current investigation.

J Biol Chem, 1986 Sep 15, 261(26), 12266 - 71
Purification and characterization of a mitochondrial isozyme of C1-tetrahydrofolate synthase from Saccharomyces cerevisiae; Shannon KW et al.; C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities . In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is encoded by the ADE3 locus, yet ade3 mutants have low but detectable levels of these enzyme activities . Synthetase, cyclohydrolase, and dehydrogenase activities in an ade3 deletion strain co-purify 4,000-fold to yield a single protein species as seen on sodium dodecyl sulfate-polyacrylamide gels . The native molecular weight of the isozyme (Mr = 200,000 by gel exclusion chromatography) and the size of its subunits (Mr = 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are similar to those of C1-tetrahydrofolate synthase . Cell fractionation experiments show that the isozyme, but not C1-tetrahydrofolate synthase, is localized in the mitochondria . Genetic studies indicate that the isozyme is encoded in the nuclear genome . Peptide mapping experiments show that C1-tetrahydrofolate synthase and the isozyme are not structurally identical . However, immunotitration experiments and amino acid sequence analysis suggest that C1-tetrahydrofolate synthase and the isozyme are structurally related . We propose to call the isozyme "mitochondrial C1-tetrahydrofolate synthase."

J Biol Chem, 1986 Sep 15, 261(26), 12066 - 73
The rate of import and assembly of F1-ATPase in Saccharomyces cerevisiae; Burns DJ et al.; Subunit specific antiserum can be employed to study the course of ATPase assembly in mitochondria isolated from bakers' yeast . Comparing rates of subunit import with rates of enzyme assembly indicated that no substantial pool of unassembled subunits exists for the three largest ATPase peptides (alpha, beta, and gamma) . Blocking import of specific ATPase subunits, however, did reveal a possible accumulation of unassembled alpha and gamma subunits in isolated mitochondria . The kinetic experiments also revealed a lag in the import of beta subunit relative to the uptake of alpha and gamma precursors . Experiments conducted in yeast cells confirmed that beta subunit is assembled soon after it is imported, but did not indicate a delay in import relative to the other subunits of F1.

J Biol Chem, 1986 Sep 5, 261(25), 11756 - 64
Transcriptional regulation of the mitochondrial genome of yeast Saccharomyces cerevisiae; Mueller DM et al.; The relative rates of transcription of several classes of the mitochondrial genes of the yeast Saccharomyces cerevisiae have been determined . The rates were measured by pulse labeling whole yeast with {32P}O4, isolating the total RNA, hybridization to single-stranded M13 DNA probes containing segments of the gene of interest, digestion with RNase A or T1, and separation of the protected fragment by gel electrophoresis . This analysis indicated that, among the genes analyzed, transcriptional promoters varied in strength by 20-fold while the rates of transcription varied by more than 50-fold . The strengths of the promoters of the genes were ordered: tRNAMetf greater than tRNAPhe greater than 14 S rRNA greater than 21 S rRNA greater than tRNAGlu greater than Oli-1 much greater than tRNACys . In addition, transcription rates were measured within polygenic transcription units . This analysis indicated that there was transcriptional attenuation within all the polygenic transcription units with the greatest attenuation factor being as much as 17-fold, occurring after the tRNAGlu and tRNAMetf genes . This analysis indicated that regulation of the rates of transcription in the yeast mitochondrial genome occurs by two distinct mechanisms, modulation of the rate of transcriptional initiation and attenuation of transcriptional elongation.

J Biol Chem, 1986 Sep 5, 261(25), 11872 - 9
Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae . Characterization of mutants in 34 complementation groups; McEwen JE et al.; To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized . In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups . Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes . One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes . These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively . Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways . A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b . These mutants fall into 17 complementation groups . The third class is represented by mutants in 14 complementation groups . These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly . The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes.

J Biol Chem, 1986 Sep 5, 261(25), 11816 - 22
Steady state analysis of mitochondrial RNA after growth of yeast Saccharomyces cerevisiae under catabolite repression and derepression; Mueller DM et al.; The steady state levels of mitochondrial rRNAs, 5 tRNAs, the 9 S RNA, and the RNA products from the genes coding for subunits 6 and 9 of the ATP synthase, cytochrome b, and subunit 1 of cytochrome oxidase have been determined after growth of yeast under conditions of respiratory repression or derepression . The analysis indicates that the mitochondrial rRNAs are present in 2000 or 9000 copies/cell in repressed or derepressed yeast, respectively . The levels of the other RNAs also differed to a similar extent, with the exception of the level of the tRNAfMet which differs by only 1.7-fold . The levels of the individual protein coding RNAs varied from 480 copies/cell for the Oli-1 RNA to 100 copies/cell for the Oli-2 RNA under derepressive conditions and from 130 copies/cell to 33 copies/cell for the same RNAs in glucose repressive conditions . The levels of the tRNAs varied even more markedly, ranging from 4200 copies/cell for the tRNAPhe to 240 copies/cell for the tRNACys after growth in derepressive conditions and from 800 copies/cell for the tRNAfMet to 30 copies/cell for the tRNACys of glucose repressed yeast . These results indicate that glucose repression uniformly decreases the levels of the individual mitochondrial RNAs studied . This decrease is related to a lower synthesis of mitochondrial RNA in the glucose repressed cells as compared to derepressed cells.

J Gen Microbiol, 1986 Sep, 132 ( Pt 9), 2605 - 10
A genetic and biochemical analysis of the role of gluconeogenesis in sporulation of Saccharomyces cerevisiae; Dickinson JR et al.; The requirement for gluconeogenesis and the pentose phosphate pathway in sporulation of Saccharomyces cerevisiae was investigated using homozygous diploids with mutations in selected portions of the respective metabolic pathways . Mutations affecting the genes FBA1 (fructose-1,6-bisphosphate aldolase), GPM1 (phosphoglycerate mutase) and ZWF1 (glucose-6-phosphate dehydrogenase) were used . Homozygous diploids bearing either fba1-11 or gpm1 mutations were asporogenous, indicating an absolute requirement for gluconeogenesis in sporulation . A strain homozygous for the zwf1 mutation sporulated, but at a reduced level compared to the wild-type . Homozygous spd1-1 mutations restored the ability to sporulate in fba1-11 homozygous diploids; this is believed to occur as a consequence of reduced NH+4 levels in spd1-1-bearing strains, the reduced intracellular NH+4 content serving to promote gluconeogenesis via the residual low levels of enzyme activity present in such mutants.

