S. cerevisiae

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 8908 - 12
Centromere DNA mutations induce a mitotic delay in Saccharomyces cerevisiae; Spencer F et al.; Cytological observations of animal cell mitoses have shown that the onset of anaphase is delayed when chromosome attachment to the spindle is spontaneously retarded or experimentally interrupted . This report demonstrates that a centromere DNA (CEN) mutation carried on a single chromosome can induce a cell cycle delay observed as retarded mitosis in the yeast Saccharomyces cerevisiae . A 31-base-pair deletion within centromere DNA element II (CDEII delta 31) that causes chromosome missegregation in only 1% of cell division elicited a dramatic mitotic delay phenotype . Other CEN DNA mutations, including mutations in centromere DNA elements I and III, similarly delayed mitosis . Single division pedigree analysis of strains containing the CDEII delta 31 CEN mutation indicated that most (and possibly all) cells experienced delay in each cell cycle and that the delay was not due to increased chromosome copy number . Furthermore, a synchronous population of cells containing the CDEII delta 31 mutation underwent DNA synthesis on schedule with wild-type kinetics, but subsequently exhibited late chromosomal separation and concomitant late cell separation . We speculate that this delay in cell cycle progression before the onset of anaphase provides a mechanism for the stabilization of chromosomes with defective kinetochore structure . Further, we suggest that the delay may be mediated by surveillance at a cell cycle checkpoint that monitors the completion of chromosomal attachment to the spindle.

Mol Cell Biol, 1992 Oct, 12(10), 4733 - 41
Localization of a DNA replication origin and termination zone on chromosome III of Saccharomyces cerevisiae; Zhu J et al.; Two-dimensional gel electrophoretic replicon mapping techniques were used to identify all functional DNA replication origins and termini in a 26.5-kbp stretch in the left arm of yeast chromosome III . Only one origin was detected; it coincided with an ARS element (ARS306), as have all previously mapped yeast origins . A replication termination region was identified in a 4.3-kbp stretch at the telomere-proximal end of the investigated region, between the origin identified in this paper and the neighboring, previously mapped, ARS305-associated origin (previously called the A6C origin) . Termination does not occur at a specific site; instead, it appears to be the consequence of replication forks converging in a stretch of DNA of at least 4.3 kbp.

Mol Cell Biol, 1992 Oct, 12(10), 4503 - 14
Involvement of the SIN4 global transcriptional regulator in the chromatin structure of Saccharomyces cerevisiae; Jiang YW et al.; We have cloned and sequenced the SIN4 gene and determined that SIN4 is identical to TSF3, identified as a negative regulator of GAL1 gene transcription (S . Chen, R.W . West, Jr., S.L . Johnson, H . Gans, and J . Ma, submitted for publication) . Yeast strains bearing a sin4 delta null mutation have been constructed and are temperature sensitive for growth and display defects in both negative and positive regulation of transcription . Transcription of the CTS1 gene is reduced in sin4 delta mutants, suggesting that Sin4 functions as a positive transcriptional regulator . Additionally, a Sin4-LexA fusion protein activates transcription from test promoters containing LexA binding sites . The sin4 delta mutant also shows phenotypes common to histone and spt mutants, including suppression of delta insertion mutations in the HIS4 and LYS2 promoters, expression of promoters lacking upstream activation sequence elements, and decreased superhelical density of circular DNA molecules . These results suggest that the sin4 delta mutation may alter the structure of chromatin, and these changes in chromatin structure may affect transcriptional regulation.

Mol Cell Biol, 1992 Oct, 12(10), 4456 - 63
Accumulation of U14 small nuclear RNA in Saccharomyces cerevisiae requires box C, box D, and a 5', 3' terminal stem; Huang GM et al.; U14 is one of several nucleolar small nuclear RNAs required for normal processing of rRNA . Functional mapping of U14 from Saccharomyces cerevisiae has yielded a number of mutants defective in U14 accumulation or function . In this study, we have further defined three structural elements required for U14 accumulation . The essential elements include the U14-conserved box C and box D sequences and a 5', 3' terminal stem . The box elements are coconserved among several nucleolar small nuclear RNAs and have been implicated in binding of the protein fibrillarin . New mutational results show that the first GA bases of the box C sequence UGAUGA are essential, and two vital bases in box D have also been identified . An intragenic suppressor of a lethal box C mutant has been isolated and shown to contain a new box C-like PyGAUG sequence two bases upstream of normal box C . The importance of the terminal stem was confirmed from new compensatory base changes and the finding that accumulation defects in the box elements can be complemented by extending the terminal stem . The results suggest that the observed defects in accumulation reflect U14 instability and that protein binding to one or more of these elements is required for metabolic stability.

Mol Cell Biol, 1992 Oct, 12(10), 4433 - 40
RPC82 encodes the highly conserved, third-largest subunit of RNA polymerase C (III) from Saccharomyces cerevisiae; Chiannilkulchai N et al.; RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes . In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa . Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82 . It maps as a single-copy gene on chromosome XVI . The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon . The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins . Disruption of the RPC82 gene was lethal . An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis . Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits . A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.

Mol Cell Biol, 1992 Oct, 12(10), 4314 - 26
RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest; Mann C et al.; RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III) . Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature . Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells . The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues . The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein . The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.

Mol Cell Biol, 1992 Oct, 12(10), 4305 - 13
The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae; Deshpande AM et al.; Replication origins have been mapped to positions that coincide, within experimental error (several hundred base pairs), with ARS elements . To determine whether the DNA sequences required for ARS function on plasmids are required for chromosomal origin function, the chromosomal copy of ARS306 was deleted and the chromosomal copy of ARS307 was replaced with mutant derivatives of ARS307 containing single point mutations in domain A within the ARS core consensus sequence . The chromosomal origin function of these derivatives was assayed by two-dimensional agarose gel electrophoresis . Deletion of ARS306 deleted the associated replication origin . The effects on chromosomal origin function of mutations in domain A paralleled their effects on ARS function, as measured by plasmid stability . These results demonstrate that chromosomal origin function is a property of the ARS element itself.

Mol Cell Biol, 1992 Oct, 12(10), 4262 - 70
Unusual aspects of in vitro RNA processing in the 3' regions of the GAL1, GAL7, and GAL10 genes in Saccharomyces cerevisiae; Sadhale PP et al.; A striking feature of the 3'-end regions in polymerase II transcripts of Saccharomyces cerevisiae adjacent to their processing and polyadenylation sites is the lack of well-defined signal elements . Nonetheless, essential signals have seemed to be confined to compact regions in vivo, and we find that a short RNA with only 70 bases of GAL7 sequence upstream and 8 to 10 bases downstream of the poly(A) addition site is processed in vitro, as is an analogous CYC1 pre-RNA . Specific polyadenylation of a precleaved species further delimits the poly(A) signal and rules out obligatory coupling between cleavage and poly(A) addition . Although little proximal and even less distal sequence is required for accurate cleavage with CYC1 and GAL7, we have been unable to identify common features to which processing could be ascribed . We therefore turned to the coregulated set of genes in the galactose cluster (GAL1, GAL7, and GAL10) to assay their corresponding pre-mRNAs in vitro, in hopes of finding a common theme . By contrast to GAL7, short pre-mRNAs corresponding to GAL1 and GAL10 fail to be cleaved detectably, and only much longer transcripts are susceptible to processing . This indicates that signals, even if preserved, are more widely dispersed than the poly(A) addition site, and these results are unchanged whether extracts are from cells grown on glucose or galactose . As a further surprise, RNAs corresponding to the antisense orientation of the 3'-end regions of all three GAL genes are also effective substrates for the processing machinery in vitro . Computer analysis reveals the presence of polydisperse dyad symmetries that might account for these observations.

J Cell Biol, 1992 Oct, 119(2), 379 - 88
Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae; Sullivan DS et al.; tub2-401 is a cold-sensitive allele of TUB2, the sole gene encoding beta-tubulin in the yeast, Saccharomyces cerevisiae . At 18 degrees C, tub2-401 cells are able to assemble spindle microtubules but lack astral microtubules . Under these conditions, movement of the spindle to the bud neck is blocked . However, spindle elongation and chromosome separation are unimpeded and occur entirely within the mother cell . Subsequent cytokinesis produces one cell with two nuclei and one cell without a nucleus . The anucleate daughter can not bud . The binucleate daughter proceeds through another cell cycle to produce a cell with four nuclei and another anucleate cell . With additional time in the cold, the number of nuclei in the nucleated cells continues to increase and the percentage of anucleate cells in the population rises . The results indicate that astral microtubules are needed to position the spindle in the bud neck but are not required for spindle elongation at anaphase B . In addition, cell cycle progression does not depend on the location or orientation of the spindle.

J Cell Biol, 1992 Oct, 119(2), 287 - 99
Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway; Klionsky DJ et al.; The Saccharomyces cerevisiae APE1 gene product, aminopeptidase I (API), is a soluble hydrolase that has been shown to be localized to the vacuole . API lacks a standard signal sequence and contains an unusual amino-terminal propeptide . We have examined the biosynthesis of API in order to elucidate the mechanism of its delivery to the vacuole . API is synthesized as an inactive precursor that is matured in a PEP4-dependent manner . The half-time for processing is approximately 45 min . The API precursor remains in the cytoplasm after synthesis and does not enter the secretory pathway . The precursor does not receive glycosyl modifications, and removal of its propeptide occurs in a sec-independent manner . Neither the precursor nor mature form of API are secreted into the extracellular fraction in vps mutants or upon overproduction, two additional characteristics of soluble vacuolar proteins that transit through the secretory pathway . Overproduction of API results in both an increase in the half-time of processing and the stable accumulation of precursor protein . These results suggest that API enters the vacuole by a posttranslational process not used by most previously studied resident vacuolar proteins and will be a useful model protein to analyze this alternative mechanism of vacuolar localization.

J Bacteriol, 1992 Oct, 174(20), 6678 - 81
Saccharomyces cerevisiae has distinct adaptive responses to both hydrogen peroxide and menadione; Jamieson DJ; Treatment of Saccharomyces cerevisiae cells with low concentrations of either hydrogen peroxide or menadione (a superoxide-generating agent) induces adaptive responses which protect cells from the lethal effects of subsequent challenge with higher concentrations of these oxidants . Pretreatment with menadione is protective against cell killing by hydrogen peroxide; however, pretreatment with hydrogen peroxide is unable to protect cells from subsequent challenge with menadione . This suggests that the adaptive responses to these two different oxidants may be distinct.

Curr Genet, 1992 Oct, 22(4), 335 - 6
A ten-minute protocol for transforming Saccharomyces cerevisiae by electroporation; Grey M et al.; We present a simplified and rapid method for the transformation of yeast cells by electroporation . Stationary cells, scraped off the agar of Petri dish cultures stored in the refrigerator for up to 6 weeks, are suspended in sorbitol buffer, spun down by gentle centrifugation, transferred into the electroporation cuvette, and immediately subjected to transformation via electroporation . Transformation efficiency of this 10-min method, which does not require the preparation of cell cultures, is about 10% of the hitherto best performing transformation procedure using cells of defined growth phase.

Curr Genet, 1992 Oct, 22(4), 277 - 82
The REV2 gene of Saccharomyces cerevisiae: cloning and DNA sequence; Ahne F et al.; The REV2 gene of Saccharomyces cerevisiae was cloned and sequenced; it contains an open reading frame of 1986 bp with a coding potential of 662 amino acids . Interruption of the chromosomal REV2 gene by integrating the URA3 gene coupled with partial deletion of the 3' terminal region produced viable haploid rev2 delta mutants . This indicates that the REV2 gene is non-essential for growth . The rev2 delta mutant is slightly more UV-sensitive than strains carrying various rev2 alleles (rev2-1, rev2x, rad5-1, rad5-8) . The putative Rev2 protein is probably a globular protein containing a highly conserved nucleotide-binding site and two zinc-finger domains.

Curr Genet, 1992 Oct, 22(4), 273 - 5
Biosynthesis of sulphur amino acids in Saccharomyces cerevisiae: regulatory roles of methionine and S-adenosylmethionine reassessed; Paszewski A et al.; cys4-1, a mutation in the reverse trans-sulphuration pathway, relieves the sulphate assimilation pathway and homocysteine synthase from methionine-mediated repression . Since the mutation blocks the synthesis of cysteine from methionine downstream from homocysteine, this indicates that neither methionine nor S-adenosylmethionine serve as low-molecular-mass effectors in this regulatory system, contradicting earlier hypotheses.

Curr Genet, 1992 Oct, 22(4), 259 - 65
Comparative analysis of spontaneous mitotic recombination in {cir0} and {cir+} strains of the yeast Saccharomyces cerevisiae; Pushnova EA et al.; The influence of the 2 microns plasmid on homologous recombination in the right arm of chromosome XV of the yeast Saccharomyces cerevisiae has been examined . No differences between spontaneous mitotic recombination rates in {cir0} and {cir+} derivatives of two yeast diploid tester strains were detected . In the course of analysis an unusually high coincident conversion frequency at ADE2, HIS3, and two RFLP loci adjacent to ADE2, was observed . The character of coincident homozygotization of linked markers argues for a "break-and-replicate" mechanism underlying the coincident conversion events.

J Cell Biol, 1992 Oct, 119(1), 153 - 62
Isolation of peroxisome assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure; Van der Leij I et al.; We have developed a positive selection system for the isolation of Saccharomyces cerevisiae mutants with disturbed peroxisomal functions . The selection is based on the lethality of hydrogen peroxide (H2O2) that is produced in wild type cells during the peroxisomal beta-oxidation of fatty acids . In total, 17 mutants having a general impairment of peroxisome biogenesis were isolated, as revealed by their inability to grow on oleic acid as the sole carbon source and their aberrant cell fractionation pattern of peroxisomal enzymes . The mutants were shown to have monogenetic defects and to fall into 12 complementation groups . Representative members of each complementation group were morphologically examined by immunocytochemistry using EM . In one mutant the induction and morphology of peroxisomes is normal but import of thiolase is abrogated, while in another the morphology differs from the wild type: stacked peroxisomal membranes are present that are able to import thiolase but not catalase . These mutants suggest the existence of multiple components involved in peroxisomal protein import . Some mutants show the phenotype characteristic of glucose-repressed cells, an indication for the interruption of a signal transduction pathway resulting in organelle proliferation . In the remaining mutants morphologically detectable peroxisomes are absent: this phenotype is also known from fibroblasts of patients suffering from Zellweger syndrome, a disorder resulting from impairment of peroxisomes.

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2035 - 43
Nutrient-induced activation of trehalase in nutrient-starved cells of the yeast Saccharomyces cerevisiae: cAMP is not involved as second messenger; Hirimburegama K et al.; Starvation of Saccharomyces cerevisiae cells for specific nutrients such as nitrogen, phosphate or sulphate causes arrest in the G1 phase of the cell cycle at a specific point called 'start' . Re-addition of different nitrogen sources, phosphate or sulphate to such starved cells causes activation of trehalase within a few minutes . Nitrogen-source- and sulphate-induced activation of trehalase were not associated with any change in the cAMP level, but in the case of phosphate there was a small transient increase . When nitrogen-source-activated trehalase was isolated by immuno-affinity chromatography from crude extracts, the purified enzyme showed the same activity profile as in the original crude extracts, indicating that post-translational modification is responsible for the activation . In the yeast mutants cdc25-5 and cdc35-10, which are temperature sensitive for cAMP synthesis, incubation at the restrictive temperature lowered but did not prevent nitrogen-, phosphate- or sulphate-induced activation of trehalase . Since under these conditions the cAMP level in the cells is very low, it is unlikely that cAMP acts as a second messenger in this nutrient-induced effect . Nitrogen-source-induced activation of trehalase requires the presence of glucose at a concentration similar to that able to stimulate the RAS-adenylate cyclase pathway . This indicates that the same glucose-sensing system might be involved in both phenomena . Nitrogen-starved cells fractionated according to cell size all showed nitrogen-source-induced activation of trehalase to the same extent, indicating that the nitrogen-induced signalling pathway involved is not dependent on the well-known cell size requirement for progression over the start point of the cell cycle.

Genetics, 1992 Oct, 132(2), 361 - 74
A mutant tRNA affects delta-mediated transcription in Saccharomyces cerevisiae; Happel AM et al.; Mutations in the SPT3, SPT7, SPT8 and SPT15 genes define one class of trans-acting mutations that are strong suppressors of insertion mutations caused by Ty elements or by the Ty long terminal repeat sequence, delta . These SPT genes are required for normal transcription of Ty elements, and their gene products are believed to be involved in initiation of Ty transcription from delta sequences . We have isolated and analyzed extragenic suppressors of spt3 mutations . These new mutations, named rsp, partially suppress the requirement for SPT3, SPT7, SPT8 and SPT15 functions . In addition, rsp mutations cause changes in transcription of some delta insertions in an SPT+ genetic background . Interactions between mutations in the four identified RSP genes show a number of interesting genetic properties, including the failure of unlinked rsp mutations to complement for recessive phenotypes . Cloning and sequencing of one rsp mutant gene, rsp4-27, showed that it encodes a frameshift suppressor glycine tRNA . Our results indicate that the other three RSP genes also encode frameshift suppressor glycine tRNAs . In addition, other types of frameshift suppressor glycine tRNAs can confer some Rsp- phenotypes.

Genetics, 1992 Oct, 132(2), 325 - 36
SPT4, SPT5 and SPT6 interactions: effects on transcription and viability in Saccharomyces cerevisiae; Swanson MS et al.; The SPT4, SPT5 and SPT6 genes of Saccharomyces cerevisiae were identified originally by mutations that suppress delta insertion mutations at HIS4 and LYS2 . Subsequent analysis has demonstrated that spt4, spt5 and spt6 mutations confer similar pleiotropic phenotypes . They suppress delta insertion mutations by altering transcription and are believed to be required for normal transcription of several other loci . We have now analyzed interactions between SPT4, SPT5 and SPT6 . First, the combination of mutations in any two of these three genes causes lethality in haploids . Second, some recessive mutations in different members of this set fail to complement each other . Third, mutations in all three genes alter transcription in similar ways . Finally, the results of coimmunoprecipitation experiments demonstrate that at least the SPT5 and SPT6 proteins interact physically . Taken together, these genetic and biochemical results indicate that SPT4, SPT5 and SPT6 function together in a transcriptional process that is essential for viability in yeast.

Mol Cell Biol, 1992 Oct, 12(10), 4441 - 8
Ectopic recombination between Ty elements in Saccharomyces cerevisiae is not induced by DNA damage; Parket A et al.; Mitotic recombination is increased when cells are treated with a variety of physical and chemical agents that cause damage to their DNA . We show here, using Saccharomyces cerevisiae strains that carry marked Ty elements, that recombination between members of this family of retrotransposons is not increased by UV irradiation or by treatment with the radiomimetic drug methyl methanesulfonate . Both ectopic recombination and mutation events were elevated by these agents for non-Ty sequences in the same strain . We discuss possible mechanisms that can prevent the induction of recombination between Ty elements.

Biochem Cell Biol, 1992 Oct-Nov, 70(10-11), 946 - 53
G1 cyclins regulate proliferation of the budding yeast Saccharomyces cerevisiae; Rowley A et al.; The eukaryotic cell cycle is regulated at two points, the G1-S and G2-M boundaries . The molecular basis for these regulatory activities has recently been elucidated, in large part by the use of molecular and genetic analyses using unicellular yeast . The molecular characterization of cell-cycle regulation has revealed striking functional conservation among evolutionarily diverse cell types . For many eukaryotic cells, regulation of cell proliferation occurs primarily in the G1 interval . The G1 regulatory step, termed START, requires the activation of a highly conserved p34 protein kinase by association with a functionally redundant family of proteins, the G1 cyclins . Here we review studies using the genetically tractable budding yeast Saccharomyces cerevisiae, which have provided insight into the role of G1 cyclins in the regulation of START.

Biochem Cell Biol, 1992 Oct-Nov, 70(10-11), 1230 - 7
Mutagenesis of Ste18, a putative G gamma subunit in the Saccharomyces cerevisiae pheromone response pathway; Whiteway M et al.; The yeast STE18 gene product has sequence and functional similarity to the gamma subunits of G proteins . The cloned STE18 gene was subjected to a saturation mutagenesis using doped oligonucleotides . The populations of mutant genes were screened for two classes of STE18 mutations, those that allowed for increased mating of a strain containing a defective STE4 gene (compensators) and those that inhibited mating even in the presence of a functional STE18 gene (dominant negatives) . Three amino acid substitutions that enhanced mating in a specific STE4 (G beta) point mutant background were identified . These compensatory mutations were allele specific and had no detectable phenotype of their own; they may define residues that mediate an association between the G beta and G gamma subunits or in the association of the G beta gamma subunit with other components of the signalling pathway . Several dominant negative mutations were also identified, including two C terminal truncations . These mutant proteins were unable to function in signal transduction by themselves, but they prevented signal transduction mediated by pheromone, as well as the constitutive signalling which is present in cells defective in the GPA1 (G alpha) gene . These mutant proteins may sequester G beta or some other component of the signalling machinery in a nonfunctional complex.

Appl Biochem Biotechnol, 1992 Oct, 37(1), 1 - 10
Effects of potassium on the ethanol production rate of Saccharomyces cerevisiae carrying the plasmid pCYG4 related with ammonia assimilation; da Silva NL et al.; The influence of potassium on ethanol production by Saccharomyces cerevisiae wild type and AR5 cells carrying the plasmid pCYG4 was investigated . This plasmid carries the glutamate dehydrogenase gene conferring an 11-fold higher level of expressed enzyme activity over the wild type cells . All experiments were carried out in batch culture with medium supplemented to different potassium concentrations up to 180 mM . Maximum ethanol production rate was observed in the AR5 cells grown in medium supplemented with 3.5 mM of potassium ions . Glucose uptake rate increased with increasing potassium up to 60 mM, but higher concentrations depressed glucose uptake rate in both strains . Furthermore, the wild type cells showed higher growth rate, ethanol production, and glucose consumption rate than the AR5 cells . These lower rates in the AR5 cells could be explained by repression of potassium uptake by an enhancement of ammonium feeding, and greater energy requirements by these cells due the presence of the plasmid.

FEBS Lett, 1992 Sep 28, 310(2), 182 - 6
Glycogen metabolism in a Saccharomyces cerevisiae phosphoglucose isomerase (pgil) disruption mutant; Corominas J et al.; Disruption of the gene pgil of Saccharomyces cerevisiae, which codes for phosphoglucose isomerase, results in a dramatic increase in the amount of intracellular glycogen in early exponential cultures . The level of glucose 6-phosphate was much higher in mutant than in wild-type cells . Phosphorylase a activity and the state of activation of glycogen synthase were also investigated . Phosphorylase a activity was rather low along the culture in wild-type cells, whereas it was consistently higher in mutants . Glycogen synthase was mostly in the active form in early-medium exponential cultures in wild-type cells whereas the activation state of this enzyme in mutant cells, although lower at the earlier steps of the culture, did not differ from wild-type cells at later stages . The fact that the intracellular levels of UDP-glucose are markedly increased in mutant cells suggest that the observed accumulation of glycogen results from a rise in substrate availability rather than from the activation of the enzyme responsible for the synthesis of the polysaccharide.

Biochim Biophys Acta, 1992 Sep 25, 1102(2), 213 - 9
Assessing hydrophobic regions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae; Seto-Young D et al.; The hydrophobic, photoactivatable probe TID {3-trifluoromethyl-3-(m-{125I}iodophenyl)diazirine} was used to label the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae . The H(+)-ATPase accounted for 43% of the total label associated with plasma membrane protein and incorporated 0.3 mol of {125I}TID per mol of 100 kDa polypeptide . The H(+)-ATPase was purified by octyl glucoside extraction and glycerol gradient centrifugation, and was cleaved by either cyanogen bromide digestion or limited tryptic proteolysis to isolate labeled fragments . Cyanogen bromide digestion resulted in numerous labeled fragments of mass less than 21 kDa . Seven fragments suitable for microsequence analysis were obtained by electrotransfer to poly(vinylidene difluoride) membranes . Five different regions of amino-acid sequence were identified, including fragments predicted to encompass both membrane-spanning and cytoplasmic protein structure domains . Most of the labeling of the cytoplasmic domain was concentrated in a region comprising amino acids 347 to 529 . This catalytic region contains the site of phosphorylation and was previously suggested to be hydrophobic in character (Goffeau, A . and De Meis, L . (1990) J . Biol . 265, 15503-15505) . Complementary labeling information was obtained from an analysis of limited tryptic fragments enriched for hydrophobic character . Six principal labeled fragments, of 29.6, 20.6, 16, 13.1, 11.4 and 9.7 kDa, were obtained . These fragments were found to comprise most of the putative transmembrane region and a portion of the cytoplasmic region that overlapped with the highly labeled active site-containing cyanogen bromide fragment . Overall, the extensive labeling of protein structure domains known to lie outside the bilayer suggests that {125I}TID labeling patterns cannot be unambiguously interpreted for the purpose of discerning membrane-embedded protein structure domains . It is proposed that caution should be applied in the interpretation of {125I}TID labeling patterns of the yeast plasma membrane H(+)-ATPase and that new and diverse approaches should be developed to provide a more definitive topology model.

