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