Mol Cell Biol, 1986 Sep, 6(9), 3166 - 72
Construction and behavior of circularly permuted and telocentric chromosomes in Saccharomyces cerevisiae; Murray AW et al.; We developed techniques that allow us to construct novel variants of Saccharomyces cerevisiae chromosomes . These modified chromosomes have precisely determined structures . A metacentric derivative of chromosome III which lacks the telomere-associated X and Y' elements, which are found at the telomeres of most yeast chromosomes, behaves normally in both mitosis and meiosis . We made a circularly permuted telocentric version of yeast chromosome III whose closest telomere was 33 kilobases from the centromere . This telocentric chromosome was lost at a frequency of 1.6 X 10(-5) per cell compared with a frequency of 4.0 X 10(-6) for the natural metacentric version of chromosome III . An extremely telocentric chromosome whose closet telomere was only 3.5 kilobases from the centromere was lost at a frequency of 6.0 X 10(-5) . The mitotic stability of telocentric chromosomes shows that the very high frequency of nondisjunction observed for short linear artificial chromosomes is not due to inadequate centromere-telomere separation.

Mol Cell Biol, 1986 Sep, 6(9), 3150 - 5
Negative regulatory gene for general control of amino acid biosynthesis in Saccharomyces cerevisiae; Myers PL et al.; In Saccharomyces cerevisiae, many amino acid biosynthetic pathways are coregulated by a complex general control system: starvation for a single amino acid results in the derepression of amino acid biosynthetic genes in multiple pathways . Derepression of these genes is mediated by positive (GCN) and negative (GCD) regulatory genes . In this paper we describe the isolation and characterization of a previously unreported negative regulatory gene, GCD3 . A gcd3 mutation is recessive to wild type, confers resistance to multiple amino acid analogs, and results in overproduction and partially constitutive elevation of mRNA levels for amino acid biosynthetic genes . Furthermore, a gcd3 mutation can overcome the derepression-deficient phenotype of mutations in the positive regulatory GCN1, GCN2, and GCN3 genes . However, the gcd3 mutation cannot overcome the derepression-deficient phenotype of a gcn4 mutation, suggesting that GCD3 acts as a negative regulator of the important GCN4 gene . Northern blot analysis confirmed this conclusion, in that the steady-state levels of GCN4 mRNA are greatly increased in a gcd3 mutant . Thus, the negative regulatory gene GCD3 plays a central role in derepression of amino acid biosynthetic genes.

Genetika, 1986 Sep, 22(9), 2235 - 43
{Mutants of Saccharomyces cerevisiae supersensitive to the mutagenic effect of 6-N-hydroxylaminopurine}; Pavlov IuI; Yeast mutants hypersensitive to the mutagenic action of 6-N-hydroxylaminopurine (HAP) were obtained by EMS mutagenesis . One of the mutants segregated monogenically and possessed reduced capacity to utilize HAP as a purine source . A set of diploids suitable for parallel study of mutagenesis and induction of recombination, and differing in the trait of mutability after exposure to HAP ("hm" trait or HAP mutability), were constructed . It was shown that a weak recombinogenic effect of HAP is not enhanced in "hm" mutants when HAP mutability increases.

Yeast, 1986 Sep, 2(3), 169 - 78
Mutations in ARS1 increase the rate of simple loss of plasmids in Saccharomyces cerevisiae; Strich R et al.; Autonomously replicating sequence (ARS) elements are DNA sequences that promote extrachromosomal maintenance of plasmids in yeast . Mutations generated in vitro in the ARS1 region were examined for their effect on plasmid maintenance in a yeast centromeric plasmid . Our data show that mutations in the regions surrounding the ARS1 consensus sequence cause increases in the frequency of simple loss (1:0) events without affecting the rate of nondisjunction (2:0) . Removal of the consensus sequence itself causes a drastic increase in the rate of simple loss . Sequences sensitive to mutagenesis were identified in each flanking region and differ with respect to their location and importance to ARS function . These results suggest that the role ARS1 plays in plasmid maintenance deals with the replication and/or localization of the plasmid in yeast.

Mol Cell Biol, 1986 Sep, 6(9), 3295 - 7
High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion; Gyuris J et al.; After a new transformation procedure, 10% of Saccharomyces cerevisiae cells were found to contain transforming DNA sequences . We used direct transfer of plasmid molecules by fusing bacterial minicell protoplasts to yeast protoplasts . Since the procedure significantly reduces the toxic effect of procaryotic protoplasm on the eucaryotic organism, it might be generally applicable in other systems in which transformation is inefficient or impossible.

Mol Gen Genet, 1986 Sep, 204(3), 363 - 6
Transcription of genes encoding enzymes involved in DNA synthesis during the cell cycle of Saccharomyces cerevisiae; McIntosh EM et al.; We have examined the pattern of transcription exhibited by four genes in the dTTP biosynthetic pathway of Saccharomyces cerevisiae . Consistent with the results reported previously by Storms et al . (1984), the TMP1 (or CDC21) gene encoding thymidylate synthase was found to be transcribed in a periodic manner during the cell cycle with maximal mRNA levels occurring just prior to the onset of DNA replication . Three other genes in this pathway DCD1, DUT1 and DFR1 encoding dCMP deaminase, dUTP pyrophosphatase and dihydrofolate reductase, respectively, exhibited relatively constant levels of transcription throughout the cell cycle . These results, particularly for DFR1, are in marked contrast with those obtained in other eukaryotic systems which have suggested that, in general, genes encoding enzymes involved in DNA precursor synthesis are subject to cell cycle regulation . Thus, periodic transcription is not a property common to all genes involved in DNA replication in this eukaryote.