Biochim Biophys Acta, 1992 Sep 24, 1132(2), 195 - 8
Yeast ribosomal proteins: XIV . Complete nucleotide sequences of the two genes encoding Saccharomyces cerevisiae YL16; Hashimoto T et al.; We isolated and sequenced YL16A and YL16B encoding ribosomal protein YL16 of Saccharomyces cerevisiae . The two nucleotide sequences within coding regions retain 91.1% identity, and their predicted sequences of 176 amino acids show 93.8% identity . Out of the ribosomal protein sequences from various organisms currently available, no counterpart to YL16 could be found.

Biochim Biophys Acta, 1992 Sep 24, 1132(2), 211 - 3
QRI8, a novel ubiquitin-conjugating enzyme in Saccharomyces cerevisiae; Vassal A et al.; We have cloned and sequenced the gene encoding a novel ubiquitin-conjugating enzyme in Saccharomyces cerevisiae . Disruption of this gene shows that it is not essential for cell viability.

Gene, 1992 Sep 21, 119(1), 57 - 63
Characterization of the tRNA(Trp) genes of Saccharomyces cerevisiae; Atkin AL et al.; The purpose of this work was to examine the tRNA(Trp)-encoding genes (tRNA(Trp)) of Saccharomyces cerevisiae to gain insight as to why tRNA(Trp) amber suppressors, isolated by conventional genetic techniques, have not been reported . The results herein indicate that the haploid yeast genome contains six tRNA(Trp) genes which map to five or six chromosomes . Not only do the six genes have identical coding sequences but their introns are also identical . Gene replacement experiments indicate that five copies of tRNA(Trp) are sufficient for cell viability . Thus, mutation of one tRNA(Trp) gene to a suppressor in vivo, lowering the functional number of tRNA(Trp) genes, would not be expected to be lethal.

Gene, 1992 Sep 21, 119(1), 49 - 56
A multisite integrative cassette for the yeast Saccharomyces cerevisiae; Kudla B et al.; We have developed a cassette for the integration of cloned DNA sequences at multiple sites in the Saccharomyces cerevisiae genome, taking advantage of the naturally repeated sigma sequences . This cassette contains one engineered sigma element which allows the targeting of an embedded gene at different genomic sigma elements by gene replacement . Two yeast genes, ARG4 and URA3, were thus integrated in the absence of any bacterial sequences, individually or sequentially on twelve chromosomes . Consequently, these studies led to the genetical tagging of individual members of the sigma family.

Gene, 1992 Sep 21, 119(1), 137 - 41
pYLZ vectors: Saccharomyces cerevisiae/Escherichia coli shuttle plasmids to analyze yeast promoters; Hermann H et al.; Two yeast/Escherichia coli shuttle vectors have been constructed to analyze promoter structures in Saccharomyces cerevisiae: the multicopy vector, pYLZ-2, and the centromere-based vector, pYLZ-6 . Both plasmids contain the coding region of lacZ from E . coli lacking the N-terminal eight amino acids . The truncated reporter gene is preceded by a short polylinker (MCS) suitable for the insertion of promoter fragments . The vectors allow for the study of expression from complete promoters containing UAS and TATA elements, transcriptional start point(s) and the original context of the ATG start codon of a yeast gene . A yeast terminator fragment has been inserted 3' of the lacZ coding region . It contains the transcription termination region of the convergently transcribed yeast genes, GCY1 and PFY1, together with sequences corresponding to the mapped 3'-ends of the respective mRNAs . As an example, reporter activity was measured with promoter fragments from three yeast genes (GCY1, PFY1 and LEO1) . The results demonstrate the efficiency of the plasmids for studying constitutive and regulated transcription, both at high and low levels of expression.

Gene, 1992 Sep 21, 119(1), 65 - 74
A new family of polymorphic metallothionein-encoding genes MTH1 (CUP1) and MTH2 in Saccharomyces cerevisiae; Naumov GI et al.; By pulsed-field gel electrophoresis of chromosomal DNA and hybridization with a cloned MTH1 (CUP1) gene, we determined the locations of metallothionein-encoding gene sequences on chromosomes in monosporic cultures of 76 natural strains of Saccharomyces cerevisiae . Most of the strains (68) exhibited a previously known location for the MTH sequence on chromosome (chr.) VIII . Seven strains (resistant or sensitive to Cu2+) showed a MTH sequence in a new locus, MTH2, on chr . XVI . One strain carried an MTH locus on both chromosomes VIII and XVI . Restriction fragment and Southern blot analyses showed that the two MTH loci were very closely related . The strains displayed heterogeneity in the size and structure of their MTH2 locus . The length of the repeat unit of MTH2 varied: a 1.9-kb or 1.7-kb unit was found, instead of the 2.0-kb unit of the MTH1 locus . The most resistant strain (resistant to 1.2 mM CuSO4) contained a 0.9-kb repeat unit in addition to those of 1.9 kb and 1.7 kb . All three sensitive (to over 0.3 mM CuSO4) strains with an mth2 locus had a repeat unit of 1.9 kb or 1.7 kb, suggesting the presence of at least two copies of the MTH2 gene, with one always being in the junction area outside of the repeat unit . A monogenic tetrad segregation of 2:2 was usually found in crosses of resistant MTH2 and sensitive mth2 strains . Hybrids between strains with different MTH loci in all combinations showed low ascospore viability, suggesting that the complete lack of an MTH locus may lead to the death of segregants on YPD medium . The MTH1 and MTH2 loci were exchangeable . Strains with a high level of Cu2+ resistance were also resistant to Cd2+ . However, these two properties did not cosegregate in heterozygotic hybrids.

J Biol Chem, 1992 Sep 5, 267(25), 18013 - 20
The 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae are regulated differentially by phosphorylation via cAMP-dependent protein kinase; Quinlan JJ et al.; Evidence is presented that demonstrated that the 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae (Morlock, K . R., McLaughlin, J . J., Lin, Y.-P., and Carman, G . M . (1991) J . Biol . Chem . 266, 3586-3593) were regulated differentially by phosphorylation . Purified 45-kDa phosphatidate phosphatase was phosphorylated by cAMP-dependent protein kinase whereas purified 104-kDa phosphatidate phosphatase was not phosphorylated . cAMP-dependent protein kinase catalyzed the phosphorylation of pure 45-kDa phosphatidate phosphatase at a serine residue which resulted in a stimulation (2.4-fold) of phosphatidate phosphatase activity . Alkaline phosphatase catalyzed the dephosphorylation of pure 45-kDa phosphatidate phosphatase which resulted in an inhibition (1.3-fold) of phosphatidate phosphatase activity . Results of studies using mutants (bcy1 and cyr1) defective in cAMP-dependent protein kinase activity corroborated the results of the phosphorylation studies using pure preparations of phosphatidate phosphatase . The 45-kDa phosphatidate phosphatase phosphorylated in vitro and in vivo had phosphopeptides in common . The activation of the GAL10-RAS2val19 allele in mutant cells resulted in an increase in the synthesis of diacylglycerols and triacylglycerols . These results were consistent with the phosphorylation and activation of 45-kDa phosphatidate phosphatase by cAMP-dependent protein kinase in vivo.

J Biol Chem, 1992 Sep 15, 267(26), 18671 - 5
Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae . II . VAM7, a gene for regulating morphogenic assembly of the vacuoles; Wada Y et al.; VAM7 gene function has shown to be required for proper morphogenesis of the vacuole in yeast . The DNA fragments that complemented the defective vacuolar morphology of the vam7-1 mutation were isolated from a yeast genomic library . An overlapping 2.5-kilobase BglII-HindIII region was found to be sufficient for complementation of the vam7-1 phenotype . This fragment was integrated at the chromosomal VAM7 locus, indicating that it contained an authentic VAM7 gene . On nucleotide sequencing of the VAM7 gene, an open reading frame of 948 base pairs, coding for a hydrophilic polypeptide of 316 amino acid residues, was identified . The deduced amino acid sequence of the carboxyl-terminal region of the VAM7 gene product has heptad repeats and shows potential ability to form a coiled-coil structure . Disruption of VAM7 was not lethal; cells with a disrupted VAM7 gene did not, however, have a prominent large vacuoles but rather numerous small compartments that accumulated the histochemical marker molecule of the vacuolar compartment . They contained mature forms of the vacuolar marker proteins carboxypeptidase Y and vacuolar glycoprotein vgp72 . A mutant with both vam7 and vam5 null mutations was constructed and shown to have neither vacuolar structures stained with ade-related fluorochrome nor mature forms of vacuolar marker proteins . These findings suggested that the VAM7 gene product functions in the process of morphogenic assembly of the vacuolar compartment but is not involved in the protein sorting and delivery to the vacuole.

J Biol Chem, 1992 Sep 15, 267(26), 18665 - 70
Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae . I . Isolation and characterization of two classes of vam mutants; Wada Y et al.; We identified nine VAM genes (for vacuolar morphology) by genetic analyses on mutants with defective vacuolar morphologies and assembly in the yeast Saccharomyces cerevisiae . The nine VAM genes were classified into two classes according to the mutant phenotypes . The class I vam mutants (vam1, vam5, vam8, and vam9) show a few small vesicles that are stained with histochemical markers for the vacuolar compartment . They also have defects in the maturation of vacuolar marker proteins, and their growth is hypersensitive to high concentrations of CaCl2 or a temperature of 37 degrees C . There are apparent genetic overlaps among the class I vam mutations and other mutations including cls, end, pep, and vps, which have been shown to be involved in the expression of the vacuolar functions . The class II vam mutants (vam2, vam3, vam4, vam6, and vam7) contain numerous small vesicles stained with the vacuolar histochemical markers and mature forms of the vacuolar proteins and do not show any apparent growth defects in the presence of CaCl2 or at 37 degrees C.

Biochemistry, 1992 Sep 15, 31(36), 8442 - 8
The C-terminus of the succinate dehydrogenase IP peptide of Saccharomyces cerevisiae is significant for assembly of complex II; Schmidt DM et al.; Site-directed mutagenesis was used to introduce mutations into the gene for the iron protein (IP) of succinate dehydrogenase (SDH) of Saccharomyces cerevisiae . Specifically, three mutations were examined which caused the synthesis of truncated IP peptides missing four, seven, or 17 amino acids from the C-terminus, respectively . The deletion of seven or more amino acids includes the loss of two lysine residues, which appear to have been highly conserved in evolution . While the deletion of four amino acids had no effect on the assembly of complex II and on its activity, the deletion including the two lysines abolished SDS activity completely and led to the failure of the imported IP peptide to be incorporated into a stable complex II or SDH complex . Replacement of one of the lysines by threonine had no effect, but replacement of both by threonine affected the specific activity of complex II but not its assembly and stability.

J Biol Chem, 1992 Sep 5, 267(25), 17738 - 42
Unbalanced regulation of the ribosomal 5 S RNA-binding protein in Saccharomyces cerevisiae expressing mutant 5 S rRNAs; Tang B et al.; A gene encoding the 5 S rRNA-binding protein (YL3) in yeast (Saccharomyces cerevisiae) was further characterized with respect to its chromosomal localization, the controlling sequence regions, and the influence of 5 S rRNA gene expression . Sequence and chromosome blot analyses localized the gene on chromosome XVI immediately downstream of a cytochrome oxidase assembly gene, COXII . S1 nuclease protection studies identified two major initiation sites, 20 and 65 nucleotides upstream of the coding sequence, and a single polyadenylation site, 98 nucleotides downstream of the stop codon . Northern blot analyses and S1 nuclease protection indicated a normal pattern of gene regulation in media supporting alternate rates of growth, but significantly unbalanced regulation was observed in the presence of mutant 5 S rRNA genes which under-produce RNA and result in reduced growth rates . The results suggest a co-ordinating regulatory mechanism which maintains appropriate levels of 5 S rRNA-protein complex; an internal control region-like sequence in the upstream region of the YL3 gene is consistent with this feedback mechanism.

J Biol Chem, 1992 Sep 5, 267(25), 17553 - 9
Purification and characterization of BiP/Kar2 protein from Saccharomyces cerevisiae; Tokunaga M et al.; Using specific anti-BiP/Kar2 antibody as the probe, we have developed an efficient purification method of BiP/Kar2 protein from the total cell extract of Saccharomyces cerevisiae . Overproduction of BiP/Kar2 protein was achieved by the cloning of the KAR2 gene on multicopy plasmids and the treatment of cells harboring the cloned KAR2 gene with tunicamycin . Freeze-thaw treatment, hydroxyapatite high pressure liquid chromatography, and ATP-agarose column chromatography of crude extract yielded homogeneous BiP/Kar2 protein (including less than 0.2% of degradative derivative) with a 430-fold purification and 28% recovery . Edman degradation of purified BiP/Kar2 suggests that the mature protein corresponds to a processed product with the removal of a 42-amino acid presequence . It is active as a homodimer and exhibits ATPase activity with a specific activity of 2 pmol/min/micrograms of protein . Protease susceptibility indicated that the ADP form of BiP/Kar2 is more resistant than the ATP form to the chymotrypsin digestion and that BiP/Kar2 required the presence of ATP to avoid the irreversible denaturation . Synthesis of BiP/Kar2 was induced by the inducible expression of an aberrant heterologous protein, yeast killer prepro-signal mouse alpha-amylase fusion protein.

Curr Genet, 1992 Sep, 22(3), 175 - 80
Mutational analysis of a variant of ARS1 from Saccharomyces cerevisiae; Kirpekar F et al.; A naturally occurring single base-pair G to A transition, creating a 10/11 near-match close to the essential 11 base-pair core consensus of ARS1, was used to investigate the importance of near-match sequences . The 10/11 near-match can not substitute for the core consensus since an ARS- phenotype is observed when the core consensus is deleted . However, deletion mutations revealed that this near-match together with a short palindromic sequence, also situated in the B-flanking region, comprise a single element crucial for optimal ARS function . The palindrome has the potential of forming a stem-loop structure . Rather precise observations concerning the borders of the B-region were achieved . The four base pairs separating the near-match from the core consensus perform a spacing function where the identity of the bases are unimportant . However, this spacing is highly important since deletion of these four base pairs leads to an ARS- phenotype.

J Bacteriol, 1992 Sep, 174(18), 5985 - 7
An ordered clone bank for chromosome I of Saccharomyces cerevisiae; Tanaka S et al.; Chromosome I of Saccharomyces cerevisiae DC5 rho 0 was dissected into segments with an average size of 14.0 kb and cloned into lambda phage vectors . The physical maps of the resultant clones, totaling 205.9 kb, were used to construct an ordered clone bank of this chromosome.

Gene, 1992 Sep 1, 118(1), 131 - 6
SDH1, the gene encoding the succinate dehydrogenase flavoprotein subunit from Saccharomyces cerevisiae; Chapman KB et al.; We describe the isolation, sequence and construction of a disruption of the yeast SDH1 gene, encoding the flavoprotein subunit of succinate dehydrogenase . This is the first eukaryotic flavoprotein subunit-encoding gene to be fully sequenced . The deduced amino acid (aa) sequence is 50% identical to the Escherichia coli enzyme sequence . The yeast gene encodes an N-terminal extension of 45 aa relative to the E . coli sequence which may act as a mitochondrial targeting signal . Disruption of the gene results in the inability to respire, assayed as the inability to utilize the nonfermentable carbon source, glycerol . This is the expected phenotype for disruption of an essential component of the yeast citric acid cycle.

Mol Cell Biol, 1992 Sep, 12(9), 4215 - 29
Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae; Heidmann S et al.; In contrast to higher eukaryotes, little is known about the nature of the sequences which direct 3'-end formation of pre-mRNAs in the yeast Saccharomyces cerevisiae . The hexanucleotide AAUAAA, which is highly conserved and crucial in mammals, does not seem to have any functional importance for 3'-end formation in yeast cells . Instead, other elements have been proposed to serve as signal sequences . We performed a detailed investigation of the yeast ACT1, ADH1, CYC1, and YPT1 cDNAs, which showed that the polyadenylation sites used in vivo can be scattered over a region spanning up to 200 nucleotides . It therefore seems very unlikely that a single signal sequence is responsible for the selection of all these polyadenylation sites . Our study also showed that in the large majority of mRNAs, polyadenylation starts directly before or after an adenosine residue and that 3'-end formation of ADH1 transcripts occurs preferentially at the sequence PyAAA . Site-directed mutagenesis of these sites in the ADH1 gene suggested that this PyAAA sequence is essential for polyadenylation site selection both in vitro and in vivo . Furthermore, the 3'-terminal regions of the yeast genes investigated here are characterized by their capacity to act as signals for 3'-end formation in vivo in either orientation.

Mol Cell Biol, 1992 Sep, 12(9), 4084 - 92
Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae; McCabe PC et al.; The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function . A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae . Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites . To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p . Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation . Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression . Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells . p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins . Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells . Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12 . Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo . A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway . These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

Mol Cell Biol, 1992 Sep, 12(9), 4026 - 37
DPH5, a methyltransferase gene required for diphthamide biosynthesis in Saccharomyces cerevisiae; Mattheakis LC et al.; A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPH5 . The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen III methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis . Both DPH5 and AdoMet:uroporphyrinogen III methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases . The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dph5 mutant . A null mutation of the chromosomal DPH5 gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival . The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fully active DT fragment A . Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.

Mol Cell Biol, 1992 Sep, 12(9), 3843 - 56
PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing; O'Connor JP et al.; We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing . PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth . pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing . Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity . pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame . The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect . Episome-borne PTA1 directs recombination to the pta1-1 locus . PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417 . No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base . Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal . Possible functions of the PTA1 gene product are discussed.

Can J Microbiol, 1992 Sep, 38(9), 969 - 74
Interaction between flocculent and nonflocculent cells of Saccharomyces cerevisiae; Soares EV et al.; Interaction between nonflocculent and flocculent cells of Saccharomyces cerevisiae was studied . Adhesion experiments were done using three types of nonflocculent cells and a flocculent one . Two types of nonflocculent cells were obtained from the flocculent strain by changing environmental growth conditions . The integration of nonflocculent cells in the flocs was observed by two different methods: measurement of the sedimentation capacity of mixtures and microscopic observation of stained nonflocculent cells blended with flocculent cells . It was possible to verify that cell-cell interaction corresponds to a true stable binding and not to a simple entrapment inside the floc matrix.

Electrophoresis, 1992 Sep-Oct, 13(9-10), 651 - 3
Chromosomal localization of the HYP2-gene in Saccharomyces cerevisiae and use of pulsed-field gel electrophoresis for detection of irregular recombination events in gene disruption experiments; Wohl T et al.; In the hypusine-containing protein (HP), a specific lysine residue is modified by spermidine to form the unusual amino acid hypusine (4-amino-2-hydroxybutyllysine) . The HP has been designated as an eucaryotic translation initiation factor--eIF-5A--because of its stimulating effect in the methionyl-puromycin in vitro assay . Nevertheless, the precise function of this protein remains to be elucidated . In the yeast Saccharomyces cerevisiae two genes, HYP1 and HYP2, coding for two different forms of the HP, are present . The HYP1-gene is identical to the ANB1-gene and has already been localized on chromosome X . However, the chromosomal localization of the HYP2-gene has not been elucidated . By using pulsed-field gel electrophoresis (PFGE) and subsequent Southern blotting, we determined the localization of the HYP2-gene to chromosome V . Furthermore, PFGE was used for the detection of irregular recombination events, such as misintegration or integration into a duplicated gene, and in gene disruption experiments using haploid and diploid yeast cells . The obtained data support the critical role of the HP for cell viability.

Yeast, 1992 Sep, 8(9), 749 - 59
DNA sequencing and analysis of a 24.7 kb segment encompassing centromere CEN11 of Saccharomyces cerevisiae reveals nine previously unknown open reading frames; Dusterhoft A et al.; A 24.7 kb segment of the cosmid clone pUKG047 containing a Sau3AI-partial fragment from the centromere region of Saccharomyces cerevisiae chromosome XI was sequenced and analysed . A mixed strategy of directed methods including exonuclease III nested deletion, restriction fragment subcloning and oligonucleotide-directed sequences was carried out . Exclusive use was made of the Applied Biosystems Taq DyeDeoxy Terminator Cycle technology and a laser-based AB1373A sequencing system for reactions, gel electrophoresis and automated reading . A total of 12 open reading frames (ORFs) was found . Nine new ORFs (YK102 to YK110) were identified, three of which (YK102, YK107, YK108) showed homologies to proteins of known function from other organisms . In addition, sequence analysis revealed three recently functionally characterized genes (MET14, VPS/SPO15, PAP1), which could be joined to the earlier published CEN11 region.

Yeast, 1992 Sep, 8(9), 743 - 8
Disruption and mapping of IDI1, the gene for isopentenyl diphosphate isomerase in Saccharomyces cerevisiae; Mayer MP et al.; Isopentenyl diphosphate isomerase catalyses an essential activation step in the isoprene biosynthetic pathway . The Saccharomyces cerevisiae gene for isomerase, IDI1, was recently isolated and characterized (Anderson et al . J . Biol . Chem . 1989a, 264, 19169-19175) . Wild-type IDI1 was disrupted with a LEU2 marker, and the resulting DNA was used to transform a yeast leucine auxotroph . Southern blots of EcoRI fragments of chromosomal DNA from the diploid strain showed the expected fragments for intact and disrupted IDI1 . Dissection and analysis of tetrads demonstrated that IDI1 is an essential single-copy gene . A CHEF gel and clone grid filter analysis, followed by chromosomal mapping indicated that the gene is located on chromosome XVI approximately 55 kb centromere proximal to PEP4.

Yeast, 1992 Sep, 8(9), 735 - 41
RHO gene products, putative small GTP-binding proteins, are important for activation of the CAL1/CDC43 gene product, a protein geranylgeranyltransferase in Saccharomyces cerevisiae; Qadota H et al.; Two multicopy suppressors of the cal1-1 mutation in the yeast Saccharomyces cerevisiae have been isolated and characterized . They are identical to the yeast RHO1 and RHO2 genes, which encode putative small GTP-binding proteins . Multiple copies of either RHO gene suppressed temperature-sensitive growth of the cal1-1 mutant but did not suppress the cal1 null mutant . Genetic analysis suggests that overproduction of either RHO gene product acts for activation of the CAL1 gene product.

Yeast, 1992 Sep, 8(9), 699 - 710
MOL1, a Saccharomyces cerevisiae gene that is highly expressed in early stationary phase during growth on molasses; Praekelt UM et al.; We have isolated a new Saccharomyces cerevisiae gene, MOL1, that is transiently expressed at high levels in the early stationary phase of batch cultures growing on industrial molasses medium . The DNA sequence of the MOL1 gene (for MOLasses-inducible) with its flanking regions was determined (EMBL accession number X61669) . It encodes a polypeptide of M(r) 35 kDa that is closely related to stress-inducible proteins of similar size from two Fusarium species . Unlike ST135 of Fusarium, MOL1 is not induced by ethanol or heat shock . MOL1 expression is absent in rich (YP) medium, and only very low levels of expression are detectable in minimal (YNB) medium . The gene is not essential, and a MOL1 disruption strain showed no apparent phenotype under a variety of growth conditions . The 5' region of MOL1 contains the complete sequence previously determined for the SUF4 locus, encoding a tRNA-gly (UCC) gene, which has been mapped to chromosome VII.

Mol Biol Cell, 1992 Sep, 3(9), 1025 - 35
Specification of sites for polarized growth in Saccharomyces cerevisiae and the influence of external factors on site selection; Madden K et al.; Many eucaryotic cell types exhibit polarized cell growth and polarized cell division at nonrandom sites . The sites of polarized growth were investigated in G1 arrested haploid Saccharomyces cerevisiae cells . When yeast cells are arrested during G1 either by treatment with alpha-factor or by shifting temperature-sensitive cdc28-1 cells to the restrictive temperature, the cells form a projection . Staining with Calcofluor reveals that in both cases the projection usually forms at axial sites (i.e., next to the previous bud scar); these are the same sites where bud formation is expected to occur . These results indicate that sites of polarized growth are specified before the end of G1 . Sites of polarized growth can be influenced by external conditions . Cells grown to stationary phase and diluted into fresh medium preferentially select sites for polarized growth opposite the previous bud scar (i.e., distal sites) . Incubation of cells in a mating mixture results in projection formation at nonaxial sites: presumably cells form projections toward their mating partner . These observations have important implications in understanding three aspects of cell polarity in yeast: 1) how yeast cell shape is influenced by growth conditions 2) how sites of polarized growth are chosen, and 3) the pathway by which polarity is affected and redirected during the mating process.