Proc Natl Acad Sci U S A, 1986 Sep, 83(17), 6352 - 6
Functional expression of rat cytochrome c in Saccharomyces cerevisiae; Scarpulla RC et al.; To determine whether a mammalian cytochrome c could efficiently replace iso-1-cytochrome c, which is encoded by the yeast CYC1 gene, the coding sequence of RC9 (a nondefective processed gene from rat) was cloned in both single- and multiple-copy expression vectors under the direction of the yeast alcohol dehydrogenase 1 (ADC1) promoter . Upon transformation of a CYC1 deletion strain, the multiple-copy construct restored a wild-type growth rate on lactate medium; such growth normally requires derepressed amounts of iso-1-cytochrome c . These transformants expressed a level of hybrid ADC1/RC9 mRNA approximately 5- to 10-fold greater than the amount of message from the endogenous ADC1 gene and produced a steady-state level of rat cytochrome c equivalent to that of the wild-type yeast protein . A requirement for the vector was evidenced by its absence in all transformants that lost the lactate growth phenotype after propagation in nonselective medium . In contrast, the level of vector-specific message in single copy was equivalent to that of the endogenous ADC1 mRNA, but transformants exhibited no significant growth on lactate . Constructions having a small deletion or a mammalian intron within the rat cytochrome c coding region failed to support lactate-dependent growth, indicating that complementation depends upon proper translation of the correct rat coding sequence . Therefore, the rat polypeptide, when expressed at normal physiological levels, is recognized by the yeast machinery involved in the multiple steps required for the processing and transport of an active cytochrome c as well as its functional interaction with the respiratory apparatus.

Eur J Biochem, 1986 Aug 15, 159(1), 31 - 8
Structure and properties of casein kinase-2 from Saccharomyces cerevisiae . A comparison with the liver enzyme; Meggio F et al.; A type-2 casein kinase (YCK-2), lacking the 25-kDa autophosphorylatable beta subunit characteristic of animal casein kinases-2, has been obtained in a nearly pure form from Saccharomyces cerevisiae and was compared with liver casein kinase-2 (LCK-2) . A 22-kDa phosphorylatable protein, copurifying with YCK-2, can be removed by ultracentrifugation at low ionic strength and is shown by several criteria to be unrelated to the beta subunit of LCK-2 . The native Mr of YCK-2, deprived of the 22-kDa phosphoprotein, is about 150 000 . Limited proteolysis experiments show that YCK-2 included 37-kDa catalytic subunits, which can be converted into still active 35-kDa proteolytic derivatives . These data are consistent with a homotetrameric quaternary structure as opposed to the heterotetrameric subunit composition alpha 2 beta 2 of LCK-2 and other animal casein kinases-2 . Although many properties of YCK-2 and LCK-2, including substrate specificity, inhibition by heparin, polyglutamic acid and quercetin and stimulation by polyamines, are similar; their stability under denaturing and dissociating conditions and their response to polybasic peptides are quite different . In particular YCK-2 is more readily denatured than LCK-2 by heating and exposure to urea, sodium dodecylsulphate and deoxycholate while its activity is inhibited by 100-150 mM NaCl, which conversely stimulates LCK-2 activity 2-3-fold . The Km value of the synthetic peptide substrate Ser-(Glu)5 for YCK-2 is not significantly changed by the addition of polylysine . On the contrary the Km value of the same peptide substrate for LCK-2 decreases approximately tenfold upon addition of polylysine, which also prevents the fast autophosphorylation of the kinase at its beta subunit . These data suggest that the beta subunit of animal CK-2 may play a role in determining both the stability of the enzyme and its regulation and that, consequently, the different properties of YCK-2 may be at least in part accounted for by its lack of beta subunits.

Biochim Biophys Acta, 1986 Aug 14, 878(1), 93 - 101
Intracellular transfer of phospholipids in the yeast, Saccharomyces cerevisiae; Daum G et al.; In Saccharomyces cerevisiae, unlike in higher eukaryotic cells, most of the reactions involved in phospholipid biosynthesis occur both in mitochondria and in the endoplasmic reticulum . Some of the key enzymes involved, however, are restricted to one compartment . Thus, the formation of phosphatidylethanolamine by decarboxylation of phosphatidylserine occurs only in mitochondria, while phosphatidylcholine synthesis via methylation of phosphatidylethanolamine is restricted to microsomes . When yeast cells were pulse labelled with {3H}serine,{3H} phosphatidylethanolamine formed in mitochondria was found not only in the organelle but also, with even higher specific radioactivity, in the endoplasmic reticulum . Translocation of phosphatidylethanolamine between organelles was blocked immediately after poisoning cells with cyanide, azide and fluoride . Part of the {3H}phosphatidylcholine formed in the endoplasmic reticulum by methylation of {3H}phosphatidylethanolamine was transferred to mitochondria . This process continued in deenergized cells, although at a lower rate as compared to metabolizing cells . This result indicates rapid movement of both phosphatidylethanolamine and phosphatidylcholine requires metabolic energy, but that phosphatidylinositol-specific phospholipid transfer protein that has been found in saccharomyces cerevisiae (Daum, G . and Paltauf, F . (1984) Biochim . Biophys . Acta 784, 385-391) . The mechanism of movement of phospholipids from internal membranes to the cell surface was studied with temperature-sensitive secretory mutants (Schekman, R . (1982) Trends Biochem . Sci . 7, 243-246) of Saccharomyces cerevisiae . A shift from the permissive to the restrictive temperature, which blocks the flow of vesicles involved in the secretion of proteins, had no effect on the transfer of phosphatidylinositol to the plasma membrane.

Nucleic Acids Res, 1986 Aug 11, 14(15), 6247 - 64
DNA sequence analysis of ARS elements from chromosome III of Saccharomyces cerevisiae: identification of a new conserved sequence; Palzkill TG et al.; Four fragments of Saccharomyces cerevisiae chromosome III DNA which carry ARS elements have been sequenced . Each fragment contains multiple copies of sequences that have at least 10 out of 11 bases of homology to a previously reported 11 bp core consensus sequence . A survey of these new ARS sequences and previously reported sequences revealed the presence of an additional 11 bp conserved element located on the 3' side of the T-rich strand of the core consensus . Subcloning analysis as well as deletion and transposon insertion mutagenesis of ARS fragments support a role for 3' conserved sequence in promoting ARS activity.

J Gen Microbiol, 1986 Aug, 132 ( Pt 8), 2147 - 53
Transport and hydrolysis of peptides in Saccharomyces cerevisiae; Moneton P et al.; The transport and hydrolysis of several radioactive di- and tripeptides in Saccharomyces cerevisiae was studied . A peptide-transport-deficient mutant isolated on the basis of its resistance to nikkomycin Z lost most of its capacity to take up di- and tripeptides . The transport kinetics of {14C}methionylglycine, {14C}methionylsarcosine and {3H}nikkomycin Z indicated that peptide transport is not dependent on intracellular hydrolysis . Intact cells had some peptidase activity towards methionylsarcosine but not towards nikkomycin Z . The relationship between this activity and peptide transport is discussed.

J Gen Microbiol, 1986 Aug, 132 ( Pt 8), 2087 - 97
Nuclear suppressors of the mitochondrial mutation oxi1-V25 in Saccharomyces cerevisiae . Genetic analysis of the suppressors: absence of complementation between non-allelic mutants; Boguta M et al.; Ten informational nuclear suppressors of the oxi1- mitochondrial mutation of Saccharomyces cerevisiae are recessive . They are linked to each other, but their allelism is uncertain . Some of them unfavourably affect functions of standard (mit+) mitochondrial genomes . One suppressor severely impairs or entirely prevents mitochondrial functions of the spore clones carrying it . The spectrum of mit- mutations on which these suppressors act is similar to that exhibited by nam3-1 . In double heterozygotes namx/NAM3+, NAM+x/nam3-1 the oxi1- (and box3-) mutation is suppressed, yet one of our suppressors (R705) and nam3-1 show independent segregation in tetrads . This indicates that there may be absence of complementation between non-allelic suppressors.

Eur J Cell Biol, 1986 Aug, 41(2), 165 - 73
Ultrastructure of two secretory mutants of Saccharomyces cerevisiae as revealed by freeze-fracture technique; Necas O et al.; Permissive and restrictive phenotypes of two secretory mutants of Saccharomyces cerevisiae, sec 1 and sec 18, were studied by freeze-fracture technique . The sec 1 mutant, in addition to accumulating secretory vesicles, was characterized by a disappearance of the plasma membrane invaginations and by an aggregation of intra-membrane particles in vacuolar membranes . A prolonged incubation of the cells at 37 degrees C led to pathological fusion of some vesicles with the plasma membrane . After the cells were transferred back to the permissive temperature the invaginations reappeared rapidly while the accumulated vesicles disappeared only after budding had been resumed . The sec 18 mutant, apart from having distended endoplasmic reticulum membranes, also lost the plasma membrane invaginations at 37 degrees C and regained them at 24 degrees C . The described ultrastructural changes are typical for the restrictive phenotypes and represent further manifestations of the pleiotropic effect of the respective sec mutations.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5973 - 7
Identification of phosphoproteins correlated with proliferation and cell cycle arrest in Saccharomyces cerevisiae: positive and negative regulation by cAMP-dependent protein kinase; Tripp ML et al.; Recent genetic and biochemical studies of two mutants of the cAMP pathway in yeast, cyr1 and bcy1, have demonstrated that cAMP-dependent protein phosphorylation plays a major regulatory role in the control of proliferation and differentiation . As a first step in examining this regulatory system in more detail and in identifying the protein substrates of cAMP-dependent protein kinase, we have analyzed phosphoprotein patterns in the mutants cyr1-2(ts) and bcy1 by two-dimensional polyacrylamide gel electrophoresis . Our analysis has revealed several proteins whose phosphorylation is controlled positively or negatively by the cAMP pathway in yeast . The presence of some of these phosphoproteins was directly associated with proliferation (positive regulation), while that of others was correlated with cell cycle arrest (negative regulation) . The phosphoprotein patterns of cyr1-2(ts) temperature-arrested cells, and nitrogen (NH+4)-starved cells, were strikingly similar, suggesting that response to NH+4 is mediated in part by adenylate cyclase . Phosphoproteins whose presence correlated with cell cycle arrest were found to be phosphorylated on serine and threonine residues, while the major phosphoproteins present predominantly in proliferating cells were phosphorylated only on serine residues . None of the greater than 20 phosphoproteins we examined contained phosphotyrosine under either growth condition.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5563 - 7
Saccharomyces cerevisiae contains two functional genes encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase; Basson ME et al.; We have isolated two genes from yeast encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase {hydroxymethylglutaryl-coenzyme A reductase (NADPH); HMG-CoA reductase; EC 1.1.1.34}, the rate-limiting enzyme of sterol biosynthesis . These genes, HMG1 and HMG2, were identified by hybridization to a cDNA clone encoding hamster HMG-CoA reductase . DNA sequence analysis reveals homology between the amino acid sequence of the proteins encoded by the two yeast genes and the carboxyl-terminal half of the hamster protein . Cells containing mutant alleles of both HMG1 and HMG2 are unable to undergo spore germination and vegetative growth . However, cells containing a mutant allele of either HMG1 or HMG2 are viable but are more sensitive to compactin, a competitive inhibitor of HMG-CoA reductase, than are wild-type cells . Assays of HMG-CoA reductase activity in extracts from hmg1- and hmg2- mutants indicate that HMG1 contributes at least 83% of the activity found in wild-type cells.

Mutat Res, 1986 Aug, 174(4), 271 - 4
Inducibility of gene conversion in Saccharomyces cerevisiae treated with MMS; Cundari E et al.; At high survival levels (85%), point mutation and gene conversion frequencies were determined in strain D7 of Saccharomyces cerevisiae after treatment with methyl methanesulfonate (MMS) either after cells were incubated in complete medium before plating or following a split-dose protocol . It is shown that induction of gene conversion by MMS post-incubation leads to an additional enhancement in frequency . This increase is not observed for point mutation . By fractionation of the MMS dose (1 mM + 1 mM) with incubation in complete medium between the 2 doses the frequency of gene conversion is twice as high as with a single equal total dose (2 mM) . This treatment does not modify the frequencies of point mutation . These data support the notion that an inducible recombinogenic function exists in wild-type yeast.

Mutat Res, 1986 Aug, 162(1), 33 - 40
DNA-repair characterization of cdc40-1, a cell-cycle mutant of Saccharomyces cerevisiae; Kupiec M et al.; The cell-cycle specific mutation cdc40-1, which has been previously shown to be sensitive to MMS at the restrictive temperature, was further characterized as a DNA-repair-deficient mutation . cdc40-1 mutants shown only slight sensitivity to UV irradiation . Double mutant studies shown that rad6-l is epistatic to cdc40-1 with respect to sensitivity to UV irradiation and MMS . rad50-1 is epistatic to cdc40-1 with respect to MMS sensitivity in G1 stationary cells, but not in logarithmic cultures . An additive effect is seen between cdc40-1 and rad50-1 with respect to UV irradiation . cdc40-1 mutants are defective in UV-induced mutagenesis at the restrictive temperature . UV-induced levels of recombination are normal at both temperatures, while MMS-induced recombination is enhanced at the restrictive temperature.