Mol Gen Genet, 1992 Sep, 234(3), 369 - 78
Translational readthrough at nonsense mutations in the HSF1 gene of Saccharomyces cerevisiae; Kopczynski JB et al.; The HSF1 gene of Saccharomyces cerevisiae directs the synthesis of the heat shock transcription factor, HSF . The gene is essential; disruption mutations are lethal . Using a plasmid shuffle screen, we isolated mutations in the HSF1 gene after in vitro mutagenesis of plasmid DNA with hydroxylamine . From a collection of both conditional (temperature-sensitive) and unconditional lethal mutations, we recovered mutations that map exclusively to the 5' half of the gene . All are nonsense mutations, including conditional mutations that map 5' to the portion of the HSF1 gene that encodes the DNA-binding domain of the transcription factor . For one such mutation, we demonstrated that the nonsense mutation is subject to translational readthrough, even though there are no known nonsense suppressors in the genetic background of our strain . Our results suggest that the HSF protein is highly tolerant of amino acid changes, a conclusion that is consistent with the very low degree of evolutionary conservation among HSF proteins . Our results also suggest that translational readthrough occurs with moderate efficiency in yeast, particularly when the terminator codon is followed immediately by an A or C residue . This result illustrates that the inference of gene function from mutant phenotype depends critically upon the analysis of a true null allele, and not merely an amber or ochre allele.

Genetics, 1992 Sep, 132(1), 9 - 21
An examination of adaptive reversion in Saccharomyces cerevisiae; Steele DF et al.; Reversion to Lys+ prototrophy in a haploid yeast strain containing a defined lys2 frameshift mutation has been examined . When cells were plated on synthetic complete medium lacking only lysine, the numbers of Lys+ revertant colonies accumulated in a time-dependent manner in the absence of any detectable increase in cell number . An examination of the distribution of the numbers of early appearing Lys+ colonies from independent cultures suggests that the mutations to prototrophy occurred randomly during nonselective growth . In contrast, an examination of the distribution of late appearing Lys+ colonies indicates that the underlying reversion events occurred after selective plating . No accumulation of Lys+ revertants occurred when cells were starved for tryptophan, leucine or both lysine and tryptophan prior to plating selectively for Lys+ revertants . These results indicate that mutations accumulate more frequently when they confer a selective advantage, and are thus consistent with the occurrence of adaptive mutations in yeast.

Genetics, 1992 Sep, 132(1), 87 - 96
MAK10, a glucose-repressible gene necessary for replication of a dsRNA virus of Saccharomyces cerevisiae, has T cell receptor alpha-subunit motifs; Lee YJ et al.; The MAK10 gene is necessary for the propagation of the L-A dsRNA virus of the yeast Saccharomyces cerevisiae . We have isolated MAK10 from selected phage lambda genomic DNA clones that map near MAK10 . This gene encodes a 733-amino acid protein with several regions of similarity to T cell receptor alpha-subunit V (variable) regions . We show that MAK10 is essential for optimal growth on nonfermentable carbon sources independent of its effect on L-A . Although loss of L-A by mak10-1 mutants is partially suppressed by loss of the mitochondrial genome, no such suppression of a mak10::URA3 mutation was observed . Using MAK10-lacZ fusions we show that MAK10 is expressed at a very low level and that it is glucose repressed . The highest levels of expression were seen in tup1 and cyc8 mutants, known to be defective in glucose repression . These results suggest that the mitochondrial genome and L-A dsRNA compete for the MAK10 protein.

Genetics, 1992 Sep, 132(1), 63 - 73
Possible cross-regulation of phosphate and sulfate metabolism in Saccharomyces cerevisiae; O'Connell KF et al.; CP1 (encoded by the gene CEP1) is a sequence-specific DNA binding protein of Saccharomyces cerevisiae that recognizes a sequence element (CDEI) found in both yeast centromeres and gene promoters . Strains lacking CP1 exhibit defects in growth, chromosome segregation and methionine biosynthesis . A YEp24-based yeast genomic library was screened for plasmids which suppressed the methionine auxotrophy of a cep1 null mutant . The suppressing plasmids contained either CEP1 or DNA derived from the PHO4 locus . Subcloning experiments confirmed that suppression correlated with increased dosage of PHO4 . PHO4c, pho80 and pho84 mutations, all of which lead to constitutive activation of the PHO4 transcription factor, also suppressed cep1 methionine auxotrophy . The suppression appeared to be a direct effect of PHO4, not a secondary effect of PHO regulon derepression, and was PHO2-dependent . Spontaneously arising extragenic suppressors of cep1 methionine auxotrophy were also isolated; approximately one-third of them were alleles of pho80 . While PHO4 overexpression suppressed the methionine auxotrophy of a cep1 mutant, CEP1 overexpression failed to suppress the phenotype of a pho4 mutant; however, a cep1 null mutation suppressed the low inorganic phosphate growth deficiency of a pho84 mutant . The results may suggest that phosphate and sulfate metabolism are cross-regulated.

Genetics, 1992 Sep, 132(1), 39 - 51
A colony color assay for Saccharomyces cerevisiae mutants defective in kinetochore structure and function; Perier F et al.; We have designed a colony color assay for monitoring centromere DNA-protein interactions in yeast (Saccharomyces cerevisiae) . The assay is based on the ability of centromere DNA sequences to block (in cis) transcription initiated from a hybrid CEN-GAL1 promoter . Using a IacZ reporter gene under control of the CEN-GAL1 promoter, we screened colonies derived from mutagenized cells for a blue color phenotype indicative of derepression of the hybrid construct . A limited screen in which a 61-bp CEN11 DNA fragment containing an intact CDEIII subregion plus flanking sequences was used as the "pseudo-operator" led to the identification of mutations (blu) in three complementation groups . The blu1 mutants exhibited a decrease in activity of the major CEN DNA-binding proteins in vitro . The BLU1 gene was shown to be identical to the previously isolated SPT3 gene, known to be involved in the transcriptional regulation of a subset of yeast genes . Our results indicate that the BLU1/SPT3 gene product may also be required to maintain optimal levels of functional centromere DNA-binding proteins.

Genetics, 1992 Sep, 132(1), 113 - 23
Analysis of a gene conversion gradient at the HIS4 locus in Saccharomyces cerevisiae; Detloff P et al.; Heteroduplexes formed between genes on homologous chromosomes are intermediates in meiotic recombination . In the HIS4 gene of Saccharomyces cerevisiae, most mutant alleles at the 5' end of the gene have a higher rate of meiotic recombination (gene conversion) than mutant alleles at the 3' end of the gene . Such gradients are usually interpreted as indicating a higher frequency of heteroduplex formation at the high conversion end of the gene . We present evidence indicating that the gradient of conversion at HIS4 primarily reflects the direction of mismatch repair rather than the frequency of heteroduplex formation . We also identify a site located between the 5' end of HIS4 and the 3' end of BIK1 that stimulates heteroduplex formation at HIS4 and BIK1.

Mutat Res, 1992 Sep, 269(1), 97 - 105
Toxicological significance of dog liver cytochrome P-450: examination with the enzyme expressed in Saccharomyces cerevisiae using recombinant expression plasmid; Fukuta H et al.; A complementary DNA (cDNA) coding for a form of beagle dog cytochrome P-450 (Dah1), which is the orthologue to the CYP1A1 cDNA of rat, mouse and human, was inserted between the alcohol dehydrogenase (ADH) promoter and terminator regions of the yeast expression vector pAAH5 . On introduction of the resulting recombinant plasmid pDC-1, Saccharomyces cerevisiae strain AH22 cells synthesized up to 1.5 x 10(5) molecules per cell of cytochrome P-450 protein (P-450(Dah1)) . The carbon monoxide-bound reduced form of P-450(Dah1) showed an absorption peak at 447 nm and specific content of P-450(Dah1) was about 0.1 nmole P-450 per mg of microsomal protein . P-450(Dah1) cross-reacted with antibodies to rat P-448-H (CYP1A2) and dog P-450-D2 (CYP1A2) . P-450(Dah1) activated 2-amino-3-methyl-imidazo{4,5-f}quinoline (IQ) and 2-amino-3,4-dimethylimidazo{4,5-f}quinoline (MeIQ) most efficiently in the umu test and exhibited a high activity of aryl hydrocarbon hydroxylase toward benzo{a}pyrene.

Yeast, 1992 Sep, 8(9), 777 - 85
The sequence of an 8 kb segment on the left arm of chromosome II from Saccharomyces cerevisiae identifies five new open reading frames of unknown functions, two tRNA genes and two transposable elements; Skala J et al.; The DNA sequence of an 8079 bp ClaI fragment located at 40 kb from the centromere on the left arm of chromosome II from Saccharomyces cerevisiae has been determined . Sequence analysis reveals five new open reading frames, tRNA(Gly) and tRNA(Leu) genes as well as sigma and truncated delta elements . The disruption of the three larger open reading frames shows that they are not essential for mitotic growth.

Mol Biol Cell, 1992 Sep, 3(9), 999 - 1013
A replication map of a 61-kb circular derivative of Saccharomyces cerevisiae chromosome III; Greenfeder SA et al.; Using two-dimensional agarose gel electrophoresis, we determined the replication map of a 61-kb circular derivative of Saccharomyces cerevisiae chromosome III . The three sites of DNA replication initiation on the ring chromosome are specific and coincide with ARS elements . The three origins are active to different degrees; two are used > 90% of the time, whereas the third is used only 10-20% of the time . The specificity of these origins is shown by the fact that only ARS elements were competent for origin function, and deletion of one of the ARS elements removed the corresponding replication origin . The activity of the least active origin was not increased by deletion of the nearby highly active origin, demonstrating that the highly active origin does not repress function of the relatively inactive origin . Replication termination on the ring chromosome does not occur at specific sites but rather occurs over stretches of DNA ranging from 3 to 10 kb . A new region of termination was created by altering the sites of initiation . The position of the new termination site indicates that termination is not controlled by specific cis-acting DNA sequences, but rather that replication termination is determined primarily by the positions at which replication initiates . In addition, two sites on the ring chromosome were found to slow the progression of replication forks through the molecule: one is at the centromere and one at the 3' end of a yeast transposable element.

Genetics, 1992 Sep, 132(1), 97 - 112
cis- and trans-acting suppressors of a translation initiation defect at the cyc1 locus of Saccharomyces cerevisiae; Pinto I et al.; The cyc1-362 mutant of Saccharomyces cerevisiae is deficient in iso-1-cytochrome c as a consequence of an aberrant ATG codon that initiates a short open reading frame (uORF) in the cyc1 transcribed leader region . We have isolated and characterized functional revertants of cyc1-362 in an effort to define cis- and trans-acting factors that can suppress the effect of the uORF . Genetic and DNA sequence analyses have defined three classes of revertants: (i) those that acquired point mutations in the upstream ATG (uATG), restoring iso-1-cytochrome c to its normal level; (ii) substitution of the normal A residue at position -1 relative to the uATG by either C or T, enhancing iso-1-cytochrome c production from approximately 2% to 6% (C) or 10% (T) of normal, indicating that the nucleotide immediately preceding the initiator codon can affect the efficiency of AUG start codon recognition and that purines are preferred over pyrimidines at this site; and (iii) extragenic suppressors that enhance iso-1-cytochrome c expression to 10-40% of normal while retaining the uATG . These suppressors are represented by five different genes, designated sua1-sua4 and sua6 . In contrast to the previously described sua7 and sua8 suppressors, they do not compensate for the uATG by affecting cyc1 transcription start site selection . Potential suppressor mechanisms are discussed.

Genetics, 1992 Sep, 132(1), 23 - 37
Analysis of mitotic and meiotic defects in Saccharomyces cerevisiae SRS2 DNA helicase mutants; Palladino F et al.; The hyper-gene conversion srs2-101 mutation of the SRS2 DNA helicase gene of Saccharomyces cerevisiae has been reported to suppress the UV sensitivity of rad18 mutants . New alleles of SRS2 were recovered using this suppressor phenotype . The alleles have been characterized with respect to suppression of rad18 UV sensitivity, hyperrecombination, reduction of meiotic viability, and definition of the mutational change within the SRS2 gene . Variability in the degree of rad18 suppression and hyperrecombination were found . The alleles that showed the severest effects were found to be missense mutations within the consensus domains of the DNA helicase family of proteins . The effect of mutations in domains I (ATP-binding) and V (proposed DNA binding) are reported . Some alleles of SRS2 reduce spore viability to 50% of wild-type levels . This phenotype is not bypassed by spo13 mutation . Although the srs2 homozygous diploids strains undergo normal commitment to meiotic recombination, this event is delayed by several hours in the mutant strains and the strains appear to stall in the progression from meiosis I to meiosis II.

Mol Cell Biol, 1992 Sep, 12(9), 3959 - 66
Substitutions in the hydrophobic core of the alpha-factor receptor of Saccharomyces cerevisiae permit response to Saccharomyces kluyveri alpha-factor and to antagonist; Marsh L; Mutations in the Saccharomyces cerevisiae alpha-factor receptor that lead to improved response to Saccharomyces kluyveri alpha-factor were identified and sequenced . Mutants were isolated from cells bearing randomly mutagenized receptor gene (STE2) plasmids by an in vivo screen . Five mutations lead to substitutions in hydrophobic segments in the core of the receptor (M54I, S145L, S145L-S219L, A229V, L255S-S288P) . Remarkably, strains expressing these mutant receptors exhibited positive pheromone responses to desTrp1,Ala3-alpha-factor, an analog that normally blocks these responses . The M54I mutation appeared to affect only ligand specificity . The other mutations conferred additional effects on signaling or recovery . Two mutants were more sensitive to alpha-factor than wild type (S145L, A229V) . One mutant was more sensitive to alpha-factor-induced cell cycle arrest initially, but then recovered more efficiently (S145L-S219L) . One mutant (L255S-S288P) conferred positive pheromone responses to alpha-factor as assayed by FUS1-lacZ reporter induction, but did not display growth arrest . The hydrophobic receptor core thus appears to control activation by some ligands and to play roles in aspects of signal transduction and recovery.

Mol Cell Biol, 1992 Sep, 12(9), 3857 - 64
A delay in the Saccharomyces cerevisiae cell cycle that is induced by a dicentric chromosome and dependent upon mitotic checkpoints; Neff MW et al.; Dicentric chromosomes are genetically unstable and depress the rate of cell division in Saccharomyces cerevisiae . We have characterized the effects of a conditionally dicentric chromosome on the cell division cycle by using microscopy, flow cytometry, and an assay for histone H1 kinase activity . Activating the dicentric chromosome induced a delay in the cell cycle after DNA replication and before anaphase . The delay occurred in the absence of RAD9, a gene required to arrest cell division in response to DNA damage . The rate of dicentric chromosome loss, however, was elevated in the rad9 mutant . A mutation in BUB2, a gene required for arrest of cell division in response to loss of microtubule function, diminished the delay . Both RAD9 and BUB2 appear to be involved in the cellular response to a dicentric chromosome, since the conditionally dicentric rad9 bub2 double mutant was highly inviable . We conclude that a dicentric chromosome results in chromosome breakage and spindle aberrations prior to nuclear division that normally activate mitotic checkpoints, thereby delaying the onset of anaphase.

Mol Cell Biol, 1992 Sep, 12(9), 3807 - 18
Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome; Johnson RE et al.; rad5 (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RAD5 is in the RAD6 epistasis group of DNA repair genes . To unambiguously define the function of RAD5, we have cloned the RAD5 gene, determined the effects of the rad5 deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein . Our genetic studies indicate that RAD5 functions primarily with RAD18 in error-free postreplication repair . We also show that RAD5 affects the rate of instability of poly(GT) repeat sequences . Genomic poly(GT) sequences normally change length at a rate of about 10(-4); this rate is approximately 10-fold lower in the rad5 deletion mutant than in the corresponding isogenic wild-type strain . RAD5 encodes a protein of 1,169 amino acids of M(r) 134,000, and it contains several interesting sequence motifs . All seven conserved domains found associated with DNA helicases are present in RAD5 . RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RAD18 and the RAG1 gene required for immunoglobin gene arrangement . A leucine zipper motif preceded by a basic region is also present in RAD5 . The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5 . Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed.

Biochim Biophys Acta, 1992 Aug 21, 1122(3), 311 - 6
Adenosine deaminase from Saccharomyces cerevisiae: kinetics and interaction with transition and ground state inhibitors; Lupidi G et al.; Several adenosine analogs, such as coformycin, 2'-deoxycoformycin and erythro-9-(3-nonyl-p-aminobenzyl)adenine (EHNA), which are strong inhibitors of mammalian adenosine deaminase, are much weaker inhibitors of the Saccharomyces cerevisiae enzyme . The specificity of the yeast enzyme is more restricted than that of mammalian adenosine deaminase, particularly towards the ribose moiety and around position 6 and 1 of the substrate . The sulphydryl group appears to be more masked in the yeast than in the mammalian enzyme . The kinetic effects of pH with adenosine substrate and with the inhibitor purine riboside are reported . The findings on specificity and pH kinetic effects can be interpreted in a model involving proton transfer from the -SH group of the enzyme to the N-1 atom of the substrate.

FEBS Lett, 1992 Aug 17, 308(2), 130 - 2
Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae; Albertyn J et al.; The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD+ oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saccharomyces cerevisiae strain H44-3D by affinity- and ion-exchange chromatography . SDS-PAGE indicated that the enzyme had a molecular mass of approximately 42,000 (+/- 1,000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer . The pH optimum for the reduction of dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pI of 7.4 . NADPH will not substitute for NADH as coenzyme in the reduction of DHAP . The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0 . Apparent Km values obtained were 0.023 and 0.54 mM for NADH and DHAP, respectively . NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity . Ki values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.

J Biol Chem, 1992 Aug 15, 267(23), 16417 - 23
Cloning and characterization of the gene encoding the IDH1 subunit of NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae; Cupp JR et al.; NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 and IDH2 . The gene encoding IDH2 was previously cloned and sequenced (Cupp, J.R., and McAlister-Henn, L . (1991) J . Biol . Chem . 266, 22199-22205), and in this paper we describe the isolation of a yeast genomic clone containing the IDH1 gene . A fragment of the IDH1 gene was amplified by the polymerase chain reaction method utilizing degenerate oligonucleotides based on tryptic peptide sequences of the purified subunit; this fragment was used to isolate a full length IDH1 clone . The nucleotide sequence of the IDH1 coding region was determined and encodes a 360-residue polypeptide including an 11-residue mitochondrial targeting presequence . Amino acid sequence comparison between IDH1 and IDH2 reveals a 42% sequence identity, and both IDH1 and IDH2 show approximately 32% identity to Escherichia coli NAD(P)(+)-dependent isocitrate dehydrogenase . To examine the function of the IDH1 subunit and to determine the metabolic role of NAD(+)-dependent isocitrate dehydrogenase the IDH1 gene was disrupted in a wild type haploid yeast strain and in a haploid strain lacking IDH2 . The IDH1 disruption strains expressed no detectable IDH1 as determined by Western blot analysis, and these strains were found to lack NAD(+)-dependent isocitrate dehydrogenase activity indicating that IDH1 is essential for a functional enzyme . Over-expression of IDH1 in a strain containing IDH2 restored wild type activity but did not result in increased levels of activity, suggesting that both IDH1 and IDH2 are required for a functional enzyme . Growth phenotype analysis of the IDH1 disruption strains revealed that they grew at a reduced rate on the nonfermentable carbon sources examined (glycerol, lactate, and acetate), consistent with NAD(+)-dependent isocitrate dehydrogenase performing a critical role in oxidative function of the citric acid cycle . In addition, the IDH1 disruption strains grew at wild type rates in the absence of glutamate, indicating that these strains are not glutamate auxotrophs.

J Biol Chem, 1992 Aug 15, 267(23), 16177 - 81
Unbalanced ribosome assembly in Saccharomyces cerevisiae expressing mutant 5 S rRNAs; Van Ryk DI et al.; Mutant 5 S rRNA genes were expressed in Saccharomyces cerevisiae to further define the function of the ribosomal 5 S RNA . RNA synthesis and utilization were assayed using previously constructed markers which have been shown to be functionally neutral and easily detected by gel electrophoresis . Most mutations were found not to affect the growth rate because they were poorly expressed or could be accommodated effectively in the ribosomal structure . Two of the mutants, Y5A99U56U57 and Y5U90i5 adversely affected cell growth as well as protein synthesis in vitro . Polyribosome profiles in both of these mutants were substantially shorter, and an analysis of the ribosomal subunit composition revealed a significant imbalance with a 25-35% excess in 40 S subunits . Kinetic analyses of RNA labeling indicated very low cellular levels of mutant RNA either because it was poorly expressed (Y5U90i5) or rapidly degraded before being incorporated into mature 60 subunits (Y5A99U56U57) . The results suggest that the 5 S RNA is required for the assembly of stable ribosomal 60 S subunits and raise the possibility that this RNA or, more likely, its corresponding ribonucleoprotein complex is critical for subunit assembly or even RNA processing.

Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7664 - 8
ATP hydrolysis by initiation factor 4A is required for translation initiation in Saccharomyces cerevisiae; Blum S et al.; Saccharomyces cerevisiae translation initiation factor eIF-4A, an RNA helicase of the Asp-Glu-Ala-Asp (DEAD) box protein family, was mutated in the putative ATP binding site and expressed in Escherichia coli . Mutant proteins with alanine at position 66 replaced by glycine {eIF-4A(A66G)} or valine {eIF-4A(A66V)} were purified from Escherichia coli extracts and analyzed in vitro for activity in ATP crosslinking, ATP hydrolysis, RNA helicase, and translation assays . The results show that in vitro ATP hydrolysis activity, RNA helicase activity, and translation activity of eIF-4A correlate with in vivo activity of the factor . Whereas eIF-4A(A66G) showed wild-type activity in all assays, eIF-4A(A66V) was active in ATP crosslinking but inactive in ATP hydrolysis and RNA helicase assays . In vitro translation was supported by wild-type eIF-4A and eIF-4A(A66G) but not by eIF-4A(A66V) . The results show that, for their translation, the majority of mRNAs from Saccharomyces cerevisiae including an mRNA with the initiator AUG positioned 8 nucleotides downstream of the cap structure require eIF-4A that is able to hydrolyze ATP.

Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7412 - 6
Purification of the heteromeric protein binding to the URS1 transcriptional repression site in Saccharomyces cerevisiae; Luche RM et al.; The protein that binds to the URS1 site situated upstream of many genes in Saccharomyces cerevisiae is a central element responsible for global negative control of transcription in this organism . Among the genes whose expression is regulated by this protein are those that participate in nitrogen metabolism, carbon metabolism, electron transport, inositol metabolism, heat shock response, meiosis, and sporulation . This factor, binding URS1 factor (BUF), has been purified and shown to be a heteromeric protein composed of 37.5- and 73.5-kDa monomers . The heteromeric form of BUF is stably maintained both in solution and bound to its DNA target site.

Mol Cell Biol, 1992 Aug, 12(8), 3563 - 72
The N-terminal 96 residues of MCM1, a regulator of cell type-specific genes in Saccharomyces cerevisiae, are sufficient for DNA binding, transcription activation, and interaction with alpha 1; Bruhn L et al.; MCM1 performs several functions necessary for its role in regulating cell type-specific gene expression in the yeast Saccharomyces cerevisiae: DNA binding, transcription activation, and interaction with coregulatory proteins such as alpha 1 . We analyzed a set of MCM1 deletion derivatives using in vivo reporter gene assays and in vitro DNA-binding studies to determine which regions of MCM1 are important for its various activities . We also analyzed a set of LexA-MCM1 hybrids to examine the ability of different segments of MCM1 to activate transcription independent of MCM1's DNA-binding function . The first third of MCM1 {MCM1(1-96)}, which includes an 80-residue segment homologous to the mammalian serum response factor, was sufficient for high-affinity DNA binding, for activation of reporter gene expression, and for interaction with alpha 1 in vitro and in vivo . However, the ability of MCM1(1-96) to activate transcription and to interact with alpha 1 was somewhat reduced compared with wild-type MCM1 {MCM1(1-286)} . Optimal interaction with alpha 1 required residues 99 to 117, in which 18 of 19 amino acids are acidic in character . Optimal transcription activation required a segment from residues 188 to 286, in which 50% of the amino acids are glutamine . Deletion of this segment from MCM1 reduced expression of reporter genes by about twofold . Moreover, LexA-MCM1 hybrids containing this segment were able to activate expression of reporter genes that rely on LexA binding sites as potential upstream activation sequences . Thus, glutamine-rich regions may contribute to the activation function of yeast transcription activators, as has been suggested for glutamine-rich mammalian proteins such as Sp1.

Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7013 - 6
Saccharomyces cerevisiae sec59 cells are deficient in dolichol kinase activity; Heller L et al.; The temperature-sensitive Saccharomyces cerevisiae mutant sec59 accumulates inactive and incompletely glycosylated protein precursors in its endoplasmic reticulum at the restrictive temperature . O-mannosylation and glycosyl phosphatidylinositol membrane anchoring of protein are also abolished, consistent with a deficiency in dolichyl phosphate mannose . Membranes prepared from sec59 cells that had been shifted to the restrictive temperature, however, made normal amounts of dolichyl phosphate mannose when exogenous dolichyl phosphate was supplied, but dolichyl phosphate mannose synthesis was severely depressed in the absence of exogenous dolichyl phosphate . Quantitative measurements of dolichyl phosphate in sec59 cells showed that the levels were decreased to 48% of wild type at the permissive temperature and to less than 10% at the restrictive temperature . Assays of enzymes from the dolichyl phosphate synthetic pathway, cis-prenyltransferase and dolichyl pyrophosphate phosphatase, gave wild-type levels . However, dolichol kinase activity was greatly decreased . When sec59 cells were transformed with a plasmid that overexpresses the wild-type gene, dolichol kinase activity increased 10-fold over wild-type levels . These results strongly suggest that the sec59 gene encodes dolichol kinase.

Nucleic Acids Res, 1992 Aug 11, 20(15), 3925 - 31
Molecular cloning of RAD16, a gene involved in differential repair in Saccharomyces cerevisiae; Bang DD et al.; We have cloned the RAD16 gene of Saccharomyces cerevisiae and determined its nucleotide sequence . The gene complements the UV sensitivity of a rad16 mutant and restores the ability to repair the transcriptionally inactive HML alpha locus that is absent in this mutant . Disruption mutants that were constructed using the cloned gene are viable and UV sensitive and show no detectable growth defect . Moreover, such a mutant is deficient for repair of the HML alpha locus . The nucleotide sequence shows that the gene codes for a protein of 790 amino acids that has two potential zinc binding domains and shares homology with two other yeast proteins: the RAD54 gene product involved in recombinational repair and SNF2, a transcription factor that possibly functions in transcription activation through an interaction with chromatin components that allows access of other factors involved in transcription . The role of RAD16 in the repair of HML alpha might be to change the chromatin structure of silenced genes to provide access for excision repair enzymes.

Nucleic Acids Res, 1992 Aug 11, 20(15), 3897 - 904
Splice site selection by intron aI3 of the COX1 gene from Saccharomyces cerevisiae; Winter AJ et al.; Interactions of the 5' and 3' splice sites with intron internal sequences of the yeast mitochondrial group I intron aI3 were studied using mutation analysis . The results can be fully explained by the splice guide model in which the splice sites are defined by the Internal Guide Sequence . No evidence was found for an alternative interaction between intron nucleotides preceding the 3' splice site and nucleotides in the vicinity of the core region as was found for the Tetrahymena intron . Our results also suggest that binding of the 5' and 3' splice site nucleotides to the IGS can not take place simultaneously . The intron must therefore undergo conformational changes as the reaction proceeds from the first step of self splicing, GTP attack at the 5' splice site, to exon ligation, the second step.

FEBS Lett, 1992 Aug 3, 307(3), 249 - 52
CDC25-dependent induction of inositol 1,4,5-trisphosphate and diacylglycerol in Saccharomyces cerevisiae by nitrogen; Schomerus C et al.; The addition of ammonium sulfate to starved yeast cells leads to a 3- to 4-fold rapid increase of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), the products of phosphoinositide-specific phospholipase C (PI-PLC) . This response is reduced by dissecting the RAS-activating Cdc25 protein, and is completely abolished by the cdc25-1 mutation even at permissive temperature . Starved cdc25-1 mutant cells have a strongly reduced IP3 content, but an at least 10-fold increased DAG level compared to the isogenic wild-type strain . NH4 does not stimulate cAMP synthesis, and glucose does not induce IP3 and DAG . Our data suggest that the Cdc25 protein controls a nitrogen-specific signalling pathway involving the effector PI-PLC, in addition to the glucose-induced activation of adenylyl cyclase (AC).

Genes Dev, 1992 Aug, 6(8), 1414 - 29
CIK1: a developmentally regulated spindle pole body-associated protein important for microtubule functions in Saccharomyces cerevisiae; Page BD et al.; A genetic screen was devised to identify genes important for spindle pole body (SPB) and/or microtubule functions . Four mutants defective in both nuclear fusion (karyogamy) and chromosome maintenance were isolated; these mutants termed cik (for chromosome instability and karyogamy) define three complementation groups . The CIK1 gene was cloned and characterized . Sequence analysis of the CIK1 gene predicts that the CIK1 protein is 594 amino acids in length and possesses a central 300-amino-acid coiled-coil domain . Two different CIK1-beta-galactosidase fusions localize to the SPB region in vegetative cells, and antibodies against the authentic protein detect CIK1 in the SPB region of alpha-factor-treated cells . Evaluation of cells deleted for CIK1 (cik1-delta) indicates that CIK1 is important for the formation or maintenance of a spindle apparatus . Longer and slightly more microtubule bundles are visible in cik1-delta strains than in wild type . Thus, CIK1 encodes a SPB-associated component that is important for proper organization of microtubule arrays and the establishment of a spindle during vegetative growth . Furthermore, the CIK1 gene is essential for karyogamy, and the level of the CIK1 protein at the SPB appears to be dramatically induced by alpha-factor treatment . These results indicate that molecular changes occur at the microtubule-organizing center (MTOC) as the yeast cell prepares for karyogamy and imply that specialization of the MTOC or its associated microtubules occurs in preparation for particular microtubule functions in the yeast life cycle.

J Cell Biol, 1992 Aug, 118(3), 619 - 29
Calmodulin concentrates at regions of cell growth in Saccharomyces cerevisiae; Brockerhoff SE et al.; Calmodulin was localized in Saccharomyces cerevisiae by indirect immunofluorescence using affinity-purified polyclonal antibodies . Calmodulin displays an asymmetric distribution that changes during the cell cycle . In unbudded cells, calmodulin concentrates at the presumptive site of bud formation approximately 10 min before bud emergence . In small budded cells, calmodulin accumulates throughout the bud . As the bud grows, calmodulin concentrates at the tip, then disperses, and finally concentrates in the neck region before cytokinesis . An identical staining pattern is observed when wild-type calmodulin is replaced with mutant forms of calmodulin impaired in binding Ca2+ . Thus, the localization of calmodulin does not depend on its ability to bind Ca2+ with a high affinity . Double labeling of yeast cells with affinity-purified anti-calmodulin antibody and rhodamine-conjugated phalloidin indicates that calmodulin and actin concentrate in overlapping regions during the cell cycle . Furthermore, disrupting calmodulin function using a temperature-sensitive calmodulin mutant delocalizes actin, and act1-4 mutants contain a random calmodulin distribution . Thus, calmodulin and actin distributions are interdependent . Finally, calmodulin localizes to the shmoo tip in cells treated with alpha-factor . This distribution, at sites of cell growth, implicates calmodulin in polarized cell growth in yeast.

Arch Biochem Biophys, 1992 Aug 1, 296(2), 569 - 74
Binding of Saccharomyces cerevisiae extracellular proteins to glucane; Mrsa V et al.; Interactions of Saccharomyces cerevisiae cell wall proteins with purified yeast glucane were studied . Using the beta-glucanase (BGL2 gene product) as the model cell wall protein, strong binding to glucane was demonstrated at pH lower than 7, while at pH higher than 8 the reaction did not occur . NaCl (2 M) did not influence the binding, while urea in concentrations higher than 4 M affected the interactions . It was also found that most other cell wall proteins, as well as intracellular proteins, reacted with glucane in the same way, showing that the interactions of proteins with glucane are rather nonspecific . Soluble periplasmic proteins invertase and acid phosphatase failed to react with glucane under the same conditions, indicating that these proteins have certain structural features preventing their interactions with glucane.

Genetics, 1992 Aug, 131(4), 851 - 66
Isolation of crt mutants constitutive for transcription of the DNA damage inducible gene RNR3 in Saccharomyces cerevisiae; Zhou Z et al.; Ribonucleotide reductase is an essential enzyme that catalyzes the rate limiting step for production of the deoxyribonucleotides required for DNA synthesis . It is encoded by three genes, RNR1, RNR2 and RNR3, each of which is inducible by agents that damage DNA or block DNA replication . To probe the signaling pathway mediating this DNA damage response, we have designed a general selection system for isolating spontaneous trans-acting mutations that alter RNR3 expression using a chromosomal RNR3-URA3 transcriptional fusion and an RNR3-lacZ reporter plasmid . Using this system, we have isolated 202 independent trans-acting crt (constitutive RNR3 transcription) mutants that express high levels of RNR3 in the absence of DNA damaging agents . Of these, 200 are recessive and fall into 9 complementation groups . In some crt groups, the expression of RNR1 and RNR2 are also elevated, suggesting that all three RNR genes share a common regulatory pathway . Mutations in most CRT genes confer additional phenotypes, among these are clumpiness, hydroxyurea sensitivity, temperature sensitivity and slow growth . Five of the CRT genes have been identified as previously cloned genes; CRT4 is TUP1, CRT5 is POL1/CDC17, CRT6 is RNR2, CRT7 is RNR1, and CRT8 is SSN6 . crt6-68 and crt7-240 are the first ts alleles of RNR2 and RNR1, respectively, and arrest with a large budded, cdc terminal phenotype at the nonpermissive temperature . The isolation of crt5-262, an additional cdc allele of POL1/CDC17, suggests for the first time that directly blocking DNA replication can provide a signal to induce the DNA damage response . crt2 mutants show a defect in basal level expression of RNR1-lacZ reporter constructs . These are the first mutants isolated in yeast that alter the regulation of DNA damage inducible genes and the identification of their functions sheds light on the DNA damage sensory network.

FEMS Microbiol Lett, 1992 Aug 1, 74(1), 99 - 104
Dual effect of monovalent cations on the glucose-induced transient increase in the rate of DMP influx into Saccharomyces cerevisiae; Borst-Pauwels GW et al.; The transient increase in the rate of 2-(4-di-methyl-aminostyryl)-1-ethylpyridinium (DMP) influx in Saccharomyces cerevisiae caused by addition of glucose to a suspension of non-metabolizing cells at relatively high pH can be prevented by monovalent cations . Their concentrations for half-maximum inhibition of DMP uptake are of the same order of magnitude as the corresponding Km values for their uptake into yeast cells . It is argued that the inhibition of DMP uptake by monovalent cations is caused by a fast depolarization of the cell membrane and a second further decrease in the rate of DMP uptake . The latter effect develops slowly with time and depends upon the extent of accumulation of the monovalent cations in the cells.

Eur J Biochem, 1992 Aug 1, 207(3), 931 - 6
Structural and functional characterisation of recombinant human haemoglobin A expressed in Saccharomyces cerevisiae; Coghlan D et al.; Recombinant human HbA, produced by co-expressing alpha-globin and beta-globin chains in the yeast Saccharomyces cerevisiae, has been characterised extensively both physically and functionally . Structural studies using N-terminal sequence analysis, peptide mapping, amino acid composition analysis and electrospray MS demonstrated that the recombinant protein was identical to standard HbA purified from erythrocytes . The functional properties of the recombinant protein were assessed using equilibrium and kinetic measurements of oxygen and carbon monoxide binding . The oxygen-binding studies demonstrated that the yeast-derived HbA behaved as a fully functional, cooperative tetramer (Hill coefficient, 2.9), exhibited a normal Bohr effect and response to phosphate, and displayed a rate of oxygen dissociation identical to that of the native human molecule . The recombinant protein also showed the same characteristics of carbon monoxide combination as the standard protein . These studies demonstrate that yeast provides an ideal system for the production of Hb for structural and functional analysis and a potentially useful source of HbA for formulation into a Hb-based oxygen carrier.

Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7227 - 31
Distinct processes mediate glycoprotein and glycopeptide export from the endoplasmic reticulum in Saccharomyces cerevisiae; Romisch K et al.; Protein and peptide export from the Saccharomyces cerevisiae endoplasmic reticulum was examined in vitro using the secretory protein pro-alpha-factor and a synthetic tripeptide containing the acceptor site for N-linked glycosylation as substrates . The release of both glycosylated pro-alpha-factor and glycotripeptide from the endoplasmic reticulum was dependent on cytosol, temperature, and ATP . Antibodies against two proteins essential for the formation of transport vesicles, Sec23p and p105, inhibited glyco-pro-alpha-factor exit from the endoplasmic reticulum but did not affect the release of the glycosylated tripeptide . Furthermore, in contrast to pro-alpha-factor, the exported glycopeptide was not associated with a membrane fraction and did not acquire Golgi-specific alpha(1-6)-linked mannose residues . We conclude that the glycosylated tripeptide leaves the yeast endoplasmic reticulum by a route different from the secretory pathway, possibly through an ATP-driven pump.

Appl Biochem Biotechnol, 1992 Aug, 36(2), 107 - 12
Uptake of ammonia by Saccharomyces cerevisiae carrying the plasmid pCYG4 related with ammonia assimilation; Lima Filho JL et al.; Batch culture experiments involving ammonia uptake in Saccharomyces cerevisiae BC55 pCYG4 have been carried out . This strain carries the plasmid pCYG4 that directs substantial overproduction of NADP-GDH, conferring an 11-fold increase in activity . The wild type cells had a specific growth rate greater than BC55 pCYG4 . The ammonia uptake was practically the same until 15 h of growth . However, the amount of ammonia hydroxide added during growth (60 h) was two and half times greater in the BC55 pCYG4 than wild type cells . The results suggest that the presence of the plasmid pCYG4 can increase the amount of ammonia taken by the cells, but not the amount of biomass.

Antonie Van Leeuwenhoek, 1992 Aug, 62(1-2), 95 - 108
The pheromone signal pathway in Saccharomyces cerevisiae; Konopka JB et al.; Haploid cells of the yeast Saccharomyces cerevisiae normally undergo a budding life cycle, but after binding the appropriate mating pheromone they undergo a different developmental pathway that leads to conjugation . This intercellular communication between the two mating types activates a signal transduction pathway that stimulates the diverse physiological changes required for conjugation, such as induction of cell surface agglutinins, cell division arrest in G1, morphogenesis to form a conjugation tube, and cell fusion . The components of this pathway include a G protein-coupled receptor, several protein kinases, and a pheromone-responsive transcription factor . The molecular mechanisms that transduce the pheromone signal are remarkably similar to the mechanisms of hormone signaling used in multicellular organisms . Thus, the analysis of the pheromone signal pathway in yeast directly contributes to the study of cell growth and development in other eukaryotic organisms.

Yeast, 1992 Aug, 8(8), 667 - 8
Efficient selection of phleomycin-resistant Saccharomyces cerevisiae transformants; Wenzel TJ et al.; The recently described dominant yeast marker Tn5ble confers phleomycin resistance on the yeast Saccharomyces cerevisiae (Gatignol, Baron and Tiraby, 1987 . Mol . Gen . Genet . 207, 342-348) . Incubation in non-selective medium prior to selection is critical, however, for getting phleomycin-resistant transformants . A 6-h incubation period was found to give optimal transformation frequencies, up to 10(5) transformants/micrograms plasmid, comparable to selection for uracil prototrophy (Ura+).

Yeast, 1992 Aug, 8(8), 629 - 33
A screening procedure for the intracellular expression of native proteins by Saccharomyces cerevisiae: discrimination of diphtheria toxin-resistant mutants; Donovan MG et al.; A general method is described for screening Saccharomyces cerevisiae colonies for the intracellular expression of native proteins . Colonies are replicated onto nitrocellulose membranes and yeast cell walls are removed enzymatically . The resulting spheroplasts are rapidly lysed by placing chromatography paper soaked in hypotonic buffer on the membranes . Intracellular proteins released by spheroplast lysis are bound in situ to the nitrocellulose under non-denaturing conditions and potentially can be examined using enzymatic or immunologic methods . For example, in the present study colonies were screened for the presence of elongation factor 2 (EF-2) that can be {32P}ADP-ribosylated by diphtheria toxin and {32P}NAD+ . Recognition by the toxin requires the presence in EF-2 of the unique post-translationally modified histidine derivative, diphthamide . The procedure described here reliably discriminates between wild-type yeast colonies and mutant colonies that do not synthesize diphthamide . In addition to facilitating the study of diphthamide biosynthesis in yeast, the more general application of this procedure will enable the screening of colonies with assays that require native proteins.

Glycoconj J, 1992 Aug, 9(4), 209 - 16
Comparison of the carbohydrate moieties of recombinant soluble Fc epsilon receptor (sFc epsilon RII/sCD23) expressed in Saccharomyces cerevisiae and Chinese hamster ovary cells . Different O-glycosylation sites are used by yeast and mammalian cells; Kalsner I et al.; Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFc epsilon RII/sCD23) was produced in Saccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis . Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases . The results revealed that sFc epsilon RII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(alpha 2-3)Gal(beta 1-3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only alpha-mannosyl residues . It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.

Glycobiology, 1992 Aug, 2(4), 369 - 72
Topology of ER processing alpha-mannosidase of Saccharomyces cerevisiae; Grondin B et al.; The yeast specific alpha-mannosidase which converts Man9GlcNAc to a single isomer of Man8GlcNAc is involved in N-linked oligosaccharide processing in the endoplasmic reticulum (ER) . Sequence analysis of the structural gene for this enzyme suggested that it is a type II transmembrane protein (Camirand et al., 1991) . To firmly establish its membrane topology, the gene was transcribed in vitro and translation was performed in a reticulocyte lysate with and without dog pancreas microsomal membranes . Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of {35S}methionine-labelled products showed that the largest band formed corresponded in size to the 63 kDa peptide expected from the alpha-mannosidase gene product . It was transformed into a 4 kDa larger endoglycosidase H-sensitive band in the presence of microsomal membranes . This glycosylated translation product was completely protected from proteinase K digestion in the absence of detergent . These results demonstrate that the yeast ER alpha-mannosidase is a type II membrane protein, like Golgi enzymes involved in N-linked glycosylation.

Mol Microbiol, 1992 Aug, 6(16), 2339 - 48
The effects of 5'-capping, 3'-polyadenylation and leader composition upon the translation and stability of mRNA in a cell-free extract derived from the yeast Saccharomyces cerevisiae; Gerstel B et al.; A new modular expression system was developed to direct the in vitro synthesis of defined transcripts that were used as templates for translation in yeast cell-free extracts . The system was used to examine the influence of 5'-capping, 3'-polyadenylation and leader sequence upon the translation and stability of the synthetic Tn9 cat (chloramphenicol acetyl transferase), yeast PGK (phosphoglycerate kinase) and yeast HSP26 (heat-shock protein 26) mRNAs . The addition of a methylated cap (m7Gppp) or of a poly(A) tail enhanced translation and stabilized the mRNA . The dependence of translation upon capping was reduced in the presence of the HSP26 leader sequence . This may indicate the existence of a translational mechanism that enhances cap-independent translation . The enhancement of the translation and stability of mRNA was relatively insensitive to changes in the position of the poly(A) tail relative to the reading frame.

Mutat Res, 1992 Aug, 282(4), 235 - 9
Mutagenicity of methyl methanesulfonate and cyclophosphamide in resting and growing Saccharomyces cerevisiae D7 cells; Monaco M et al.; In order to evaluate the optimal experimental conditions and to identify the best growth phase for yeast genotoxicity studies, comparative experiments were performed with stationary and growing cells . Methyl methanesulfonate (MMS) and cyclophosphamide (CP) were used as chemical mutagens and strain D7 of Saccharomyces cerevisiae as detector of induced mitotic gene conversion (trp+ convertants) and point reverse mutation (ilv+ revertants) in log or stationary phase cells after either 4 or 16 h of treatment . The highest MMS-induced toxicity and genotoxicity were observed after 16 h of exposure in a suspension test with log phase cells, which is consistent with the greater permeability and sensitivity of growing yeast cells . The maximal induction of genetic effects and toxicity by CP was conversely obtained after 16 h of treatment in stationary phase cells . This may be ascribed to the greater ability of detoxication of growing cells as compared to resting cells . Our results suggest that in evaluating the mutagenicity of chemicals in yeast systems it is important to consider factors such as growth phase and exposure time.

Genetics, 1992 Aug, 131(4), 821 - 32
Ty element-induced temperature-sensitive mutations of Saccharomyces cerevisiae; Kawakami K et al.; Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3 . In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures . Five mutations were isolated and named hit for high temperature growth . The hit1-1 mutation was located on chromosome X and conferred the pet phenotype . Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures . The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene . The hit4-1 mutation was located on chromosome XIII . These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response . The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein . Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family . In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus . The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes . The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome . This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein . The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.

Genetics, 1992 Aug, 131(4), 791 - 801
Isolation and characterization of SUA5, a novel gene required for normal growth in Saccharomyces cerevisiae; Na JG et al.; We have identified the sua5 locus as a suppressor of an aberrant ATG codon located in the leader region of the cyc1 gene . The sua5-1 allele enhances the iso-1-cytochrome c steady state level in the cyc1-1019 mutant from 2% to approximately 60% of normal (Cyc+) and also confers a marked slow growth (Slg-) phenotype . Suppression is not a consequence of altered transcription initiation at the cyc1 locus . The SUA5 wild-type gene was isolated and sequenced, revealing an open reading frame (ORF) encoding a potential protein of 46,537 Da . SUA5 transcript analyses were consistent with expression of the predicted ORF and Sua5 antisera detected a protein with an apparent molecular mass of 44 kDa . SUA5 was mapped to chromosome VII, immediately adjacent to the PMR1 gene . Hybridization analysis revealed the presence of a related gene on chromosome XII . Neither the SUA5 DNA sequence nor deduced amino acid sequence showed homology to any sequences in the data banks . Disruption of SUA5 conferred the same Cyc+ and Slg- phenotypes as the sua5-1 suppressor, which is the result of a missense mutation, encoding a Ser107----Phe replacement . In addition, sua5 null mutants lack cytochrome a.a3 and fail to grow on lactate or glycerol medium . These results define SUA5 as a new gene encoding a novel protein that is necessary for normal cell growth.

J Cell Biol, 1992 Aug, 118(3), 531 - 40
Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae; Seeger M et al.; The role of clathrin in retention of Golgi membrane proteins has been investigated . Prior work showed that a precursor form of the peptide mating pheromone alpha-factor is secreted by Saccharomyces cerevisiae cells which lack the clathrin heavy chain gene (CHC1) . This defect can be accounted for by the observation that the Golgi membrane protein Kex2p, which initiates maturation of alpha-factor precursor, is mislocalized to the cell surface of mutant cells . We have examined the localization of two additional Golgi membrane proteins, dipeptidyl aminopeptidase A (DPAP A) and guanosine diphosphatase (GDPase) in clathrin-deficient yeast strains . Our findings indicate that DPAP A is aberrantly transported to the cell surface but GDPase is not . In mutant cells carrying a temperature-sensitive allele of CHC1 (chc1-ts), alpha-factor precursor appears in the culture medium within 15 min, and Kex2p and DPAP A reach the cell surface within 30 min, after imposing the nonpermissive temperature . In contrast to these immediate effects, a growth defect is apparent only after 2 h at the nonpermissive temperature . Also, sorting of the vacuolar membrane protein, alkaline phosphatase, is not affected in chc1-ts cells until 2 h after the temperature shift . A temperature-sensitive mutation which blocks a late stage of the secretory pathway, sec1, prevents the appearance of mislocalized Kex2p at the cell surface of chc1-ts cells . We propose that clathrin plays a direct role in the retention of specific proteins in the yeast Golgi apparatus, thereby preventing their transport to the cell surface.

Yeast, 1992 Aug, 8(8), 673 - 80
Saccharomyces cerevisiae contains a homolog of human FKBP-13, a membrane-associated FK506/rapamycin binding protein; Partaledis JA et al.; FKB2 encodes a homolog of human FKBP-13, a membrane-associated binding protein for the immunosuppressants FK506 and rapamycin . FKB2 is located on the right arm of chromosome IV and contains an open reading frame of 135 amino acids, of which the first 17 residues comprise a putative hydrophobic leader peptide . Yeast FKBP-13 is homologous to human FKBP-13 (52% amino acid identity) and to FKBP-12, the major cytosolic receptor for FK506 . In the alignment of FKBP-13 and FKBP-12 sequences, there are 28 invariant residues . Among these conserved residues are those that comprise the drug binding and peptidyl-prolyl cis-trans isomerase active site of FKBP-12 . The phylogenetic conservation of the FKBP family suggest that the proteins are involved in a basic cellular function.