J Gen Microbiol, 1986 Aug, 132 ( Pt 8), 2187 - 93
Effect of mitochondrial cytochromes and haem content on cytochrome P450 in Saccharomyces cerevisiae; Meussdoerffer F et al.; It is well established that the mitochondrial and the microsomal cytochromes in Saccharomyces cerevisiae are regulated differently . Mutations affecting the mitochondrial cytochromes aa3 or c had no effect on the concentration of the microsomal cytochrome P450 even during haem limitation . Moreover, a defect in the cytochrome P450 gene did not affect mitochondrial cytochromes . However, a regulatory mutation present in strain SG1 decreased both mitochondrial and microsomal cytochrome contents . This mutation also affected the intracellular haem concentration . The haem precursor 5-aminolaevulinate increased both mitochondrial and microsomal cytochrome contents . Our results indicate that carbon source and haem concentration are involved in the regulation of cytochrome P450.

Mol Gen Genet, 1986 Aug, 204(2), 310 - 6
Deletion of the phosphoglucose isomerase structural gene makes growth and sporulation glucose dependent in Saccharomyces cerevisiae; Aguilera A; The structural gene PGI1 coding for phosphoglucose isomerase was replaced by the LEU2 gene in the genome of Saccharomyces cerevisiae . Plasmids carrying the LEU2 gene between genomic regions flanking the PGI1 gene were constructed and used to transform a PGI1/pgi1 diploid strain . Stable transformants lacking the PGI1 allele were isolated . Southern analysis of their meiotic products showed that haploid strains with a deletion of 1.6 kb within the 2.2 kb PGI1 coding region were viable . Thus, the PGI1 gene is not essential in yeasts . However, unlike pgi1 mutants with residual phosphoglucose isomerase activity, no growth was detected in the pgi1 delta haploid strains when fructose was supplied as sole carbon source . The wild-type growth rate could be restored by adding 0.1% glucose to the medium . Furthermore, pgi1 mutants with residual enzymatic activity grew very slowly on fructose-supplemented media containing up to 2% glucose . Strains carrying the deletion allele, however, failed to grow at glucose concentrations higher than 0.5% . Also the pgi1 delta strains did not grow in glucose as sole carbon source . On the other hand pgi1 delta/pgi1 delta diploid strains did not sporulate on the usual acetate medium . This defect could be alleviated by the addition of 0.05% glucose to the sporulation medium . Under these conditions the pgi1 delta mutants sporulated with an efficiency of 25% compared with the wild type . These results suggest that the phosphoglucose isomerase reaction is the only step catalysing the interconversion of glucose-6-P and fructose-6-P, glucose-6-P is essential in yeasts, and the oxidation of glucose-6-P through the glucose-6-P dehydrogenase reaction is not sufficient to support growth in yeasts.

Mol Gen Genet, 1986 Aug, 204(2), 249 - 57
Purified Saccharomyces cerevisiae RNA polymerase II interacts homologously with two different promoters as revealed by P1 endonuclease analysis; Camilloni G et al.; The intergenic region of the Saccharomyces cerevisiae GAL1-GAL10 divergent promoters has been circularized in vitro in different topological states . In defined conditions, purified homologous RNA polymerase II forms two stable complexes (half-life approximately equal to 5 h) with this DNA in the presence of the four ribonucleotides, as determined by measurement (Gamper and Hearst 1983) of the amount and stability of the resulting unwinding . Each stable complex induces in the closed DNA domain a region of hypersensitivity to P1 endonuclease . The two induced hypersensitive regions are very similar: each maps on one promoter, spans over the 100 bp DNA sequence that encompasses the RNA Initiation Sites (RIS) and the TATA box, is composed by three subregions (one on the RIS, one proximal or overlapping the TATA sequence, one intermediate) . We show that this promoter-localized interaction is supercoil-dependent.

J Virol Methods, 1986 Aug, 14(1), 25 - 35
Hepatitis B surface antigen polypeptide micelles from antigen expressed in Saccharomyces cerevisiae; Howard CR et al.; Hepatitis B micelles containing the p25 component of hepatitis B surface antigen (HBsAg) have been produced by Triton X-100 solubilization followed by ultracentrifugation in linear sucrose gradients . The product was found to resemble micelle forms prepared from plasma-derived HBsAg with the surface being composed of discrete globular and stranded sub-units . The degree of immunochemical relatedness of the micellular preparation was compared to the native 22-nm HBsAg particle present in either plasma or yeast cell extracts . The yeast micelle preparation competed for anti-HBs in a similar manner as intact HBsAg of plasma origin . Enhanced immunogenicity may be expected for micelles containing a recombinant HBsAg protein as has previously been shown for the plasma-derived antigen.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5919 - 23
Ribonuclease H(70) from Saccharomyces cerevisiae possesses cryptic reverse transcriptase activity; Karwan R et al.; Yeast cells contain a protein of molecular size 70 kDa that possesses RNase H activity . A polyclonal antibody against it reacts in addition with proteins of molecular sizes 160 kDa from yeast extracts . All these immunologically related proteins exhibit reverse transcriptase activity and in this respect they resemble the products of retroviral pol genes, relatives of which reside in Ty elements and mitochondrial introns of yeast . Experimental evidence, however, indicates that the protein described here that combines RNase H and reverse transcriptase activity is not coded for by a known element of the retrotransposon family . It may originate from a cellular gene distantly related to retrotransposon sequences.

Biochem Biophys Res Commun, 1986 Jul 31, 138(2), 679 - 86
Induction of polypeptides in Saccharomyces cerevisiae after ultraviolet irradiation; Angulo JF et al.; Alterations in the synthesis of proteins following exposure of Saccharomyces cerevisiae to UV light were investigated using radioactive labelling and two dimensional electrophoresis . UV-irradiation induced the synthesis of various proteins . Among them the analogue of the RecA protein of Escherichia coli (Angulo et al . 1985) and two other polypeptides (34 Kd and 35 Kd, pI 5.8) were observed in all four strains analyzed namely two DNA-repair deficient (rad-) strains: (rad6-1 and pso2-1) and their isogenic wild type RAD+ strains.

J Biol Chem, 1986 Jul 25, 261(21), 9703 - 9
Isolation and characterization of the TRM1 locus, a gene essential for the N2,N2-dimethylguanosine modification of both mitochondrial and cytoplasmic tRNA in Saccharomyces cerevisiae; Ellis SR et al.; The trm1 mutation of Saccharomyces cerevisiae is a single nuclear mutation that affects a specific base modification of both cytoplasmic and mitochondrial tRNA . Transfer RNA isolated from trm1 cells lacks the modified base N2,N2-dimethylguanosine, and extracts from these cells do not have detectable N2,N2-dimethylguanosine-specific tRNA methyltransferase activity . As part of our efforts to determine how this mutation affects enzyme activities in two different cellular compartments we have isolated the TRM1 locus by genetic complementation . The TRM1 locus restores the N2,N2-dimethylguanosine modification to both cytoplasmic and mitochondrial tRNA in trm1 cells . An open reading frame in this TRM1 gene is essential for complementation of the trm1 phenotype . Expression of this open reading frame in Escherichia coli converts the organism from one that neither makes N2,N2-dimethylguanosine nor has N2,N2-dimethylguanosine-specific tRNA methyltransferase activity into one that does . This result suggests that the TRM1 locus is the structural gene for the tRNA modification enzyme and that both nuclear/cytoplasmic and mitochondrial forms of the methyltransferase are produced from the same gene.

Biochem Biophys Res Commun, 1986 Jul 16, 138(1), 78 - 86
Localisation of the hydrophilic C terminal part of the ATP synthase subunit 8 of Saccharomyces cerevisiae; Velours J et al.; The hydrophobic subunit 8 of the yeast ATP synthase was modified using the non-penetrating amino reactive specific reagent: isethionylacetimidate . The polypeptide was modified when using the isolated ATP synthase and sodium bromide-treated submitochondrial particles . It is shown that the only lysine of the protein was modified by the reagent . It is concluded that the hydrophilic C terminal part of the protein containing lysine 47 is located on the inner side of the inner mitochondrial membrane.

Eur J Biochem, 1986 Jul 15, 158(2), 345 - 50
Detection and partial characterization of the chromatin-associated proteases of yeast Saccharomyces cerevisiae; Motizuki M et al.; The chromatin fraction was prepared from yeast Saccharomyces cerevisiae free from cytoplasmic contamination except for a trace of mitochondria . When the yeast chromatin was incubated with histones as a substrate it showed three peaks of proteolytic activity as approximately pH 4, pH 7 and pH 11 . These activities were separated from each other by differential extractions from chromatin and successive gel filtration through Sephadex G-100 . Proteases were partially characterized by affinity labeling with {3H}diisopropylfluorophosphate (iPr2P-F) and by various protease inhibitors . The neutral and the alkaline proteases were serine proteases with a molecular mass of 35 kDa and 25 kDa respectively . The acidic protease showed a molecular size larger than 100 kDa on the gel filtration, and was probably an aspartyl protease because it was most strongly inhibited by pepstatin . A iPr2P-F-binding protein with a molecular mass of 66 kDa, found in chromatin, was likely to be converted to the alkaline protease of 25 kDa when chromatin was incubated at pH 10 or in 6 M urea/0.1 M phosphoric acid at the extraction . The distribution of proteolytic activities and iPr2P-F-binding proteins were compared among chromatins from different strains and from cells in different growth phases and it was found that these three proteases were present in all of them but with different proportions . Considering that rat liver chromatin contains equivalents to these proteases {Tsurugi, K . and Ogata, K . (1982) J . Biochem . (Tokyo) 92, 1369-1381}, the results suggested that they play some important roles in the function of eukaryotic chromatin.

J Biol Chem, 1986 Jul 15, 261(20), 9328 - 32
A nuclear mutant of Saccharomyces cerevisiae deficient in mitochondrial DNA replication and polymerase activity; Genga A et al.; We have isolated a thermosensitive mutant which is transformed into a population of cells devoid of mitochondrial DNA (rho 0 cells) at 35 degrees C and is deficient in mitochondrial (mt) DNA polymerase activity . A single recessive nuclear mutation (mip1) is responsible for rho 0 phenotype and mtDNA polymerase deficiency in vitro . At 25 degrees C (or 30 degrees C) a dominant suppressor mutation (SUP) masks the deficiency in vivo . The meiotic segregants (mip1 sup) which do not harbor the suppressor have a rho 0 phenotype both at 25 and 35 degrees C . They have no mtDNA polymerase activity, in contrast with MIP rho 0 mutants of mitochondrial inheritance which do exhibit mtDNA polymerase activity . In the thermosensitive mutant (mip1 SUP), the replication of mtDNA observed in vivo at 30 degrees C is completely abolished at 35 degrees C . In the meiotic segregants (mip1 sup), no mtDNA replication takes place at 30 and 35 degrees C . The synthesis of nuclear DNA is not affected . DNA polymerases may have replicative and/or repair activity . There is no evidence that mip mutants are deficient in mtDNA repair . In contrast the MIP gene product is strictly required for the replication of mtDNA and for the expression of the mtDNA polymerase activity . This enzyme might be the replicase of mtDNA.

J Biol Chem, 1986 Jul 15, 261(20), 9144 - 9
Mutants of Saccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase . Simultaneous loss of dihydroxyacetone phosphate acyltransferase indicates a common gene; Tillman TS et al.; Fourteen independent mutants of Saccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase activity were isolated using a colony autoradiographic screening technique . All 14 mutants were similarly defective in dihydroxyacetone phosphate acyltransferase activity . The mutations were recessive and fell into a single complementation group . Tetrad analysis gave results consistent with mutations in a single nuclear gene affecting both activities . sn-Glycerol-3-phosphate acyltransferase activity from different mutant strains exhibited different substrate dependencies and differing responses to temperature, detergent, and pH . In each case, the response of the dihydroxyacetone phosphate acyltransferase activity was similar to that of the sn-glycerol-3-phosphate acyltransferase . These results are consistent with the mutations occurring in the structural gene . The data also establish that the predominant dihydroxyacetone phosphate acyltransferase activity in yeast is a second activity of the sn-glycerol-3-phosphate acyltransferase.