Nucleic Acids Res, 1992 Jul 25, 20(14), 3725 - 30
The PCF1-1 mutation increases the activity of the transcription factor (TF) IIIB fraction from Saccharomyces cerevisiae; Willis I et al.; PCF1-1 is a dominant suppressor of a tRNA gene A block promoter mutation (A19) in Saccharomyces cerevisiae . Transcriptional activation by PCF1-1 was examined in vitro using whole-cell extracts and purified factors derived from mutant and wild-type strains . These experiments show that PCF1 is a general activator of RNA polymerase III (pol III) gene transcription . The transcription of all pol III genes analyzed to date, including type I and numerous type II genes, is increased 3-7 fold in mutant cell extracts . Single round transcription assays indicate that the PCF1-1 mutation increases the number of functional preinitiation complexes and suggest that this is achieved by increasing the intrinsic activity of the encoded product rather than its amount . Point mutations throughout the A block of the sup3-e gene and numerous B block mutations fail to abolish transcriptional activation suggesting that interactions between TFIIIC and the internal promoter are unaffected by PCF1-1 . Moreover, TFIIIC purified from the mutant strain is incapable of conferring PCF1-1 transcriptional activity to a reaction in which the remaining components are wild-type . In contrast, the activity of the TFIIIB fraction is increased in PCF1-1 extracts and can reconstitute mutant levels of transcription when added to wild-type TFIIIC and polymerase . We conclude that PCF1 is a component or regulator of TFIIIB.

J Biol Chem, 1992 Jul 25, 267(21), 15224 - 8
Coordinate regulation of glycogen metabolism in the yeast Saccharomyces cerevisiae . Induction of glycogen branching enzyme; Thon VJ et al.; The yeast glycogen branching enzyme (EC 2.4.1.18) is shown to be induced in batch culture simultaneously with the onset of intracellular glycogen accumulation . The branching enzyme structural gene (GLC3) has been cloned . Its predicted amino acid sequence is very similar to procaryotic branching enzymes . Northern analysis indicates that GLC3 mRNA abundance increases in late exponential growth phase coincident with glycogen accumulation . Disruption of the branching enzyme structural gene establishes that branching enzyme activity is an absolute requirement for maximal glycogen synthesis.

J Biol Chem, 1992 Jul 25, 267(21), 14879 - 83
Cytoplasmic and mitochondrial tRNA nucleotidyltransferase activities are derived from the same gene in the yeast Saccharomyces cerevisiae; Chen JY et al.; ATP (CTP):tRNA-specific tRNA nucleotidyltransferase is an enzyme required for the synthesis of functional tRNAs in eukaryotic cells . Neither the tRNA genes in the nucleus nor in organelles encode the CCA end, so it must be added post-transcriptionally . The gene that codes for the enzyme that adds the CCA end to nuclear coded tRNAs in Saccharomyces cerevisiae has been isolated (Aebi, M., Kirchner, G., Chen, J.-Y., Vijayraghavan, U., Jacobson, A., Martin, N . C., and Abelson, J . (1990) J . Biol . Chem . 265, 16216-16220) . We now demonstrate that there is a mitochondrial tRNA nucleotidyltransferase activity in yeast and that it is a matrix enzyme . A comparison of purified mitochondrial enzyme with its cytoplasmic counterpart revealed no differences . These results suggest that proteins responsible for this step in the maturation of tRNAs in the nucleus and mitochondria might be identical and coded by the same nuclear gene . Accumulation of shortened mitochondrial as well as cytoplasmic tRNAs in a strain with a temperature-sensitive tRNA nucleotidyltransferase is consistent with this hypothesis . Alteration of the wild type gene such that amino-terminal truncated proteins are produced leads to a defect in mitochondrial function and a decrease in mitochondrial nucleotidyltransferase activity . This provides a direct demonstration that one gene provides this enzyme activity for the biosynthesis of tRNAs in both the nuclear/cytoplasmic and mitochondrial compartments in yeast.

J Mol Biol, 1992 Jul 20, 226(2), 335 - 47
Transcriptional commitment of mitochondrial RNA polymerase from Saccharomyces cerevisiae; Biswas TK; The transcriptional commitment of mitochondrial RNA (mtRNA) polymerase and the conditions required for the formation of a stable ternary complex have been determined by in vitro transcription study . Four different transcription complexes were made in vitro by incubating purified mtRNA polymerase, cloned synthetic mitochondrial promoters and selective ribonucleotides . The responses of these complexes to heparin, an inhibitor of unbound mtRNA polymerase, have been examined to determine their involvement in transcription . This study leads to the following observations . (1) Under normal reaction conditions, 40 nM-heparin completely inhibited mitochondrial transcription . (2) A preinitiation mitochondrial DNA-RNA polymerase complex (complex 0) showed partial resistance to heparin (approximately 25% resistant to 40 nm-heparin) when heparin and ribonucleoside triphosphates (rNTPs) were added together to the preformed complex . This complex was rapidly inactivated when preincubated with heparin before the addition of rNTPs . (3) The early initiation (complexes 2 and 4) containing DNA template, RNA polymerase and a short RNA product showed more resistance (approx . 40 to 50%) to 40 nM-heparin but destabilized upon further incubation with heparin before addition of the rest of the rNTPs . (4) After generation of ten or more phosphodiester bonds (complex 11), the early transcription complex is converted into a stable initiation complex, leading to the polymerase consignment to elongation . On the basis of stability and heparin sensitivity, three initial steps of mitochondrial transcription have been defined: polymerase-promoter interaction, initiation, and the transition from initiation to elongation . The formation of preinitiation complex is the rate-limiting step t 1/2 approx . 50 s), whereas the initiation and elongation reactions are very fast processes (t 1/2 greater than 5 s) in mitochondrial transcription.

Biochem Biophys Res Commun, 1992 Jul 15, 186(1), 467 - 74
In vitro interaction between Saccharomyces cerevisiae CDC25 and RAS2 proteins; Baroni MD et al.; In Saccharomyces cerevisiae the CDC25 protein is a positive regulator of RAS/cAMP pathway {1-4}, enhancing the GDP-releasing rate of RAS2 protein {5} . In this work we have tried to detect a direct interaction between CDC25 and RAS2 gene products . The results indicate that both the whole RAS2 protein and a truncated version that lacks approximately 25 C-terminal residues interact specifically with the CDC25 protein . On the contrary, a derivative of RAS2 that lacks the 112 C-terminal residues as well as the p21TI-ras is not able to bind the CDC25 protein in our assay conditions . The 310 C-terminal aminoacids of CDC25 bind RAS2 while a C-terminus deletion within this aminoacid stretch abolishes the binding . The possible physiological significance of these findings is discussed.

J Photochem Photobiol B, 1992 Jul 15, 14(3), 231 - 45
Inactivation and mutation induction in Saccharomyces cerevisiae exposed to simulated sunlight: evaluation of action spectra; Schenk-Meuser K et al.; The effectiveness of polychromatic light irradiation was investigated for haploid yeast cells . Inactivation and mutation induction were measured in both a RAD-wildtype strain and an excision-repair defective strain . The behaviour of vegetative "wet" cells was compared to that of dehydrated cells . The aim of the study was to assess the interaction of UVC with other wavelengths in cells of different states of humidity . The irradiation procedure was therefore carried out using a solar simulator either with full spectrum or with a UVC-blocking filter (modified sunlight) added . The results were analysed on the basis of separately determined action spectra . The summation of the efficiency of individual wavelengths was compared to the values obtained from polychromatic irradiation . It is shown that the effects caused by the whole-spectrum irradiation in wet cells can be predicted sufficiently from the calculation, while dried wildtype cells exhibit higher mutation rates . Thus it can be assumed that drying-specific damage leads to lethal and mutagenic lesions which are processed in different ways, causing a synergistic behaviour in mutation induction . Irradiation of vegetative cells with modified sunlight (UVC-) results in less inactivation and lower mutation rates than were calculated . From these results it can be concluded that this antagonistic behaviour is caused by the interaction of near-UV photoproducts.

FEMS Microbiol Lett, 1992 Jul 15, 73(3), 271 - 6
Construction of Saccharomyces cerevisiae strains that accumulate relatively low concentrations of trehalose, and their application in testing the contribution of the disaccharide to stress tolerance; Attfield PV et al.; Genetically related diploid strains of Saccharomyces cerevisiae that accumulate varied amounts of trehalose during starvation for nitrogen have been constructed . Strains that produced greater than 5% trehalose (dry cell weight) were more tolerant of thermal, or freeze-thaw stresses than strains that produced less than 4% trehalose . Thus trehalose appears to play a role in stress tolerance of yeast . The significance of these results is that, for the first time, a series of related, unmutated strains have been used to test the effect of trehalose on thermotolerance . Previous studies employed either heat shock treatment, or mutated strains to provide trehalose variations, and as such the contribution of the disaccharide to stress tolerance could not necessarily be separated from other factors such as heat shock proteins.

Biochim Biophys Acta, 1992 Jul 13, 1122(1), 85 - 92
Purification, characterization and preliminary X-ray study of fumarase from Saccharomyces cerevisiae; Keruchenko JS et al.; Fumarase (fumarate hydratase, EC 4.2.1.2) from Saccharomyces cerevisiae has been purified to homogeneity by a method including acetone fractionation, DEAE ion-exchange and dye-sorbent affinity chromatography . The suggested method allows fumarase purification with a yield higher than 60% and may be used to obtain large enzyme quantities . The native protein consists of four subunits with a approximately 50 kDa molecular mass each and has an isoelectric point at pH 6.5 +/- 0.3 . The equilibrium constant for fumarate hydration is about 4.3 (25 degrees C, pH 7.5), the Michaelis constants for fumarate and 1-malate are approximately 30 microM and approximately 250 microM, respectively . The enzyme is activated by substrates and multivalent anions, the activation seems to be of a non-competitive type . The fumarase complex with meso-tartaric acid has been crystallized by the vapor diffusion method . The unit cell parameters are a = 93.30, b = 94.05 and c = 106.07 A, space group P2(1)2(1)2(1) . The unit cell contains 2 protein molecules . The crystals diffract to at least 2.6 A resolution and are suitable for X-ray structure analysis.

FEBS Lett, 1992 Jul 6, 305(3), 244 - 8
Upstream activation element of the PH03 gene encoding for thiamine-repressible acid phosphatase in Saccharomyces cerevisiae; Nosaka K et al.; The PH03 gene of Saccharomyces cerevisiae encodes thiamine-repressible acid phosphatase and requires the positively acting regulatory protein THI2 for its expression . Deletion analysis of the 5'-flanking region of PH03 gene revealed that an activating region located at nucleotide position -234 to -215 relative to the translation initiation codon is required for the expression and sensitivity to thiamine . A chemically synthesized DNA fragment covering -234 to -215 showed a significant level of expression when inserted in front of the PH03 promoter lacking the activating region . Electrophoretic mobility shift assay demonstrated the presence of proteins that bound to the above DNA fragment in the nuclear extract from cells grown in thiamine-free medium . These findings suggested that this region between -234 and -215 acts as an upstream activation element of the PH03 gene that can interact with regulatory proteins.

J Biol Chem, 1992 Jul 5, 267(19), 13200 - 4
Purification, gene cloning, and gene disruption of the transcription elongation factor S-II in Saccharomyces cerevisiae; Nakanishi T et al.; Saccharomyces cerevisiae S-II was purified to near homogeneity as a protein stimulating RNA polymerase II . Four of seven lysyl endopeptidase-digested fragments of S-II were located in the PPR2 sequence reported previously . Analysis of a genomic clone of S-II revealed that S-II and PPR2 are the same protein consisting of 309 amino acid residues, and frame shifts were found in the sequence of PPR2 gene reported previously . Yeast S-II and mouse S-II showed high similarity in their amino acid sequences, especially in their amino-terminal and carboxyl-terminal regions . A gene disruption experiment showed that an S-II null mutant was not lethal under usual growth conditions, indicating that S-II is not essential for the growth of yeast.

J Biol Chem, 1992 Jul 5, 267(19), 13175 - 9
Active site topology of Saccharomyces cerevisiae lanosterol 14 alpha-demethylase (CYP51) and its G310D mutant (cytochrome P-450SG1); Tuck SF et al.; Incubation of phenyldiazene (PhN = NH) with lanosterol 14 alpha-demethylase, a cytochrome P-450 enzyme (CYP51) that oxidatively removes the 14 alpha-methyl group of lanosterol, results in the appearance of a 478-nm band indicative of phenyl-iron complex formation . In situ oxidation of the phenyl-iron complex by ferricyanide yields exclusively the N-phenylprotoporphyrin IX regioisomer with the phenyl group on the nitrogen of pyrrole ring C (NC) . The biphenyl-iron complex formed in the analogous reaction of the enzyme with biphenyldiazene similarly rearranges on treatment with ferricyanide to the NC regioisomer of N-biphenylprotoporphyrin IX . The active site cavity must therefore be at least 10 A high directly above the iron atom and pyrrole ring C of the heme group, and lanosterol binds to the enzyme in the region above pyrrole ring C . Phenyl-iron complex formation is not detected spectroscopically with cytochrome P-450SG1, a catalytically inactive G310D mutant of lanosterol 14 alpha-demethylase in which the sixth iron coordination site is thought to be occupied by an imidazole ligand . Nevertheless, oxidation of the phenyldiazene-treated enzyme with ferricyanide provides the NA and NC regioisomers of N-phenylprotoporphyrin IX in a 40:60 ratio . The single amino acid substitution in cytochrome P-450SG1 thus causes a conformational change that retracts the amino acid residues that cover pyrrole ring A and moves an imidazole ligand into the active site.

Genes Dev, 1992 Jul, 6(7), 1319 - 31
SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae; Eisenmann DM et al.; Mutations in the Saccharomyces cerevisiae gene SPT15, which encodes the TATA-binding protein TFIID, have been shown to cause pleiotropic phenotypes and to lead to changes in transcription in vivo . Here, we report the cloning and analysis of one such mutation, spt15-21, which causes a single-amino-acid substitution in a conserved residue of TFIID . Surprisingly, the spt15-21 mutation does not affect the stability of TFIID, its ability to bind to DNA or to support basal transcription in vitro, or the ability of an upstream activator to function in vivo . To study further the spt15-21 defect, extragenic suppressors of this mutation were isolated and analyzed . All of the extragenic suppressors of spt15-21 are mutations in the previously identified SPT3 gene . Suppression of spt15-21 by these spt3 mutations is allele-specific, suggesting that TFIID and SPT3 interact and that spt15-21 impairs this interaction in some way . Consistent with these genetic data, coimmunoprecipitation experiments demonstrate that the TFIID and SPT3 proteins are physically associated in yeast extracts . Taken together, these results suggest that SPT3 is a TFIID-associated protein, required for TFIID to function at particular promoters in vivo.

Genes Dev, 1992 Jul, 6(7), 1280 - 92
Signal transduction in Saccharomyces cerevisiae requires tyrosine and threonine phosphorylation of FUS3 and KSS1; Gartner A et al.; The FUS3 and KSS1 kinases are components of the pheromone-dependent signal transduction pathway in yeast . We show that FUS3 and KSS1 become rapidly phosphorylated after pheromone treatment . Similar to mammalian MAP kinases, this modification occurs at two amino acids of FUS3, threonine-180 and tyrosine-182 . A mutation introduced at either position results in complete loss of function in vivo . Amino acid substitutions that destroy catalytic activity of the kinase do not prevent phosphorylation of the mutant products, a result that excludes an autocatalytic activation pathway . The modification of FUS3 is dependent on kinases encoded by the STE11 and STE7 genes . Furthermore, a hyperactive allele of STE11 causes increased phosphorylation of FUS3 in the absence of pheromone stimulation . Thus, either STE7 or STE11 could be the kinase responsible for the phosphorylation of FUS3.

Genes Dev, 1992 Jul, 6(7), 1173 - 89
Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA; Amberg DC et al.; We have combined techniques of genetics and histochemistry to identify genes required for the nucleocytoplasmic export of mRNA in the budding yeast Saccharomyces cerevisiae . We adapted in situ hybridization using a digoxigenin-labeled oligo(dT)50 probe to localize poly(A)+ RNA in fixed yeast cells and used yeast strains carrying the rna1-1 mutation to develop an assay . The rna1-1 mutation is the only previously described mutation that causes defects in mRNA export . As visualized with this RNA localization assay, rna1-1 strains accumulated poly(A)+ RNA at the nuclear periphery at the nonpermissive temperature . This was in contrast to the RNA localization pattern of wild-type cells or rna1-1 cells grown at permissive temperature . Wild-type cells showed bright uniform cytoplasmic staining with little detectable RNA in the nuclei . We used this RNA localization assay to screen a bank of temperature-sensitive yeast strains for mutants with inducible defects in mRNA trafficking . Strains identified in this manner are designated RAT mutants for ribonucleic acid trafficking . The rat1-1 allele conferred temperature-sensitive accumulation of poly(A)+ RNA in one to several intranuclear spots that appear to lie at the nuclear periphery . RNA processing was unaffected in rat1-1 strains, except for an inducible defect in trimming the 5' end of the 5.8S rRNA . The wild-type RAT1 gene was cloned by complementation; it encodes an essential 116-kD protein with regions of homology to the protein encoded by SEP1 (also known as DST2, XRN1, KEM1, and RAR5) . Sep1p is a nucleic acid binding protein, a 5'----3' exonuclease, and catalyzes DNA strand transfer reactions in vitro . We discuss the possible significance of the Rat1p/Sep1p homology for RNA trafficking . We also discuss the potential of this RNA localization assay to identify genes involved in nuclear structure and RNA metabolism.

Genetics, 1992 Jul, 131(3), 559 - 74
The structure and evolution of subtelomeric Y' repeats in Saccharomyces cerevisiae; Louis EJ et al.; The subtelomeric Y' family of repeated DNA sequences in the yeast Saccharomyces cerevisiae is of unknown origin and function . Y's vary in copy number and location among strains . Eight Y's, from two strains, were cloned and sequenced over the same 3.2-kb interval in order to assess the within- and between-strain variation as well as address their origin and function . One entire Y' sequence was reconstructed from two clones presented here and a previously sequenced 833-bp region . It contains two large overlapping open reading frames (ORFs) . The putative protein sequences have no strong homologies to any known proteins except for one region that has 27% identity with RNA helicases . RNA homologous to each ORF was detected . Comparison of the sequences revealed that the known long (Y'-L) and short (Y'-S) size classes, which coexist within cells, differ by several insertions and/or deletions within this region . The Y'-Ls from strain Y55 also differ from those of strain YP1 by several short deletions in the same region . Most of these deletions appear to have occurred between short (2-10 bp) direct repeats . The single base pair polymorphisms and the deletions are clustered in the first half of the interval compared . There is 0.30-1.13% divergence among Y'-Ls within a strain and 1.15-1.75% divergence between strains in the interval . This is similar to known unique sequence variation but contrasts with the 8-18% divergence among the adjacent subtelomeric repeats, X . Subsets of Y's exhibit concerted evolution; however, more than one variant appears to be maintained within strains . The observed sequence variation disrupts the first ORF in many Y's while most of the second ORF including the putative helicase region is unaffected . The structure and distribution of the Y' elements are consistent with having originated as a mobile element . However, they now appear to move via recombination . Recombination can account for the homogenization within subsets of Y's but does not account for the maintenance of different variants.

Genetics, 1992 Jul, 131(3), 551 - 8
Genes required for vacuolar acidity in Saccharomyces cerevisiae; Preston RA et al.; Mutations that cause loss of acidity in the vacuole (lysosome) of Saccharomyces cerevisiae were identified by screening colonies labeled with the fluorescent, pH-sensitive, vacuolar labeling agent, 6-carboxyfluorescein . Thirty nine vacuolar pH (Vph-) mutants were identified . Four of these contained mutant alleles of the previously described PEP3, PEP5, PEP6 and PEP7 genes . The remaining mutants defined eight complementation groups of vph mutations . No alleles of the VAT2 or TFP1 genes (known to encode subunits of the vacuolar H(+)-ATPase) were identified in the Vph- screen . Strains bearing mutations in any of six of the VPH genes failed to grow on medium buffered at neutral pH; otherwise, none of the vph mutations caused notable growth inhibition on standard yeast media . Expression of the vacuolar protease, carboxypeptidase Y, was defective in strains bearing vph4 mutations but was apparently normal in strains bearing any of the other vph mutations . Defects in vacuolar morphology at the light microscope level were evident in all Vph- mutants . Strains that contained representative mutant alleles of the 17 previously described PEP genes were assayed for vacuolar pH; mutations in seven of the PEP genes (including PEP3, PEP5, PEP6 and PEP7) caused loss of vacuolar acidity.

Genetics, 1992 Jul, 131(3), 531 - 9
Serine and threonine catabolism in Saccharomyces cerevisiae: the CHA1 polypeptide is homologous with other serine and threonine dehydratases; Bornaes C et al.; The catabolic L-serine (L-threonine) dehydratase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source . Previously we have cloned the CHA1 gene by complementation of a mutant, cha1, lacking the dehydratase activity . Here we present the DNA sequence of a 1,766-bp fragment of the CHA1 region encompassing an open reading frame of 1080 bp . Comparison of the predicted amino acid sequence of the CHA1 polypeptide with that of other serine/threonine dehydratases revealed several blocks of sequence homology . Thus, the amino acid sequence of rat liver serine dehydratase (SDH2) and the CHA1 polypeptide are 44% homologous allowing for conservative substitutions, while 36% similarity is found between the catabolic threonine dehydratase (tdcB) of Escherichia coli and the CHA1 protein . This strongly suggests that CHA1 is the structural gene for the yeast catabolic serine (threonine) dehydratase . S1-nuclease mapping of the CHA1 mRNA ends showed a major transcription initiation site corresponding to an untranslated leader of about 19 nucleotides, while a major polyadenylation site was located about 86 nucleotides downstream from the open reading frame . Furthermore, we have mapped the chromosomal position of the CHA1 gene to less than 0.5 kb centromere proximal to HML on the left arm of chromosome III.

Mol Cell Biol, 1992 Jul, 12(7), 3224 - 34
Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins; Aboussekhra A et al.; Eleven suppressors of the radiation sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase were analyzed and found to contain codominant mutations in the RAD51 gene known to be involved in recombinational repair and in genetic recombination . These mutant alleles confer an almost complete block in recombinational repair, as does deletion of RAD51, but heterozygous mutant alleles suppress the defects of srs2::LEU2 cells and are semidominant in Srs2+ cells . The results of this study are interpreted to mean that wild-type Rad51 protein binds to single-stranded DNA and that the semidominant mutations do not prevent this binding . The cloning and sequencing of RAD51 indicated that the gene encodes a predicted 400-amino-acid protein with a molecular mass of 43 kDa . Sequence comparisons revealed homologies to domains of Escherichia coli RecA protein predicted to be involved in DNA binding, ATP binding, and ATP hydrolysis . The expression of RAD51, measured with a RAD51-lacZ gene fusion, was found to be UV- and gamma-ray-inducible, with dose-dependent responses.

Mol Cell Biol, 1992 Jul, 12(7), 2958 - 66
Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae; Bun-Ya M et al.; We have found an open reading frame which is 1.1 kb upstream of PHO84 (which encodes a Pi transporter) and is transcribed from the opposite strand . In Saccharomyces cerevisiae, this gene is distal to the TUB3 locus on the left arm of chromosome XIII and is named GTR1 . GTR1 encodes a protein consisting of 310 amino acid residues containing, in its N-terminal region, the characteristic tripartite consensus elements for binding GTP conserved in GTP-binding proteins, except for histidine in place of a widely conserved aspargine residue in element III . Disruption of the GTR1 gene resulted in slow growth at 30 degrees C and no growth at 15 degrees C; other phenotypes resembled those of pho84 mutants and included constitutive synthesis of repressible acid phosphatase, reduced Pi transport activity, and resistance to arsenate . The latter phenotypes were shown to be due to a defect in Pi uptake, and the Gtr1 protein was found to be functionally associated with the Pho84 Pi transporter . Recombination between chromosome V (at the URA3 locus) and chromosome XIII (in the GTR1-PHO84-TUB3 region) by using a plasmid-encoded site-specific recombination system indicated that the order of these genes was telomere-TUB3-PHO84-GTR1-CENXIII.

Mol Cell Biol, 1992 Jul, 12(7), 2941 - 8
Control of mRNA turnover as a mechanism of glucose repression in Saccharomyces cerevisiae; Lombardo A et al.; We have examined the expression of the gene encoding the iron-protein subunit (Ip) of succinate dehydrogenase in Saccharomyces cerevisiae . The gene had been cloned by us and shown to be subject to glucose regulation (A . Lombardo, K . Carine, and I . E . Scheffler, J . Biol . Chem . 265:10419-10423, 1990) . We discovered that a significant part of the regulation of the Ip mRNA levels by glucose involves the regulation of the turnover rate of this mRNA . In the presence of glucose, the half-life appears to be less than 5 min, while in glycerol medium, the half-life is greater than 60 min . The gene is also regulated transcriptionally by glucose . The upstream promoter sequence appeared to have four regulatory elements with consensus sequences shown to be responsible for the interaction with the HAP2/3/4 regulatory complex . A deletion analysis has shown that the two distal elements are redundant . These measurements were carried out by Northern (RNA) analyses of Ip mRNA transcripts as well as by assays of beta-galactosidase activity in cells carrying constructs of the Ip promoter linked to the lacZ coding sequence . These observations on the regulation of mRNA stability were also extended to the mRNA of the flavoprotein subunit of succinate dehydrogenase and in some experiments of iso-1-cytochrome c.

J Cell Biol, 1992 Jul, 118(1), 109 - 20
Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly; Hoyt MA et al.; Two Saccharomyces cerevisiae genes, CIN8 and KIP1 (a.k.a . CIN9), were identified by their requirement for normal chromosome segregation . Both genes encode polypeptides related to the heavy chain of the microtubule-based force-generating enzyme kinesin . Cin8p was found to be required for pole separation during mitotic spindle assembly at 37 degrees C, although overproduced Kip1p could substitute . At lower temperatures, the activity of at least one of these proteins was required for cell viability, indicating that they perform an essential but redundant function . Cin8p was observed to be a component of the mitotic spindle, colocalizing with the microtubules that lie between the poles . Taken together, these findings suggest that these proteins interact with spindle microtubules to produce an outwardly directed force acting upon the poles.

Curr Genet, 1992 Jul, 22(1), 13 - 9
Adaptation and major chromosomal changes in populations of Saccharomyces cerevisiae; Adams J et al.; Thirteen independent populations of Saccharomyces cerevisiae (nine haploid and four diploid) were maintained in continuous culture for up to approximately 1000 generations, with growth limited by the concentration of organic phosphates in medium buffered at pH 6 . Analysis of clones isolated from these populations showed that a number (17) of large-scale chromosomal-length variants and rearrangements were present in the populations at their termination . Nine of the 16 yeast chromosomes were involved in such changes . Few of the changes could be explained by copy-number increases in the structural loci for acid phosphatase . Several considerations concerning the nature and frequency of the chromosome-length variants observed lead us to conclude that they are selectively advantageous.

Curr Genet, 1992 Jul, 22(1), 1 - 7
Analysis of the chromosomal DNA polymorphism of wine strains of Saccharomyces cerevisiae; Bidenne C et al.; Wine yeast strains are characterized by a high chromosomal DNA polymorphism . This can be explained partly by a size difference of different variants of specific chromosomes . This difference can reach up to 45% of the size of the chromosome in question . Two strains, SB1 and Eg8, have a very complex chromosomal pattern and show one band hybridizing with probes from two different chromosomes derived from a reference strain . This is an indication of the presence of "hybrid" chromosomes in these wine strains . The most astonishing result concerns chromosome VIII, frequently present in wine strains in two variant forms . The first normal form has a size of about 580 kb while the second is around 1000 kb . These two forms segregate at meiosis and recombine with a normal chromosome VIII from a laboratory strain . Wine yeasts are thus very different from haploid laboratory strains.

Yeast, 1992 Jul, 8(7), 569 - 75
The complete sequence of a 6146 bp fragment of Saccharomyces cerevisiae chromosome III contains two new open reading frames; Wilson C et al.; As part of the EEC project to sequence the entire chromosome III of Saccharomyces cerevisiae we have sequenced a total of 11,040 bp from near the right end of the chromosome . A new protein kinase gene was found at one extremity of the sequenced region (Wilson et al., 1992), while the previously sequenced actin binding protein gene, ABP1, (Drubin et al., 1990) was found at the other extremity . We present here the sequence of the region between these two genes which has the potential to code for two new open reading frames (ORFs).

Yeast, 1992 Jul, 8(7), 559 - 67
Analysis of the MSS51 region on chromosome XII of Saccharomyces cerevisiae; Simon M et al.; We have localized gene MSS51 on chromosome XII of Saccharomyces cerevisiae between the RDN1 and CDC42 loci . 'Head to head' with MSS51 is another gene, QRI5, the function of which is unknown . However, the proximity of these genes, the structure of the intergenic region and the presence of an ABF1 binding site right in the middle of this region suggest that the MSS51 and QRI5 expressions are submitted to a common regulatory process.

Yeast, 1992 Jul, 8(7), 535 - 47
Isolation of new temperature-sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation; Nagasu T et al.; We have isolated two temperature-sensitive Saccharomyces cerevisiae mutants which exhibit a deficiency in mannose outer chain elongation of asparagine-linked oligosaccharide . The size of yeast glycoprotein, secretory form of invertase, of one mutant (och1) was slightly larger than that of the sec18 mutant at the non-permissive temperature, while that of the other mutant (och2) was almost the same as that of the sec18 mutant . Unlike sec mutants, the och mutants were not deficient in secretion of invertase . The och1 mutant showed a 2+:2- cosegregation with regard to the temperature sensitivity and mannose outer chain deficiency, suggesting that a single gene designated as OCH1 is responsible for these two phenotypes . The och1 mutant stopped its growth at the early stage of bud formation and rapidly lost its viability at the non-permissive temperature . The och1 mutation was mapped near the ole1 on the left arm of chromosome VII . The och1 mutant cells accumulated the external invertase containing a large amount of core-like oligosaccharides (Man9-10GlcNAc2) and a small amount of high mannose oligosaccharides (greater than Man50GlcNAc2) at the non-permissive temperature . Production of the active form of human tissue-type plasminogen activator was increased in the och1 mutant compared with the parental strain, suggesting the potential advantage of this mutant for the production of mammalian-type glycoproteins which lack mannose outer chains in yeast.

Yeast, 1992 Jul, 8(7), 489 - 99
Dosage-dependent translational suppression in yeast Saccharomyces cerevisiae; Chernoff YO et al.; The overexpression of SUP35 (SUP2) wild-type gene, caused by increase of its copy number, induces an omnipotent suppression similar to the phenotype of mutants for this gene . The effect of extra-SUP35 was detected for moderate or even low copy number . Moreover, overdosage of the fragment including only the 5'-flanking region and N-terminal 100 bp of protein-coding sequence of SUP35 leads to allosuppression . Multi-SUP35 gene was also incompatible with extrachromosomal suppressor factor psi, presumably because of a high level of mistranslation . The suppressor effect caused by overdosage of another gene, SUP45 (SUP1), is much lower and can be detected only for one construction which is derived from high copy number plasmid . Suppression induced by extra-SUP35 and especially by extra-SUP45 is affected by the cell environment . A model predicting that the balance of gene products is a key for regulation of translational fidelity is discussed.

Mol Gen Genet, 1992 Jul, 234(1), 22 - 32
The role of promoter elements of a ribosomal protein gene in Saccharomyces cerevisiae under various physiological conditions; Papciak SM et al.; Previous work in our laboratory has shown that the 5' nontranscribed promoter region of the gene for ribosomal protein (rp) S16A-1 of Saccharomyces cerevisiae, when fused to a lacZ gene, is necessary and sufficient to cause an increase in expression of the heterologous lacZ gene fusion product after cells have been shifted from a glycerol to glucose carbon source . This increase in expression is characteristic of that observed with the native rp gene . We have sought to define more precisely those areas of the promoter that may be involved in the differential expression/regulation of RPS16A-1 when host cells are subjected to a variety of nutritional environments . It has already been demonstrated by others that the promoter regions of most rp genes contain at least one consensus element, designated UASrpg, which is necessary for the transcriptional activation and maintenance of expression of the gene during steady-state growth in rich media . Our main experimental approach has been to create a series of 5' end deletions in the promoter region of RPS16A-1 . The individual truncated promoter fragments were then ligated to a lacZ fusion reporter construct . By assaying the cells for production of beta-galactosidase and determining the abundance of lacZ mRNA, we have been able to determined the extent of fusion product expression . We assayed cells under three physiological conditions: steady-state growth in glucose, steady-state growth in glycerol and during sporulation . We report four main findings of our work.

Mol Gen Genet, 1992 Jul, 234(1), 164 - 7
NPK1, a nonessential protein kinase gene in Saccharomyces cerevisiae with similarity to Aspergillus nidulans nimA; Schweitzer B et al.; A new protein kinase gene {called NPK1 (for nonessential protein kinase)} has been found on chromosome I of Saccharomyces cerevisiae between CDC15 and ADE1 . The 435 amino acid/48 kDa gene product is very similar to known protein kinases . It is most closely related to the nimA protein of Aspergillus nidulans, displaying 45.9% identity and 63.5% similarity in the protein kinase domain . A 1.4 kb transcript of the NPK1 gene was detected . Disruption of the NPK1 gene impedes neither growth on glucose or a variety of other carbon sources, nor mating or sporulation.

Genetika, 1992 Jul, 28(7), 54 - 65
{Effect of hms mutations increasing spontaneous mutability on induced mutagenesis and mitotic recombination in the yeast Saccharomyces cerevisiae}; Fedorova IV et al.; The influence of five nonallelic mutations hsm-1-hsm-5 on the frequency of mutations induced by UV-light, 6-hydroxyl-aminopurine (GAP) and nitrosomethylurea (NMM) at the ADE1 and ADE2 loci was studied . All hsm mutants were resistant to the lethal effect of these mutagens . The frequency of mutations induced by UV-light was increased in hsm2-1, hsm3-1, hsm5-1 and especially in hsm1-1 mutants, the hsm4-1 mutant not differing from the HSM strain . GAP-induced mutagenesis was elevated in all hsm mutants and, particularly, in hsm3-1 . No influence of hsm mutations on the frequency of NMM-induced mutations was observed . The frequency of spontaneous mitotic gene conversion was studied in the diploids heteroallelic for mutations in the gene ADE2 (ade2-58 ade2-i) and homo- and heterozygous for the hsm mutations (HSMHSM and HSMhsm) . The mutations hsm2-1, hsm3-1 and especially hsm5-1 strongly increased the conversion frequency for all heteroallelic combinations studied . The mutations hsm1-1, hsm4-1 affected this process weakly . The properties of the hsm mutations under study demonstrated common genetic control of spontaneous and induced mutagenesis and recombination in the yeast . Possible belonging of hsm mutations to the class of mutations destroying the repair pathway for mismatch correction is under discussion.

Mol Biol Cell, 1992 Jul, 3(7), 805 - 18
Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae; Fitch I et al.; The previously described CLB1 and CLB2 genes encode a closely related pair of B-type cyclins . Here we present the sequences of another related pair of B-type cyclin genes, which we term CLB3 and CLB4 . Although CLB1 and CLB2 mRNAs rise in abundance at the time of nuclear division, CLB3 and CLB4 are turned on earlier, rising early in S phase and declining near the end of nuclear division . When all possible single and multiple deletion mutants were constructed, some multiple mutations were lethal, whereas all single mutants were viable . All lethal combinations included the clb2 deletion, whereas the clb1 clb3 clb4 triple mutant was viable, suggesting a key role for CLB2 . The inviable multiple clb mutants appeared to have a defect in mitosis . Conditional clb mutants arrested as large budded cells with a G2 DNA content but without any mitotic spindle . Electron microscopy showed that the spindle pole bodies had duplicated but not separated, and no spindle had formed . This suggests that the Clb/Cdc28 kinase may have a relatively direct role in spindle formation . The two groups of Clbs may have distinct roles in spindle formation and elongation.

Biotechnol Prog, 1992 Jul-Aug, 8(4), 298 - 306
A model for beta-galactosidase production with a recombinant yeast Saccharomyces cerevisiae in fed-batch culture; Hardjito L et al.; An unstructured model is developed to describe the growth and product formation behavior of a recombinant yeast Saccharomyces cerevisiae using beta-galactosidase as a model protein . The model shows good agreement with the experimental data over a range of conditions . It also accurately predicts the effect of growth rate on yield coefficient and gene expression . The simplicity and accuracy of the model make it suitable for designing and implementing control and optimization strategies for the production of recombinant proteins at large scale.

Int J Syst Bacteriol, 1992 Jul, 42(3), 499 - 502
Ribosomal DNA restriction analysis reveals genetic heterogeneity in Saccharomyces cerevisiae Meyen ex Hansen; Molina FI et al.; A ribosomal DNA restriction analysis of 17 strains belonging to the Saccharomyces cerevisiae complex revealed two major groups, one of which corresponded to Saccharomyces bayanus . Our results generally correlate with previously reported genetic and molecular data and support the conclusion that S . bayanus should be reinstated as a separate taxon.

Proc Natl Acad Sci U S A, 1992 Jun 15, 89(12), 5296 - 300
Meiotic recombination and segregation of human-derived artificial chromosomes in Saccharomyces cerevisiae; Sears DD et al.; We have developed a system that utilizes human DNA-derived yeast artificial chromosomes (YACs) as marker chromosomes to study factors that contribute to the fidelity of meiotic chromosome transmission . Since aneuploidy for the YACs does not affect spore viability, different classes of meiotic missegregation can be scored accurately in four-viable-spore tetrads including precocious sister separation, meiosis I nondisjunction, meiotic chromatid loss, and meiosis II nondisjunction . Segregation of the homologous pair of 360-kilobase marker YACs was shown to occur with high fidelity in the first meiotic division and was associated with a high frequency of recombination within the human DNA segment . By using this experimental system, a series of YAC deletion derivatives ranging in size from 50 to 225 kilobases was analyzed to directly assess the relationship between meiotic recombination and meiosis I disjunction in a genotypically wild-type background . The relationship between physical distance and recombination frequency within the human DNA segment was measured to be comparable to that of endogenous yeast chromosomal DNA--ranging from less than 2.0 to 7.7 kilobases/centimorgan . Physical analysis of recombinant chromosomes detected no unequal crossing-over at dispersed repetitive elements distributed along the YACs . Recombination between YACs containing unrelated DNA segments was not observed . Furthermore, the segregational data indicated that meioses in which YAC pairs failed to recombine exhibited dramatically increased levels of meiosis I missegregation, including both precocious sister chromatid separation and nondisjunction.

Biochim Biophys Acta, 1992 Jun 11, 1107(1), 126 - 30
Proton potential-dependent polyamine transport by vacuolar membrane vesicles of Saccharomyces cerevisiae; Kakinuma Y et al.; Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulated spermine and spermidine in the presence of ATP, not in the presence of ADP . Spermine and spermidine transport at pH 7.4 showed saturation kinetics with Km values of 0.2 mM and 0.7 mM, respectively . Spermine uptake was competitively inhibited by spermidine and putrescine, but was not affected by seven amino acids, substrates of active transport systems of vacuolar membrane . Spermine transport was inhibited by the H(+)-ATPase-specific inhibitors bafilomycin A1 and N,N'-dicyclohexylcarbodiimide, but not by vanadate . It was also sensitive to Cu2+ or Zn2+ ions, inhibitors of vacuolar H(+)-ATPase . Both 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847) and nigericin blocked completely the spermine uptake, but valinomycin did not . {14C}Spermine accumulated in the vesicles was exchangeable with unlabeled spermine and spermidine . However, it was released by a protonophore only in the presence of a counterion such as Ca2+ . These results indicate that a polyamine-specific transport system depending on a proton potential functions in the vacuolar membrane of this organism.

J Biol Chem, 1992 Jun 5, 267(16), 11274 - 80
CDC14 of Saccharomyces cerevisiae . Cloning, sequence analysis, and transcription during the cell cycle; Wan J et al.; We have cloned, mapped and sequenced the complete CDC14 gene of Saccharomyces cerevisiae and characterized its transcription during the cell cycle . CDC14 was found within a 3.5-kilobase pair XhoI-XbaI fragment of chromosome VI . The DNA sequence reveals an open reading frame capable of encoding a 423-amino acid polypeptide . Protein sequence comparisons through the Prosite, GenBank and EMBL databases allowed us to identify a conserved protein tyrosine phosphatase active site in the encoded CDC14 protein beginning at amino acid 153 . Disruption demonstrates that CDC14 is an essential gene . The level of the CDC14 transcript appears to be weakly cell cycle-regulated and has a periodicity which lags approximately 15 min behind histone HTB1 mRNA accumulation levels . DNA sequence analysis has identified a region within the CDC14 promoter which bears a striking resemblance (15 out of 21 base pairs identity) to the cell cycle regulation region of the promoter of the histone H2A1-H2B1 (HTA1-HTB1) gene pair . The cell cycle regulation sequence is responsible for the periodic accumulation and hydroxyurea sensitivity of the histone HTA1-HTB1 message . However, unlike histone mRNA, which is repressed upon hydroxyurea arrest, CDC14 mRNA appears to be unaffected . This suggests that CDC14 and histone genes are regulated by different mechanisms during the cell cycle . Furthermore, superhelical density measurements suggest that CDC14 is not involved in nucleosome assembly.

Genetics, 1992 Jun, 131(2), 277 - 85
Isolation and characterization of extragenic suppressors of mutations in the SSA hsp70 genes of Saccharomyces cerevisiae; Nelson RJ et al.; Saccharomyces cerevisiae strains that contain null alleles of two hsp70 genes, SSA1 and SSA2, are temperature sensitive for growth . In this study, extragenic suppressors of ssa1 ssa2 have been isolated . Suppression is due to mutations at nuclear loci designated EXA1, EXA2 and EXA3 for EXtragenic suppressor hsp70 subfamily A . Two of the four EXA1 alleles are dominant as is EXA3-1 . The other two EXA1 alleles as well as the sole EXA2 allele are recessive . EXA1 mutations lead to accumulation of a previously uncharacterized form of hsp70 . EXA2 and EXA3 mutations affect the regulation of the stress response . In exa2-1 ssa1 ssa2 strains the gene products of the remaining SSA hsp70 genes, SSA3 and SSA4 (Ssa3/4p), accumulate to higher levels . The EXA3-1 mutation results in increased accumulation of both Ssa3/4p and the hsp70s encoded by the SSB1 and SSB2 genes (Ssb1/2p), suggesting that the EXA3 gene product plays a central role in the yeast stress response . Consistent with this hypothesis, EXA3-1 is tightly linked to HSF1, the gene encoding the transcriptional regulatory protein known as "heat shock factor." All of the genes identified in this study seem to be involved in regulating the expression of SSA3 and SSA4 or the activity of their protein products.

Genetics, 1992 Jun, 131(2), 261 - 76
Plasmid recombination in a rad52 mutant of Saccharomyces cerevisiae; Dornfeld KJ et al.; Using plasmids capable of undergoing intramolecular recombination, we have compared the rates and the molecular outcomes of recombination events in a wild-type and a rad52 strain of Saccharomyces cerevisiae . The plasmids contain his3 heteroalleles oriented in either an inverted or a direct repeat . Inverted repeat plasmids recombine approximately 20-fold less frequently in the mutant than in the wild-type strain . Most events from both cell types have continuous coconversion tracts extending along one of the homologous segments . Reciprocal exchange occurs in fewer than 30% of events . Direct repeat plasmids recombine at rates comparable to those of inverted repeat plasmids in wild-type cells . Direct repeat conversion tracts are similar to inverted repeat conversion tracts in their continuity and length . Inverted and direct repeat plasmid recombination differ in two respects . First, rad52 does not affect the rate of direct repeat recombination as drastically as the rate of inverted repeat recombination . Second, direct repeat plasmids undergo crossing over more frequently than inverted repeat plasmids . In addition, crossovers constitute a larger fraction of mutant than wild-type direct repeat events . Many crossover events from both cell types are unusual in that the crossover HIS3 allele is within a plasmid containing the parental his3 heteroalleles.

Mol Microbiol, 1992 Jun, 6(11), 1441 - 6
IRE1 encodes a putative protein kinase containing a membrane-spanning domain and is required for inositol phototrophy in Saccharomyces cerevisiae; Nikawa J et al.; A novel gene, IRE1, of Saccharomyces cerevisiae was cloned through genetic complementation of a myoinositol auxotrophic mutant . The predicted amino acid sequence indicated that IRE1 encodes a protein of 126983 Da with two highly hydrophobic regions, probably a signal sequence and a membrane-spanning region . The carboxy-terminal region of IRE1 showed close sequence similarity to the catalytic domains of protein kinases . Disruption of the IRE1 locus caused myo-inositol auxotrophy . The IRE1 product is very likely a protein kinase required for myo-inositol synthesis.

Mol Gen Genet, 1992 Jun, 233(3), 487 - 90
The two genes encoding protein synthesis initiation factor eIF-5A in Saccharomyces cerevisiae are members of a duplicated gene cluster; Kang HA et al.; Translation initiation factor eIF-5A is an abundant protein in which a lysine residue is modified by spermidine to form the amino acid derivative, hypusine . The factor is encoded by two genes in Saccharomyces cerevisiae, called TIF51A and TIF51B, which are regulated reciprocally by oxygen and by heme . TIF51B, also called ANB1, is located on chromosome X in a region called COR . We physically mapped TIF51A and its associated serine tRNA2 gene by the method of chromosome fragmentation and pulsed-field gel electrophoresis . TIF51A maps 90 kb from the left end of chromosome V in a region called ARC . The COR and ARC regions contain CYC1 and CYC7, respectively, and appear to be duplications carrying numerous related genes . The arrangements of related genes in the two regions are incompatible with a duplication mechanism involving a circular intermediate.

Mol Gen Genet, 1992 Jun, 233(3), 355 - 62
Identification of a site required for DNA replication fork blocking activity in the rRNA gene cluster in Saccharomyces cerevisiae; Kobayashi T et al.; The yeast genome has DNA replication fork blocking sites, that we have named sog sites, in the ribosomal RNA gene (rDNA) cluster . These are located at the 3' end of the 35S rRNA transcription unit and they block replication fork movement in a direction opposite to that of RNA polymerase I . We cloned this replication blocking site into a YEp-type plasmid and analyzed DNA replication intermediates, using two-dimensional (2D) agarose gel electrophoresis . The blocking activity remained even on a plasmid not involved in 35S rRNA transcription and inhibited fork movement in the same polar fashion as on the yeast chromosome . To define the site further, smaller fragments were subcloned into the YEp-type plasmid . A small 109 bp region exhibited sog activity and was located near the enhancer region for 35S rRNA transcription . It overlaps an essential element of the recombinational hot spot HOT1.

J Bacteriol, 1992 Jun, 174(12), 4183 - 8
Glucose-induced regulatory defects in the Saccharomyces cerevisiae byp1 growth initiation mutant and identification of MIG1 as a partial suppressor; Hohmann S et al.; Saccharomyces cerevisiae byp1-3 mutants displayed a long lag phase when shifted from a nonfermentable carbon source to a medium containing glucose . The byp1-3 mutation also caused several defects in regulatory phenomena which occur during the transition from the derepressed state to the repressed state . As opposed to wild-type cells, the addition of glucose to cells of the byp1-3 mutant grown on nonfermentable carbon sources did not induce a cyclic AMP signal . Fructose-2,6-bisphosphate formation and inactivation of fructose-1,6-bisphosphatase were severely delayed, but trehalase activation was not affected . In addition, the induction of pyruvate decarboxylase both at the level of activity and that of transcription was very slow compared with that in wild-type cells . These pleotropic defects in glucose-induced regulatory phenomena might be responsible for the very long lag phase of byp1-3 cells and the inability of ascospores to initiate growth after germination on glucose media . Screening of a yeast gene library for clones complementing the byp1-3 phenotype resulted in the isolation of a truncated form of the previously described zinc finger transcription repressor MIG1 . The entire MIG1 gene and the truncated form suppressed even on a single-copy vector the growth initiation defect but not the regulatory abnormalities of the byp1-3 mutant . MIG1 is not allelic to byp1-3.

J Bacteriol, 1992 Jun, 174(11), 3782 - 8
Proline biosynthesis in Saccharomyces cerevisiae: analysis of the PRO3 gene, which encodes delta 1-pyrroline-5-carboxylate reductase; Brandriss MC et al.; The PRO3 gene of Saccharomyces cerevisiae encodes the 286-amino-acid protein delta 1-pyrroline-5-carboxylate reductase {L-proline:NAD(P+) 5-oxidoreductase; EC 1.5.1.2}, which catalyzes the final step in proline biosynthesis . The protein has substantial similarity to the pyrroline carboxylate reductases of diverse bacterial species, soybean, and humans . Using RNA hybridization and measurements of enzyme activity, we have determined that the expression of the PRO3 gene appears to be constitutive . It is not repressed by the pathway end product (proline), induced by the initial substrate (glutamate), or regulated by the general control system . Its expression is not detectably altered when cells are grown in a wide range of nitrogen sources or when glycerol and ethanol replace glucose as the carbon source . The possibility that this enzyme has other functions in addition to proline biosynthesis is discussed.