Nucleic Acids Res, 1986 Jul 11, 14(13), 5145 - 58
Regular distribution of length heterogeneities within non-transcribed spacer regions of cloned and genomic rDNA of Saccharomyces cerevisiae; Jemtland R et al.; A length difference of about 50 bp in the EcoRI fragment B of the rDNA from two different strains of Saccharomyces cerevisiae has been mapped in detail by sequencing of cloned fragments . This 2.4 kb EcoRI fragment contains the start of the 35S rRNA gene at one end and the 5S rRNA gene in the middle flanked by non-transcribed spacers, NTS1 and NTS2 . The difference appeared as short deletions or insertions in five regularly spaced regions within the 1 kb NTS1, 3' to the 5S rRNA gene . The same regions of heterogeneities were displayed when all available sequence data of the NTS1 were compared . Four of the variable regions are located 160-170 bp apart, indicating that they might represent linker sequences between phased nucleosomes . Two variant clones, differing in the length of one subfragment of NTS1, were isolated for each strain . In both cases these represented the major variants among chromosomal NTS1 as revealed by sequencing of genomic fragments.

J Immunol Methods, 1986 Jul 11, 91(1), 1 - 10
A rapid radiometric assay for measuring phagocytosis of Saccharomyces cerevisiae in macrophage cultures; Becker J et al.; A new assay was developed to measure yeast phagocytosis in cultures of murine resident peritoneal macrophages . Saccharomyces cerevisiae was radiolabeled during exponential growth in nutrient broth supplemented with {3H}glucose . Following ingestion of the radiolabeled heat-killed yeast particles for 15 min, phagocytic capacities were measured in harvested macrophage lysates by liquid scintillation spectrometry . The new procedure compares favorably with light microscopic techniques and appears to be a more sensitive method for quantitating phagocytic function . Dose-response studies indicate, that over a wide range of dexamethasone concentrations, the radiometric procedure consistently measures greater inhibitory effects for the steroid induced suppression of phagocytosis.

Mol Cell Biol, 1986 Jul, 6(7), 2500 - 10
The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases; Woolford CA et al.; pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases . The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation . Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment . This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide . Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts . Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI . Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family . A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol . Cell . Biol . 6:2490-2499, 1986) . Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.

Mol Cell Biol, 1986 Jul, 6(7), 2482 - 9
Interaction of the FLP recombinase of the Saccharomyces cerevisiae 2 micron plasmid with mutated target sequences; Andrews BJ et al.; The 2 micron plasmid of Saccharomyces cerevisiae codes for a site-specific recombinase, the FLP protein, that catalyzes efficient recombination across two 599-base-pair (bp) inverted repeats of the plasmid DNA both in vivo and in vitro . We analyzed the interaction of the purified FLP protein with the target sequences of two point mutants that exhibit impaired FLP-mediated recombination in vivo . One mutation lies in one of the 13-bp repeat elements that had been previously shown to be protected from DNase digestion by the FLP protein . This mutation dramatically reduces FLP-mediated recombination in vitro and appears to act by reducing the binding of FLP protein to its target sequence . The second mutation lies within the 8-bp core region of the FLP target sequence . The FLP protein introduces staggered nicks surrounding this 8-bp region, and these nicks are thought to define the sites of strand exchange . The mutation in the core region abolishes recombination with a wild-type site . However, recombination between two mutated sites is very efficient . This result suggests that proper base pairing between the two recombining sites is an important feature of FLP-mediated recombination.

Mol Cell Biol, 1986 Jul, 6(7), 2382 - 91
Secretion-defective mutations in the signal sequence for Saccharomyces cerevisiae invertase; Kaiser CA et al.; Nine mutations in the signal sequence region of the gene specifying the secreted Saccharomyces cerevisiae enzyme invertase were constructed in vitro . The consequences of these mutations were studied after returning the mutated genes to yeast cells . Short deletions and two extensive substitution mutations allowed normal expression and secretion of invertase . Other substitution mutations and longer deletions blocked the formation of extracellular invertase . Yeast cells carrying this second class of mutant gene expressed novel active internal forms of invertase that exhibited the following properties . The new internal proteins had the mobilities in denaturing gels expected of invertase polypeptides that had retained a defective signal sequence and were otherwise unmodified . The large increase in molecular weight characteristic of glycosylation was not seen . On nondenaturing gels the mutant enzymes were found as heterodimers with a normal form of invertase that is known to be cytoplasmic, showing that the mutant forms of the enzyme are assembled in the same compartment as the cytoplasmic enzyme . All of the mutant enzymes were soluble and not associated with the membrane components after fractionation of crude cell extracts on sucrose gradients . Therefore, these signal sequence mutations result in the production of active internal invertase that has lost the ability to enter the secretory pathway . This demonstrates that the signal sequence is required for the earliest steps in membrane translocation.

Mol Cell Biol, 1986 Jul, 6(7), 2287 - 97
Identification of a regulatory region that mediates glucose-dependent induction of the Saccharomyces cerevisiae enolase gene ENO2; Cohen R et al.; There are two yeast enolase genes, designated ENO1 and ENO2, which are expressed differentially in vegetative cells grown on glucose and in cells grown on gluconeogenic carbon sources . ENO2 is induced more than 20-fold in cells grown on glucose, whereas ENO1 expression is similar in cells grown on glucose and in cells grown on gluconeogenic carbon sources . Sequences within the 5' flanking region of ENO2 which are required for glucose-dependent induction were identified by deletion mapping analysis . These studies were carried out by using a fused gene containing the ENO2 5' flanking sequences and the ENO1 coding sequences . This fused gene undergoes glucose-dependent induction and is expressed at the same level as the resident ENO2 gene in cells grown on glucose or gluconeogenic carbon sources . Expression of fused genes containing deletion mutations within the ENO2 5' flanking region was monitored after integration at the ENO1 locus of a strain carrying a deletion of the resident ENO1 coding sequences . This analysis showed that there are two upstream activation sites located immediately upstream and downstream from a position 461 base pairs upstream from the transcriptional initiation site . Either one of these upstream activation sites is sufficient for glucose-dependent induction and normal gene expression in the presence of gluconeogenic carbon sources . Deletion of both regulatory regions results in a complete loss of gene expression . The regulatory regions function normally in both orientations relative to the coding sequences . Mutant fused genes containing small deletions within the regulatory regions were constructed; these genes were expressed normally in gluconeogenic carbon sources but were not induced in the presence of glucose . Based on this analysis, ENO2 contains a cis-acting regulatory region which is required for gene expression and mediates glucose-dependent induction of gene expression.