Mol Cell Biol, 1992 Jun, 12(6), 2749 - 57
Instability of simple sequence DNA in Saccharomyces cerevisiae; Henderson ST et al.; All eukaryotic genomes thus far examined contain simple sequence repeats . A particularly common simple sequence in many organisms (including humans) consists of tracts of alternating GT residues on one strand . Allelic poly(GT) tracts are often of different lengths in different individuals, indicating that they are likely to be unstable . We examined the instability of poly(GT) and poly(G) tracts in the yeast Saccharomyces cerevisiae . We found that these tracts were dramatically unstable, altering length at a minimal rate of 10(-4) events per division . Most of the changes involved one or two repeat unit additions or deletions, although one alteration involved an interaction with the yeast telomeres.

Mol Cell Biol, 1992 Jun, 12(6), 2690 - 700
Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae; Huie MA et al.; GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae . Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site . Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide . DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC . A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains . The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain . These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo . Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains . In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression . However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1 . Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.

Mol Cell Biol, 1992 Jun, 12(6), 2616 - 23
HAP1 and ROX1 form a regulatory pathway in the repression of HEM13 transcription in Saccharomyces cerevisiae; Keng T; HEM13 of Saccharomyces cerevisiae encodes coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway . Expression of HEM13 is repressed by oxygen and heme . This study investigated the regulatory pathway responsible for the regulation of HEM13 expression . The transcriptional activator HAP1 is demonstrated to be required for the full-level expression of HEM13 in the absence of heme . It is also shown that the repression of HEM13 transcription caused by heme involves the HAP1 and ROX1 gene products; a mutation in either gene results in derepression of HEM13 expression . The heme-dependent expression of ROX1 was found to require functional HAP1, leading one to propose that repression of HEM13 results from a pathway involving HAP1-mediated regulation of ROX1 transcription in response to heme levels followed by ROX1-mediated repression of HEM13 transcription . In support of this model, expression of ROX1 under control of the GAL promoter was found to result in repression of HEM13 transcription in a hap1 mutant strain . The ability of ROX1 encoded by the galactose-inducible ROX1 construct to function in the absence of HAP1 indicates that the only role of HAP1 in repression of HEM13 is to activate ROX1 transcription.

Mol Cell Biol, 1992 Jun, 12(6), 2561 - 9
Saccharomyces cerevisiae contains an RNase MRP that cleaves at a conserved mitochondrial RNA sequence implicated in replication priming; Stohl LL et al.; Yeast mitochondrial DNA contains multiple promoters that sponsor different levels of transcription . Several promoters are individually located immediately adjacent to presumed origins of replication and have been suggested to play a role in priming of DNA replication . Although yeast mitochondrial DNA replication origins have not been extensively characterized at the primary sequence level, a common feature of these putative origins is the occurrence of a short guanosine-rich region in the priming strand downstream of the transcriptional start site . This situation is reminiscent of vertebrate mitochondrial DNA origins and raises the possibility of common features of origin function . In the case of human and mouse cells, there exists an RNA processing activity with the capacity to cleave at a guanosine-rich mitochondrial RNA sequence at an origin; we therefore sought the existence of a yeast endoribonuclease that had such a specificity . Whole cell and mitochondrial extracts of Saccharomyces cerevisiae contain an RNase that cleaves yeast mitochondrial RNA in a site-specific manner similar to that of the human and mouse RNA processing activity RNase MRP . The exact location of cleavage within yeast mitochondrial RNA corresponds to a mapped site of transition from RNA to DNA synthesis . The yeast activity also cleaved mammalian mitochondrial RNA in a fashion similar to that of the mammalian RNase MRPs . The yeast endonuclease is a ribonucleoprotein, as judged by its sensitivity to nucleases and proteinase, and it was present in yeast strains lacking mitochondrial DNA, which demonstrated that all components required for in vitro cleavage are encoded by nuclear genes . We conclude that this RNase is the yeast RNase MRP.

Biokhimiia, 1992 Jun, 57(6), 845 - 55
{Substrate specificity of phosphoribosyl-aminoimidazole-succinocarboxamide synthetase (SAICAR-synthetase) from Saccharomyces cerevisiae yeast}; Alenin VV et al.; The substrate specificity of phosphoribosyl-aminoimidazole-succinocarboxamide-synthetase (SAICAR-synthetase, EC 6.3.2.6) of the yeast Saccharomyces cerevisiae towards a set of carboxyaminoimidazole ribotide (CAIR) analogs with modifications in the imidazole ring, ribose and phosphate moieties, as well as aspartic acid analogs has been studied . It was found, in particular, that: i) the presence of double charged phosphate group, 2'- and 3'-hydroxyl groups in the ribose fragment and of an amino group in the imidazole ring of the CAIR molecule is not the absolute requirement for the enzymatic reaction; ii) 3'-carboxy-1.2.4-triazole analog of CAIR is a competitive inhibitor of the enzyme; iii) 2'-deoxy-CAIR is a substrate for both yeast SAICAR-synthetase and its avian liver and human erythrocyte counterparts . A new method designed to determine the SAICAR-synthetase activity with the help of bifunctional enzymes possessing, in addition to the SAICAR-synthetase activity, also a phosphoribosyl-aminoimidazole-carboxylase activity, is proposed; this method is based on the use of 2'-deoxy-CAIR . Some aspartic acid analogs (L-malic acid, beta-threo-oxy-, and beta-threo-fluoro-aspartic acids and alanosine) are substrates for yeast SAICAR-synthetase . The possible involvement of malate as an alternative substrate for the SAICAR-synthetase reaction in vivo is discussed . The results of a comparative analysis of already established primary structures of yeast, bacterial, human, and chicken SAICAR-synthetases are presented.

EMBO J, 1992 Jun, 11(6), 2151 - 7
Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator; Martegani E et al.; In the yeast Saccharomyces cerevisiae genetic and biochemical evidence indicates that the product of the CDC25 gene activates the RAS/adenylyl cyclase/protein kinase A pathway by acting as a guanine nucleotide protein . Here we report the isolation of a mouse brain cDNA homologous to CDC25 . The mouse cDNA, called CDC25Mm, complements specifically point mutations and deletion/disruptions of the CDC25 gene . In addition, it restores the cAMP levels and CDC25-dependent glucose-induced cAMP signalling in a yeast strain bearing a disruption of the CDC25 gene . The CDC25Mm-encoded protein is 34% identical with the catalytic carboxy terminal part of the CDC25 protein and shares significant homology with other proteins belonging to the same family . The protein encoded by CDC25Mm, prepared as a glutathione S-transferase fusion in Escherichia coli cells, activates adenylyl cyclase in yeast membranes in a RAS2-dependent manner . Northern blot analysis of mouse brain poly(A)+ RNA reveals two major transcripts of approximately 1700 and 5200 nucleotides . Transcripts were found also in mouse heart and at a lower level in liver and spleen.

Mutat Res, 1992 Jun, 274(1), 1 - 9
Psoralen damage-induced plasmid recombination in Saccharomyces cerevisiae: dependence on RAD1 and RAD52; Saffran WA et al.; Photoreaction with psoralen, a DNA-crosslinking reagent, induces mitotic recombination in the yeast Saccharomyces cerevisiae . Psoralen damage-induced recombination was studied with non-replicating plasmids, which transform yeast cells by undergoing recombination events with chromosomal DNA . When plasmid DNA was photoreacted with psoralen in vitro and transformed into yeast cells, transformation was stimulated by psoralen modification in a dose-dependent manner . The stimulation by psoralen damage requires RAD52 gene function and is partially dependent on RAD1 . Analysis of transformants indicates that plasmid integration occurs at the homologous chromosomal loci . Multiple tandem integrations are common in repair-proficient cells, with more than 20 copies of integrated plasmid seen in some transformants . Multiple integration depends on RAD1 function; only 9% of rad1 transformants, compared to 80% of RAD transformants, contained multiple plasmid copies, while 52% of the rad1 transformants were produced by gene conversion.

Appl Microbiol Biotechnol, 1992 Jun, 37(3), 342 - 51
Expression and secretion of pea-seed lipoxygenase isoenzymes in Saccharomyces cerevisiae; Knust B et al.; Lipoxygenases (EC 1.13.11.12) catalyse the oxygenation of polyunsaturated fatty acids such as linoleic and arachidonic acid into reactive cis/trans hydroperoxidiene intermediates, which then serve as substrates for other enzymes leading to the production of a variety of secondary metabolites . In order to explore the characteristics of the individual lipoxygenase isoenzymes in more detail larger amounts of the pure enzymes are needed and their production in a heterologous host is therefore desirable . Full-length cDNAs encoding pea-seed lipoxygenase isoenzymes 2 and 3 were expressed in Saccharomyces cerevisiae with the aid of yeast-Escherichia coli shuttle vectors . Expression of the cDNA for lipoxygenase 2 under the control of the constitutive phosphoglycerate kinase (PGK) gene promoter yielded significant amounts of active enzyme inside the cell, both with yeast transformants carrying the cDNA gene on high-copy-number plasmids or integrated in chromosome V . Addition of the yeast invertase signal sequence in front of the pea lipoxygenase 3 yielded secreted active pea-seed lipoxygenase in the medium, but large amounts of inactive lipoxygenase 3 remained inside the yeast cell . Expression of the LOX3 cDNA can be achieved either constitutively with the PGK promoter or inducibly with the GAL1 promoter.

J Biotechnol, 1992 Jun, 24(2), 159 - 68
Invertase activity of Saccharomyces cerevisiae cells entrapped in poly (2-hydroxyethyl methacrylate) gels: kinetic and thermostability study in membrane reactors; Cantarella M et al.; Films of poly (2-hydroxyethyl methacrylate) with entrapped yeast cells have been prepared and characterized in membrane reactors . Two concentrations, 5 and 10% w/w, of crosslinking agent ethylene dimethacrylate are used . The invertase activity is monitored between 30 and 55 degrees C in the range of sucrose concentration from 40 to 200 mM and during almost 600 h of operation . Comparison is also made with the behaviour of free cells and small size particles (less than 115 mesh) of poly-HEMA immobilized cells . The results show that the whole membrane volume is not involved in substrate permeation and a combined diffusion-reaction rate controlling mechanism holds . An unusual dependence of reaction rate on bulk pH is observed . In the range of pH from 4.0 to 6.0, invertase activity in films continuously increases while two distinctive maxima for free yeast cells (pH 4.75) and for small particles of poly-HEMA-immobilized yeast cells (pH 4.5) are observed.

Biosci Biotechnol Biochem, 1992 Jun, 56(6), 884 - 9
Construction of an alpha-amylase/glucoamylase fusion gene and its expression in Saccharomyces cerevisiae; Shibuya I et al.; A fusion gene which encoded a polypeptide comprised of 1116 amino acids was constructed using the alpha-amylase and glucoamylase cDNAs of Aspergillus shirousamii . When the fusion gene was expressed in Saccharomyces cerevisiae using a yeast expression plasmid under the control of the yeast ADH1 promoter, a bifunctional fusion protein (145 kDa) having both alpha-amylase and glucoamylase activities was secreted into the culture medium . The fusion protein had higher raw-starch-digesting activity than those of the original alpha-amylase and glucoamylase, and adsorbed onto raw starch like the glucoamylase . It was suggested that the characteristics are a result of the raw-starch-affinity site in the glucoamylase domain of the fusion protein.

Biochem J, 1992 Jun 1, 284 ( Pt 2), 441 - 5
Maltose/proton co-transport in Saccharomyces cerevisiae . Comparative study with cells and plasma membrane vesicles; Van Leeuwen CC et al.; Maltose/proton co-transport was studied in intact cells and in plasma membrane vesicles of the yeast Saccharomyces cerevisiae . In order to determine uphill transport in vesicles, plasma membranes were fused with proteoliposomes containing cytochrome c oxidase as a proton-motive force-generating system . Maltose accumulation, dependent on the electrical and pH gradients, was observed . The initial uptake velocity and accumulation ratio in vesicles proved to be dependent on the external pH . Moreover, kinetic analysis of maltose transport showed that Vmax . values greatly decreased with increasing pH, whereas the Km remained virtually constant . These observations were in good agreement with results obtained with intact cells, and suggest that proton binding to the carrier proceeds with an apparent pK of 5.7 . The observation with intact cells that maltose is co-transported with protons in a one-to-one stoichiometry was ascertained in the vesicle system by measuring the balance between proton-motive force and the chemical maltose gradient . These results show that maltose transport in vesicles prepared by fusion of plasma membranes with cytochrome c oxidase proteoliposomes behaves in a similar way as in intact cells . It is therefore concluded that this vesicle model system offers a wide range of new possibilities for the study of maltose/proton co-transport in more detail.

Mol Cell Biol, 1992 Jun, 12(6), 2872 - 83
Global regulation of mitochondrial biogenesis in Saccharomyces cerevisiae: ABF1 and CPF1 play opposite roles in regulating expression of the QCR8 gene, which encodes subunit VIII of the mitochondrial ubiquinol-cytochrome c oxidoreductase; de Winde JH et al.; The multifunctional DNA-binding proteins ABF1 and CPF1 bind in a mutually exclusive manner to the promoter region of the QCR8 gene, which encodes 11-kDa subunit VIII of the Saccharomyces cerevisiae mitochondrial ubiquinol-cytochrome c oxidoreductase (QCR) . We investigated the roles that the two factors play in transcriptional regulation of this gene . To this end, the overlapping binding sites for ABF1 and CPF1 were mutated and placed in the chromosomal context of the QCR8 promoter . The effects on transcription of the QCR8 gene were analyzed both under steady-state conditions and during nutritional shifts . We found that ABF1 is required for repressed and derepressed transcription levels and for efficient induction of transcription upon escape from catabolite repression, independently of DNA replication . CPF1 acts as a negative regulator, modulating the overall induction response . Alleviation of repression through CPF1 requires passage through the S phase . Implications of these findings for the roles played by ABF1 and CPF1 in global regulation of mitochondrial biogenesis are discussed.

J Cell Biol, 1992 Jun, 117(5), 1067 - 76
Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein; Amatruda JF et al.; Capping protein binds the barbed ends of actin filaments and nucleates actin filament assembly in vitro . We purified capping protein from Saccharomyces cervisiae . One of the two subunits is the product of the CAP2 gene, which we previously identified as the gene encoding the beta subunit of capping protein based on its sequence similarity to capping protein beta subunits in chicken and Dictyostelium (Amatruda, J . F., J . F . Cannon, K . Tatchell, C . Hug, and J . A . Cooper . 1990 . Nature (Lond.) 344:352-354) . Yeast capping protein has activity in critical concentration and low-shear viscometry assays consistent with barbed-end capping activity . Like chicken capping protein, yeast capping protein is inhibited by PIP2 . By immunofluorescence microscopy yeast capping protein colocalizes with cortical actin spots at the site of bud emergence and at the tips of growing buds and shmoos . In contrast, capping protein does not colocalize with actin cables or with actin rings at the site of cytokinesis.

Nature, 1992 May 28, 357(6376), 301 - 6
Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae; Gimble FS et al.; An unusual protein splicing reaction joins the N-terminal segment (A) and the C-terminal segment (C) of the 119K primary translation product (ABC) of the yeast VMA1 gene to yield a 69K vacuolar H(+)-ATPase subunit (AC) and an internal 50K polypeptide (B) . This 50K protein is a site-specific DNA endonuclease that shares 34% identity with the homothallic switching endonuclease . The site cleaved by the VMA1-derived endonuclease exists in a VMA1 allele that lacks the derived endonuclease segment of the open reading frame . Cleavage at this site only occurs during meiosis and initiates 'homing', a genetic event that converts a VMA1 allele lacking the endonuclease coding sequence into one that contains it.

J Biol Chem, 1992 May 25, 267(15), 10457 - 64
Maturation of isoprenylated proteins in Saccharomyces cerevisiae . Multiple activities catalyze the cleavage of the three carboxyl-terminal amino acids from farnesylated substrates in vitro; Hrycyna CA et al.; Eukaryotic polypeptides containing COOH-terminal-CXXX sequences can be posttranslationally modified by isoprenylation of the cysteine residue via a thioether linkage, proteolytic removal of the three terminal amino acids, and alpha-carboxyl methylation of the cysteine residue . Through the development of an indirect coupled assay, we have identified three in vitro activities in the yeast Saccharomyces cerevisiae that can catalyze the proteolytic cleavage of the three COOH-terminal amino acids of the synthetic peptide substrate N-acetyl-KSKTK{S-farnesyl-Cys}VIM . One of these is the vacuolar protease carboxypeptidase Y . Using a mutant strain deficient in this enzyme, we find evidence for an additional soluble activity as well as for a membrane-associated activity . These latter activities are candidates for roles in the physiological processing of isoprenylated protein precursors . They are both insensitive to inhibitors of serine and aspartyl proteinases but are sensitive to sulfhydryl reagents and 0.5 mM ZnCl2 . The soluble activity appears to be a metalloenzyme, inhibitable by 2 mM o-phenanthroline but not by 1 mM N-ethylmaleimide, whereas the membrane-associated enzyme is inhibitable by 1 mM N-ethylmaleimide but not 2 mM o-phenanthroline . We show that the membrane-bound protease is not an activity of the membrane-bound methyltransferase, because protease activity is observed in membrane preparations that lack the STE14-encoded methyltransferase . The soluble activity appears to be a novel carboxypeptidase of approximately 110 kDa that catalyzes a processive removal of amino acids from the COOH terminus from both the farnesylated and non-farnesylated substrate, but not from three other unrelated peptides . Finally, we find no evidence for non-vacuolar membrane or soluble activities that catalyze the ester hydrolysis of N-acetyl-S-farnesyl-L-cysteine methyl ester.

J Mol Biol, 1992 May 20, 225(2), 313 - 26
Mutagenesis of a conserved region of the gene encoding the FLP recombinase of Saccharomyces cerevisiae . A role for arginine 191 in binding and ligation; Friesen H et al.; The FLP recombinase from the 2 microns plasmid of Saccharomyces cerevisiae contains a region from amino acid 185 to 203 that is conserved among several FLP-like proteins from different yeasts . Using site-directed mutagenesis, we have made mutations in this region of the FLP gene . Five of twelve mutations in the region yielded proteins that were unable to bind to the FLP recombination target (FRT) site . A change of arginine at position 191 to lysine resulted in a protein (FLP-R191K) that could bind to the FRT site but could not catalyze recombination . This mutant protein accumulated as a stable protein-DNA complex in which one of the two bound FLP proteins was covalently attached to the DNA . FLP-R191K was defective in strand exchange and ligation and was unable to promote protein-protein interaction with half-FRT sites . The conservation of three residues in all members of the integrase family of site-specific recombinases (His305, Arg308, Tyr343 in FLP) implies a common mechanism of recombination . The conservation of arginine 191 and the properties of the FLP-R191K mutant protein suggest that this arginine also plays an important role in the mechanism of FLP-mediated site-specific recombination.

Biochemistry, 1992 May 19, 31(19), 4729 - 35
Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae; Davies SE et al.; The influence of 6-phosphofructo-1-kinase on glycolytic flux in the yeast Saccharomyces cerevisiae was assessed by measuring the effects of enzyme overexpression on glucose consumption, ethanol production, and glycolytic intermediate levels under aerobic and anaerobic conditions . Enzyme overexpression had no effect on glycolytic flux under anaerobic conditions, but under aerobic conditions, it increased glycolytic flux up to the anaerobic level . The Pasteur effect was thus abolished in these cells . The increased glycolytic flux was accompanied by a compensatory decrease in flux in oxidative phosphorylation . The concentrations of the enzyme substrates showed only small or insignificant changes . These data imply that the enzyme has a low flux control coefficient for glycolysis . However, in cells overexpressing the enzyme, there was a compensatory decrease in 6-phosphofructo-2-kinase activity which was accompanied by a corresponding decrease in fructose 2,6-bisphosphate concentration . Measurements in vitro showed that the decrease in the concentration of this positive allosteric effector of 6-phosphofructo-1-kinase could significantly lower its specific activity in the cell and that this could compensate for the increased enzyme concentration in the overproducer.

Mutat Res, 1992 May 16, 279(2), 121 - 8
Protective effect of two sunscreens against lethal and genotoxic effects of UVB in V79 Chinese hamster cells and Saccharomyces cerevisiae strains XV185-14C and D5; Mondon P et al.; Effects of p-aminobenzoic acid (PABA) and of 4-{(2-oxo-3-bornylidene)methyl}-phenyl trimethylammonium methylsulfate (OMM), two components used in sunscreen formulations, on the mutagenicity of UVB irradiation are compared in three genetic assay systems . A haploid strain of Saccharomyces cerevisiae XV185-14C was used to measure reverse mutations at three loci . The diploid strain D5 of Saccharomyces cerevisiae was used to screen for reciprocal mitotic recombination . The induction of forward mutations was measured in Chinese hamster V79 cells . Our results indicate that UVB irradiation induced HGPRT- mutants in V79 cells, reverse mutations in Saccharomyces cerevisiae strain XV185-14C, and mitotic crossing over and other genetic alterations in Saccharomyces cerevisiae strain D5 . V79 Chinese hamster lung cells were the most sensitive to UVB irradiation, followed by Saccharomyces cerevisiae haploid strain XV185-14C and the diploid strain D5 . PABA and OMM were both capable of protecting all three types of cells from UVB irradiation regarding both lethality and induction of various types of genetic alterations . At higher concentrations (above 10(-5) M), OMM was more effective in its photoprotective effect toward UVB irradiation than PABA.

FEMS Microbiol Lett, 1992 May 15, 72(1), 13 - 6
Fatty acid alterations in Saccharomyces cerevisiae exposed to ethanol stress; Sajbidor J et al.; The influence of ethanol concentration on fatty acid alterations in total phospholipids (PL), phosphatidylcholine (PCH), phosphatidylethanolamine (PE), phosphatidylinositol (PI), sterol esters (ES) and triacylglycerols (TAG) of Saccharomyces cerevisiae was studied . Ethanol induced the elevation of palmitic and oleic acid level in major membrane phospholipids (PCH and PE) and also the palmitoleic acid content in ES and TAG.

Eur J Biochem, 1992 May 15, 206(1), 259 - 67
Specific protein kinase from Saccharomyces cerevisiae cells phosphorylating 60S ribosomal proteins; Pilecki M et al.; A protein kinase, specific for 60S ribosomal proteins, has been isolated from Saccharomyces cerevisiae cells, purified to almost homogeneity and characterized . The isolated enzyme is not related to other known protein kinases . Enzyme purification comprised three chromatography steps; DEAE-cellulose, phosphocellulose and heparin-Sepharose . SDS/PAGE analysis of the purified enzyme, indicated a molecular mass of around 71 kDa for the stained single protein band . The specific activity of the protein kinase was directed towards the 60S ribosomal proteins L44, L44', L45 and a 38 kDa protein . All the proteins are phosphorylated only at the serine residues . None of the 40S ribosomal proteins were phosphorylated in the presence of the kinase . For that reason we have named the enzyme the 60S kinase . An analysis of the phosphopeptide maps of acidic ribosomal proteins, phosphorylated at either the 60S kinase or casein kinase II, showed almost identical patterns . Using the immunoblotting technique, the presence of the kinase has been detected in extracts obtained from intensively growing cells . These findings suggest an important role played by the 60S kinase in the regulation of ribosomal activity during protein synthesis.

J Biol Chem, 1992 May 15, 267(14), 10101 - 7
Isolation and nucleotide sequence of the Saccharomyces cerevisiae gene for the succinate dehydrogenase flavoprotein subunit; Robinson KM et al.; Succinate dehydrogenase (EC 1.3.99.1) of the mitochondrial inner membrane is a four-subunit membrane-bound enzyme that catalyzes the oxidation of succinate to fumarate and the transfer of electrons into the electron transport chain to oxygen . The catalytic domain of the enzyme is composed of a flavoprotein subunit which contains a covalently attached FAD cofactor and an iron-sulfur subunit with three nonidentical iron-sulfur clusters . We have isolated a complete genomic clone for the flavoprotein subunit of the succinate dehydrogenase from Saccharomyces cerevisiae and determined its nucleotide sequence . The sequence predicts a protein of 70,185 Da (640 amino acids) that shows more similarity to the Escherichia coli succinate dehydrogenase flavoprotein subunit than it does to the only other mitochondrial homologue, the human flavoprotein subunit . The yeast flavoprotein subunit precursor was synthesized in a cell-free translation system and shown to possess a mitochondrial targeting sequence that directs its import into isolated, energized mitochondria where it is processed by the matrix-localized protease . The genes for the flavoprotein and the iron-sulfur subunits reside on different chromosomes and hence form different transcriptional units.