Radiobiologiia, 1986 Jul-Aug, 26(4), 460 - 4
{Photoreactivation in Saccharomyces cerevisiae cells after irradiation with electrons (25 MeV) . The role of photoreactivated damage in the manifestation of oxygen effects in radio- and UV-sensitive mutants of Saccharomyces cerevisiae}; Tsyb TS et al.; Significant photoreactivation was noted in radio- and UV-sensitive rad-mutants of Saccharomyces cerevisiae cells exposed to 25 MeV electrons . In order to make the photoreactivable damage be manifest anoxic conditions of irradiation should be chosen as optimal ones . It was shown that the low oxygen effect was partially associated with the photoreactivable damage involved in the lethal effect of ionizing radiation.

Mol Gen Genet, 1986 Jul, 204(1), 103 - 7
A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae; Dickinson JR et al.; In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified . Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source . The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD+-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%-70% of the wild-type levels . The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain . Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase . Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.

Genetika, 1986 Jul, 22(7), 1104 - 11
{Isolation and characteristics of Saccharomyces cerevisiae strains with increased sensitivity to detergents}; Sarsenova SZh et al.; The yeast mutants possessing enhanced sensitivity to detergents were obtained after treatment with ethyl methanesulfonate . The whole set of mutants may be divided into three groups, according to sensitivity to: cetylthreemethylammonium chloride detergents and dyes of the ethidium bromide type, detergents, dyes and antibiotics (gramycidin C and actinomycin D) . The genetic analysis performed indicated that more than one gene are responsible for sensitivity . On the basis of the test for allelism mutants were distributed into three groups . It was shown that ethidium bromide is far more potent inducer of cytoplasmic petites in detergent-sensitive than in wild-type strains.

Mutat Res, 1986 Jul, 174(3), 195 - 7
Effect of saccharin on the meiotic division of Saccharomyces cerevisiae; Persic L; The effect of saccharin on the occurrence of meiotic diploid and disomic products in Saccharomyces cerevisiae was investigated . It was found that this substance inhibits the sporulation process in a dose-dependent manner, is ineffectual on recombination frequency, and slightly increases the occurrence of diploid and disomic meiotic products . It is demonstrated that the formation of diploid meiotic products is a consequence of random nuclear fusions at the end of the meiotic process or of endomitosis preceding meiosis.

J Virol, 1986 Jul, 59(1), 176 - 80
Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: synthesis of two polypeptides translated from different initiation codons; Miyanohara A et al.; Two recombinant plasmids were constructed that allow expression of the hepatitis B core (HBc) antigen gene in the yeast Saccharomyces cerevisiae under the control of the repressible acid phosphatase promoter . One plasmid was designed to produce polypeptide I, which consists of 183 amino acids, and the other plasmid was designed to produce polypeptide II, which has an additional 29-amino-acid sequence at the amino terminus of polypeptide I . The viral genome may code for either one or both of these two polypeptides, depending upon the selection of initiation codons . Both polypeptides produced in yeast cells reacted with anti-HBc antibody and were assembled into spherical particles approximately 27 nm in diameter . Particles made of polypeptide I were stable, whereas those made of polypeptide II readily dissociated when exposed to high salt levels . The antigenicity of the HBc (as defined by its reactivity to anti-HBc antibody in the reversed passive hemagglutination assay) disappeared as the particle dissociated, leaving materials that sedimented slowly and that reacted to anti-hepatitis B e antibody . These observations strongly suggest that native viral cores are mostly (if not all) made of polypeptide I, because it is reasonably stable, and that the N-terminal portion of this polypeptide has some, but not a profound, influence on the assembly of polypeptides into particles.

Exp Cell Res, 1986 Jul, 165(1), 29 - 40
Characterization of DNA sequences associated with residual nuclei of Saccharomyces cerevisiae; Potashkin JA et al.; We have used two different approaches to determine whether particular DNA sequences are specifically associated with high-salt-treated residual nuclei of Saccharomyces cerevisiae . First, libraries of yeast DNA in phage lambda were probed with nick-translated total nuclear or residual nuclear DNA from unsynchronized yeast cells . None of the plaques gave a significantly stronger or weaker signal with the residual nuclear probe than with the total nuclear probe . Second, DNA was purified from whole nuclei or residual nuclei which had been isolated from cells in G1, G1/S, early S, or nuclear division . This DNA was "dot-blotted" and then probed with specific yeast DNA sequences . Ribosomal DNA was 2- to 3-fold enriched in residual nuclei in late G1, G1/S, and early S, and 2 microns plasmid DNA sequences were 3- to 5-fold depleted during nuclear division and early G1 . However, ARS1, TRP1, CEN6, and a telomere sequence were neither enriched nor depleted at any time during the cell cycle.

Mol Cell Biol, 1986 Jul, 6(7), 2324 - 33
Short repeated elements in the upstream regulatory region of the SUC2 gene of Saccharomyces cerevisiae; Sarokin L et al.; Expression of secreted invertase from the SUC2 gene is regulated by carbon catabolite repression . Previously, an upstream regulatory region that is required for derepression of secreted invertase was identified and shown to confer glucose-repressible expression to the heterologous promoter of a LEU2-lacZ fusion . In this paper we show that tandem copies of a 32-base pair (bp) sequence from the upstream regulatory region activate expression of the same LEU2-lacZ fusion . The level of expression increased with the number of copies of the element, but was independent of their orientation; the expression from constructions containing four copies of the sequence was only twofold lower than that when the entire SUC2 upstream regulatory region was present . This activation was not significantly glucose repressible . The 32-bp sequence includes a 7-bp motif with the consensus sequence (A/C)(A/G)GAAAT that is repeated at five sites within the upstream regulatory region . Genetic evidence supporting the functional significance of this repeated motif was obtained by pseudoreversion of a SUC2 deletion mutant lacking part of the upstream region, including two copies of the 7-bp element . In three of five pseudorevertants, the mutations that restored high-level SUC2 expression altered one of the remaining copies of the 7-bp element.

What Is Antibiotic?, What Is Activated Sludge?, What Is Listeria Monocytogenes?, What Is Prokaryote?, What Is MIC?, c, Microbes, n, Bacterium, e, Microorganisms, o, Microbiology, a, Bacteriology, a, Bacillus, r, Botulism, n, Shigella, r, Antimicrobial, e, Escherichia coli, c, Microorganisms, i, Streptococci, e, Pseudomonas aeruginosa, i, Cell cultures, n, Multidrug resistant, o, Growth media, r, Antimicrobials, o, Microorganisms, r, Micrococci, n, Micrococci, n, Proteus, s, Escherichia coli, s, Bacillus, a, Bacteriophage, s, Streptococcal, c, Salmonella

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005