J Biol Chem, 1992 May 15, 267(14), 9521 - 8
mRNA capping enzyme . Isolation and characterization of the gene encoding mRNA guanylytransferase subunit from Saccharomyces cerevisiae; Shibagaki Y et al.; The highly purified yeast mRNA capping enzyme is composed of two separate chains of 52 (alpha) and 80 kDa (beta), responsible for the activities of mRNA guanylyltransferase and RNA 5'-triphosphatase, respectively (Itoh, N., Yamada, H., Kaziro, Y., and Mizumoto, K . (1987) J . Biol . Chem . 262, 1989-1995) . The gene encoding the mRNA guanylyltransferase subunit (alpha subunit), CEG1, has been isolated by immunological screening of a yeast genomic expression library in lambda gt11 with polyclonal antibodies directed against purified yeast capping enzyme . The identity of CEG1 was confirmed by epitope selection and by expressing the gene in Escherichia coli to give a catalytically active mRNA guanylyltransferase . The gene is present in one copy per haploid genome, and encodes a polypeptide of 459 amino acid residues . From its primary structure as well as its mRNA size, it was concluded that the alpha and the beta subunits of yeast mRNA capping enzyme are encoded by two separate genes, not as a fused protein . CEG1 is located on the chromosome VII by a pulse-field gel electrophoresis . Gene disruption experiment indicated that CEG1 is essential for the growth of yeast . We have also found another open reading frame (ORF2) which lies in close proximity to CEG1 in our clones and encodes a 450 amino acid-polypeptide of yet unknown function.

J Mol Biol, 1992 May 5, 225(1), 53 - 65
Molecular analysis of Saccharomyces cerevisiae chromosome I . On the number of genes and the identification of essential genes using temperature-sensitive-lethal mutations; Harris SD et al.; Previous analyses of Saccharomyces cerevisiae chromosome I have suggested that the majority (greater than 75%) of single-copy essential genes on this chromosome are difficult or impossible to identify using temperature-sensitive (Ts-) lethal mutations . To investigate whether this situation reflects intrinsic difficulties in generating temperature-sensitive proteins or constraints on mutagenesis in yeast, we subjected three cloned essential genes from chromosome I to mutagenesis in an Escherichia coli mutator strain and screened for Ts- lethal mutations in yeast using the "plasmid-shuffle" technique . We failed to obtain Ts- lethal mutations in two of the genes (FUN12 and FUN20), while the third gene yielded such mutations, but only at a low frequency . DNA sequence analysis of these mutant alleles and of the corresponding wild-type region revealed that each mutation was a single substitution not in the previously identified gene FUN19, but in the adjacent, newly identified essential gene FUN53 . FUN19 itself proved to be non-essential . These results suggest that many essential proteins encoded by genes on chromosome I cannot be rendered thermolabile by single mutations . However, the results obtained with FUN53 suggest that there may also be significant constraints on mutagenesis in yeast . The 5046 base-pair interval sequenced contains the complete FUN19, FUN53 and FUN20 coding regions, as well as a portion of the adjacent non-essential FUN21 coding region . In all, 68 to 75% of this interval is open reading frame . None of the four predicted products shows significant homologies to known proteins in the available databases.

Can J Microbiol, 1992 May, 38(5), 405 - 9
Fluorescence study of lectinlike receptors involved in the flocculation of the yeast Saccharomyces cerevisiae; Masy CL et al.; Flocculation of some yeasts involves lectinlike receptors with two different patterns of inhibition by sugars: mannose sensitive (MS) and glucose-mannose sensitive (GMS) . The visualization and quantification of these receptors were performed using neoglycoproteins fluorescent probes . Fluorescence microscopy showed a homogeneous distribution of surface receptors for the strain belonging to the MS group and a polar distribution for cells belonging to the GMS group . Affinity constants, estimated by fluorimetry, were shown to have different values (MS, 2.6 +/- 0.7 x 10(5) M-1; GMS, 2 +/- 1 x 10(6) M-1), but the number of sites was estimated to be smaller for strain NCYC 1195 which belongs to the GMS group than for strain NCYC 869 from the MS group (MS, 2.4 +/- 0.2 x 10(7) sites/cell; GMS, 3.9 +/- 0.8 x 10(6) sites/cell).

Genetika, 1992 May, 28(5), 56 - 65
{Participation of the HIM1 gene from Saccharomyces cerevisiae in correction of heteroduplex DNA . Molecular cloning of the gene}; Gracheva LM et al.; A group of Saccharomyces cerevisiae mutants deficient in repair of induced premutation lesions (him mutants) were previously isolated in our laboratory . Recessive him1 mutant had enhanced level of spontaneous and induced mutagenesis as well as specific altered mitotic conversion . This HIM1 gene was supposed to be involved in the process of mismatch correction of heteroduplexes . In this paper the correction efficiency of in vitro constructed heteroduplex DNA in wild-type cells and him1 mutant was studied . In the former cells heteroduplex DNA was repaired highly efficiently (about 90%), this repair efficiency being reduced in him cells approx . two times as compared with the wild-type cells . Molecular cloning of yeast chromosomal DNA fragments containing HIM1 gene was carried out . The clones were selected from the bank of yeast DNA fragments by complementing him1-1 mutation which enhances conversion frequency in ADE2 gene . One of the DNA fragments was analysed by restriction endonuclease digestion and shown to contain an insert of 6 Kb . Chromosomal integrants were obtained by homologous recombination between the plasmid and chromosomal gene him1.

Genetika, 1992 May, 28(5), 47 - 55
{Isolation and characteristics of new mutants of Saccharomyces cerevisiae with increased spontaneous mutability}; Ivanov EL et al.; To isolate some new genes controlling the process of spontaneous mutagenesis, a collection of 16 yeast strains with enhanced rate of spontaneous canavanine resistant mutations was obtained . Genetical analysis allowed to define that the mutator phenotype of these strains is due to a single nuclear mutation . Such mutations were called hsm (high spontaneous mutagenesis) . Recombinational test showed that 5 mutants under study carried 5 nonallelic mutations . It was revealed that the mutation hsm3-1 is a nonspecific mutator elevating the rate of both spontaneous canavanine resistant mutations and the frequency of reversions in mutations lys1-1 and his1-7 . Genetical analysis revealed that mutation hsm3-1 is recessive . The study of cross sensitivity of mutator strains to physical and chemical mutagens demonstrated that 12 of 16 hsm mutants were resistant to the lethal action of UV, gamma rays and methylmethanesulfonate, and 4 mutants were only sensitive to these factors . Possible nature of hsm mutations is discussed.

Genetika, 1992 May, 28(5), 11 - 8
{PHO2 and GCN4 transcription activators in the regulation of Saccharomyces cerevisiae acid phosphatase synthesis}; Belova IV et al.; The possibility of substitution of the PHO2 activator protein by GCN4 protein in transcriptional regulation of acid phosphatase genes PHO5 and PHO11 was demonstrated in the yeast Saccharomyces cerevisiae . We observed the increase in acid phosphatase synthesis in the case of gcn4-delta 1 pho2-delta 1 mutant transformed with YCp88 (GCN4-wt) plasmid in comparison with that in nontransformed cells . The mode of repression of acid phosphatase synthesis in two types of transformants gcn4-delta 1 pho2-delta 1 (GCN4-wt) and gcn4-delta 1 pho2-delta 1 (PHO2-wt) was studied . It wat demonstrated that repression of acid phosphatase synthesis in the first type of transformants took place at lower concentration of Pi than was necessary for the second type of transformants . The model of interactions between the GCN4 activator protein and PHO regulatory factors is proposed.

Yeast, 1992 May, 8(5), 409 - 17
The complete sequence of a 10.8 kb segment distal of SUF2 on the right arm of chromosome III from Saccharomyces cerevisiae reveals seven open reading frames including the RVS161, ADP1 and PGK genes; Skala J et al.; We have entirely sequenced a 10,835 bp segment of the right arm from chromosome III contained in the J11D and J11D-K3B GF clones . The segment contains seven open reading frames longer then 100 amino acids . Three of them, RVS161 (Urdaci et al., 1990; Crouzet et al., 1991), ADP1 (Purnelle et al., 1991) and PGK1 (Hitzeman et al., 1982) have been described previously . YCR10C encodes a putative membrane protein . YCR8W (encoding a putative protein kinase) and YCR14C extend inside the D10H (Skala et al., 1991) and 62B5-2D clones respectively . Four ARS elements previously reported by Palzkill et al . (1986) are located between RVS161 and YCR10C.

Yeast, 1992 May, 8(5), 353 - 9
N-linked glycosylation of proteinase B precursors of the yeast Saccharomyces cerevisiae is not required for proper targeting or processing of the enzyme; Nebes VL et al.; Proteinase B precursors are modified by an N-linked carbohydrate side chain at Asn 314 . Glycosylation at this position is not required for proper localization, processing, or activation of the enzyme.

Curr Genet, 1992 May, 21(6), 437 - 42
Multiple copies of SUC4 regulatory regions may cause partial de-repression of invertase synthesis in Saccharomyces cerevisiae; Gozalbo D; Transformation to generate multiple copies of regulatory DNA sequences has been used to study the interactions between regulatory proteins and their target sequences, since a high copy number of these sequences may titrate trans-acting regulatory proteins . We have analyzed the synthesis of invertase in yeast strains carrying different SUC genes transformed with the multiple-copy plasmid pSH143, a derivative of pJDB207 containing the promoter and upstream regulatory sequences of SUC4 . The results obtained seem to be strain dependent . Under repressing conditions a high copy number of SUC4 promoter regions may cause increased expression of the invertase genes resulting in the synthesis of external glycosylated protein . A similar result was obtained under de-repressing conditions since transformants from some strains showed higher levels of activity . These results suggest that transcriptional regulatory (negative) factors may become limiting when the copy number of their target DNA sequences is increased . This effect may depend on the amount of active repressor molecules as well as on their affinity for SUC4 upstream sequences . This is discussed on the basis of the nucleotide sequences of SUC promoters.

Mol Gen Genet, 1992 May, 233(1-2), 327 - 30
The Saccharomyces cerevisiae GAM2/SIN3 protein plays a role in both activation and repression of transcription; Yoshimoto H et al.; We have cloned GAM2, which is required for transcription of STA1, a gene encoding an extracellular glucoamylase in Saccharomyces cerevisiae var . diastaticus . DNA sequence analysis revealed that GAM2 is the same gene as SIN3, known to be a general negative regulator of yeast genes . RNA blot analysis indicated that GAM2/SIN3 also acts as a positive regulator of GAM3/ADR6, which in turn is required for transcription of STA1 and ADH2 . These results suggest that GAM2 regulates STA1 expression through transcriptional activation of GAM3 and indicate that GAM2/SIN3 protein is a transcriptional regulator that can play a role in both activation and repression of transcription.

Mol Gen Genet, 1992 May, 233(1-2), 136 - 44
The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed; Ekwall K et al.; We have characterized the nuclear gene NAM8 in Saccharomyces cerevisiae . It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid . The suppressed mutations affect RNA folding and are located in both group I and group II introns . The gene is weakly transcribed in wild-type strains, its overexpression is a prerequisite for the suppressor action . Inactivation of the NAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability . The NAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms . This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.

Cell Prolif, 1992 May, 25(3), 217 - 23
Clonal heterogeneity in specific growth rate of Saccharomyces cerevisiae cells; Wheals AE et al.; The cell volume increase in individual clones of cells of the yeast Saccharomyces cerevisiae has been measured using time lapse cinematography in populations showing steady state balanced exponential growth . There were significant differences in clonal specific growth rates within the population in each of 10 experiments using different strains on different media supporting different growth rates . The results suggest that specific growth rates of cells which are either genetically identical or very closely related can be different and this difference can be propagated over at least three generations . Since the proliferation rate in yeast is determined by growth rate, these observed differences provide an additional source of cell cycle variability for yeast cells that has not been considered before . The implications for the theoretical analysis of cell cycle kinetics are examined.

Genetics, 1992 May, 131(1), 43 - 53
Meiosis in Saccharomyces cerevisiae mutants lacking the centromere-binding protein CP1; Masison DC et al.; CP1 (encoded by the CEP1 gene) is a centromere binding protein of Saccharomyces cerevisiae that binds to the conserved DNA element I (CDEI) of yeast centromeres . To investigate the function of CP1 in yeast meiosis, we analyzed the meiotic segregation of CEN plasmids, nonessential chromosome fragments (CFs) and chromosomes in cep1 null mutants . Plasmids and CFs missegregated in 10-20% of meioses with the most frequent type of aberrant event being precocious sister segregation at the first meiotic division; paired and unpaired CFs behaved similarly . An unpaired chromosome I homolog (2N + 1) also missegregated at high frequency in the cep1 mutant (7.6%); however, missegregation of other chromosomes was not detected by tetrad analysis . Spore viability of cep1 tetrads was significantly reduced, and the pattern of spore death was nonrandom . The inviability could not be explained solely by chromosome missegregation and is probably a pleiotropic effect of cep1 . Mitotic chromosome loss in cep1 strains was also analyzed . Both simple loss (1:0 segregation) and nondisjunction (2:0 segregation) were increased, but the majority of loss events resulted from nondisjunction . We interpret the results to suggest that CP1 generally promotes chromatid-kinetochore adhesion.

Genetics, 1992 May, 131(1), 21 - 9
Temperature-sensitive cdc7 mutations of Saccharomyces cerevisiae are suppressed by the DBF4 gene, which is required for the G1/S cell cycle transition; Kitada K et al.; When present on a multicopy plasmid, a gene from a Saccharomyces cerevisiae genomic library suppresses the temperature-sensitive cdc7-1 mutation . The gene was identified as DBF4, which was previously isolated by complementation in dbf4-1 mutant cells and is required for the G1----S phase progression of the cell cycle . DBF4 has an open reading frame encoding 695 amino acid residues and the predicted molecular mass of the gene product is 80 kD . The suppression is allele-specific because a CDC7 deletion is not suppressed by DBF4 . Suppression is mitosis-specific and the sporulation defect of cdc7 mutations is not suppressed by DBF4 . Conversely, CDC7 on a multicopy plasmid suppresses the dbf4-1, -2, -3 and -4 mutations but not dbf4-5 and DBF4 deletion mutations . Furthermore, cdc7 mutations are incompatible with the temperature-sensitive dbf4 mutations . These results suggest that the CDC7 and DBF4 polypeptides interact directly or indirectly to permit initiation of yeast chromosome replication.

Gene, 1992 May 1, 114(1), 43 - 9
Isolation and characterization of two novel ras superfamily genes in Saccharomyces cerevisiae; Matsui Y et al.; We have isolated two novel genes, designated as RHO3 (on chromosome IX) and RHO4 (on chromosome XI), belonging to the ras superfamily, from the yeast Saccharomyces cerevisiae . Deletion of RHO4 did not affect cell growth, but deletion of RHO3 caused a severe growth delay and a decrease in cell viability . The rho3 and rho4 disrupted genes displayed synthetic lethality at 30 degrees C . RHO3 and RHO4 seem to have overlapping functions in cell growth, because the growth defect in rho3 cells was suppressed by overexpression of RHO4 despite the low homology (about 35%) at the amino acid sequence level between the RHO3 and RHO4 proteins.

Gene, 1992 May 1, 114(1), 145 - 8
A putative second adenylate kinase-encoding gene from the yeast Saccharomyces cerevisiae; Cooper AJ et al.; Sequencing of the region upstream from the yeast RAD3 gene has revealed an open reading frame (ORF) of 225 amino acids (aa) that could encode a 25.3-kDa polypeptide . The predicted aa sequence of this ORF is homologous with that of several eukaryotic adenylate kinase (Adk)-encoding genes, including the yeast gene, ADK1 . These findings suggest that the yeast Saccharomyces cerevisiae has a second Adk-encoding gene, tentatively designated as ADK2.

J Bacteriol, 1992 May, 174(10), 3411 - 5
Allelism of IMP1 and GAL2 genes of Saccharomyces cerevisiae; Donnini C et al.; Cloning and characterization of the previously described Saccharomyces cerevisiae IMP1 gene, which was assumed to be a nuclear determinant involved in the nucleomitochondrial control of the utilization of galactose, demonstrate allelism to the GAL2 gene . Galactose metabolism does not necessarily involve the induction of the specific transport system coded by GAL2/IMP1, because a null mutant takes up galactose and grows on it . Data on galactose uptake are presented, and the dependence on ATP for constitutive and inducible galactose transport is discussed . These results can account for the inability of imp1/gal2 mutants to grow on galactose in a respiration-deficient background . Under these conditions, uptake was affected at the functional level but not at the biosynthetic level.

Eur J Biochem, 1992 May 1, 205(3), 1011 - 6
Uroporphyrinogen decarboxylase in Saccharomyces cerevisiae . HEM12 gene sequence and evidence for two conserved glycines essential for enzymatic activity; Garey JR et al.; The HEM12 gene from Saccharomyces cerevisiae encodes uroporphyrinogen decarboxylase which catalyzes the sequential decarboxylation of the four acetyl side chains of uroporphyrinogen to yield coproporphyrinogen, an intermediate in protoheme biosynthesis . The gene was isolated by functional complementation of a hem12 mutant . Sequencing revealed that the HEM12 gene encodes a protein of 362 amino acids with a calculated molecular mass of 41,348 Da . The amino acid sequence shares 50% identity with human and rat uroporphyrinogen decarboxylase and shows 40% identity with the N-terminus of an open reading frame described in Synechococcus sp . We determined the sequence of two hem12 mutations which lead to a totally inactive enzyme . They correspond to the amino acid changes Gly33----Asp and Gly300----Asp, located in two evolutionarily conserved regions . Each of these substitutions impairs binding of substrates without affecting the overall conformation of the protein . These results argue that a single active center exists in uroporphyrinogen decarboxylase.

Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 3869 - 73
Ferric reductase of Saccharomyces cerevisiae: molecular characterization, role in iron uptake, and transcriptional control by iron; Dancis A et al.; The principal iron uptake system of Saccharomyces cerevisiae utilizes a reductase activity that acts on ferric iron chelates external to the cell . The FRE1 gene product is required for this activity . The deduced amino acid sequence of the FRE1 protein exhibits hydrophobic regions compatible with transmembrane domains and has significant similarity to the sequence of the plasma membrane cytochrome b558 (the X-CGD protein), a critical component of a human phagocyte oxidoreductase, suggesting that FRE1 is a structural component of the yeast ferric reductase . FRE1 mRNA levels are repressed by iron . Fusion of 977 base pairs of FRE1 DNA upstream from the translation start site of an Escherichia coli lacZ reporter gene confers iron-dependent regulation on expression of beta-galactosidase in yeast . An 85-base-pair segment of FRE1 5' noncoding sequence contains a RAP1 binding site and a repeated sequence, TTTTTGCTCAYC; this segment is sufficient to confer iron-repressible transcriptional activity on heterologous downstream promoter elements.

Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 3736 - 40
Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae; Anderson JA et al.; We report the isolation of a cDNA (KAT1) from Arabidopsis thaliana that encodes a probable K+ channel . KAT1 was cloned by its ability to suppress a K+ transport-defective phenotype in mutant Saccharomyces cerevisiae cells . This suppression is sensitive to known K+ channel blockers, including tetraethylammonium and Ba2+ ions . The KAT1 cDNA contains an open reading frame capable of encoding a 78-kDa protein that shares structural features found in the Shaker superfamily of K+ channels . These include a cluster of six putative membrane-spanning helices (S1-S6) at the amino terminus of the protein, a presumed voltage-sensing region containing Arg/Lys-Xaa-Xaa-Arg/Lys repeats within S4, and the highly conserved pore-forming region (known as H5 or SS1-SS2) . Our results suggest that the structural motif for K+ channels has been conserved between plants and animals.

Mol Cell Biol, 1992 May, 12(5), 2165 - 77
Gene products that promote mRNA turnover in Saccharomyces cerevisiae; Leeds P et al.; We showed previously that the increased rate of mRNA turnover associated with premature translational termination in the yeast Saccharomyces cerevisiae requires a functional UPF1 gene product . In this study, we show that the UPF1 gene codes for a 109-kDa primary translation product whose function is not essential for growth . The protein contains a potential zinc-dependent nucleic acid-binding domain and a nucleoside triphosphate-binding domain . A 300-amino-acid segment of the UPF1 protein is 36% identical to a segment of the yeast SEN1 protein, which is required for endonucleolytic processing of intron-containing pre-tRNAs . The same region is 32% identical to a segment of Mov-10, a mouse protein of unknown function . Dominant-negative upf1 mutations were isolated following in vitro mutagenesis of a plasmid containing the UPF1 gene . They mapped exclusively at conserved positions within the sequence element common to all three proteins, whereas the recessive upf1-2 mutation maps outside this region . The clustering of dominant-negative mutations suggests the presence of a functional domain in UPF1 that may be shared by all three proteins . We also identified upf mutations in three other genes designated UPF2, UPF3, and UPF4 . When alleles of each gene were screened for effects on mRNA accumulation, we found that the recessive mutation upf3-1 causes increased accumulation of mRNA containing a premature stop codon . When mRNA half-lives were measured, we found that excess mRNA accumulation was due to mRNA stabilization . On the basis of these results, we suggest that the products of at least two genes, UPF1 and UPF3, are responsible for the accelerated rate of mRNA decay associated with premature translational termination.

Mol Cell Biol, 1992 May, 12(5), 2154 - 64
SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae; DeMarini DJ et al.; The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs . A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity . Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex . We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles . The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa) . Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1 . The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence . The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable . A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown . The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

Mol Cell Biol, 1992 May, 12(5), 2091 - 9
The Saccharomyces cerevisiae CDC25 gene product binds specifically to catalytically inactive ras proteins in vivo; Munder T et al.; Genetic data suggest that the yeast cell cycle control gene CDC25 is an upstream regulator of RAS2 . We have been able to show for the first time that the guanine nucleotide exchange proteins Cdc25 and Sdc25 from Saccharomyces cerevisiae bind directly to their targets Ras1 and Ras2 in vivo . Using the characteristics of the yeast Ace1 transcriptional activator to probe for protein-protein interaction, we found that the CDC25 gene product binds specifically to wild-type Ras2 but not to the mutated Ras2Val-19 and Ras2 delta Val-19 proteins . The binding properties of Cdc25 to Ras2 were strongly diminished in yeast cells expressing an inactive Ira1 protein, which normally acts as a negative regulator of Ras activity . On the basis of these data, we propose that the ability of Cdc25 to interact with Ras2 proteins is strongly dependent on the activation state of Ras2 . Cdc25 binds predominantly to the catalytically inactive GDP-bound form of Ras2, whereas a conformational change of Ras2 to its activated GTP-bound state results in its loss of binding affinity to Cdc25.

Mol Cell Biol, 1992 May, 12(5), 2017 - 28
Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae; Walworth NC et al.; Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae . To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP . The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels . A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein . The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain . The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells . However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function . While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.

Exp Cell Res, 1992 May, 200(1), 48 - 57
A new essential gene of Saccharomyces cerevisiae, a defect in it may result in instability of nucleus; Nisogi H et al.; We identified a new essential gene of Saccharomyces cerevisiae, designated NIN1 (nuclear integrity) . The nin1-1 mutant cells showed temperature-sensitive growth, and their nuclei underwent deformation and eventually were broken down upon a temperature shift . When the nin1-1 cells growing at 25 degrees C were transferred to a nonpermissive temperature, the nucleus moved to the isthmus and cells with a large bud accumulated . Flow cytometry proved that G2 nuclei accumulated during incubation at a nonpermissive temperature . NIN1 is mapped on chromosome VI, 16 cM centromere-distal to PHO4 . Cloning and sequencing of NIN1 revealed that this is a new gene . Disruption of NIN1 resulted in cell death; however, a dead spore clone contained about 30 dead cells, indicating that spores that received the disrupted nin1 allele divided approximately five times before their death . Western blotting using anti-Nin1 antibody reveals that NIN1 produces a doublet consisting of 32 and 34 kDA whose size is close enough to that calculated from the predicted amino acid sequence of Nin1.

Photochem Photobiol, 1992 May, 55(5), 713 - 21
Comparative studies on the lethal, mutagenic, and recombinogenic effects of ultraviolet -A, -B, -C, and visible light with and without 8-methoxypsoralen in Saccharomyces cerevisiae; Mondon P et al.; Genetic effects of UV-A, UV-B, UV-C, and the combination of 8-methoxypsoralen (8-MOP) with UV-A or visible light were studied in the haploid strain XV185-14C and diploid strain D5 of Saccharomyces cerevisiae . The induction of his+, lys+, and hom+ reverse mutations was measured in strain XV185-14C . In strain D5 we measured the induction of genetically altered colonies, particularly twin spot colonies arising from a mitotic crossing-over . UV-C and UV-B induced point mutations at the three loci in the haploid strain and mitotic crossing-over and other genetic alterations in the diploid strain . UV-C was more mutagenic and recombinogenic than UV-B . UV-A or visible light alone did not induce genotoxic effects at the doses tested . However, UV-A plus 8-MOP produced lethal and mutagenic effects in the haploid strain XV185-14C, although mutagenic activity was less than that of UV-B . Visible light plus 8-MOP also induced genotoxic effects in strain XV185-14C . In the diploid strain D5, UV-A plus 8-MOP induced a higher frequency of genetic alterations than UV-B at comparative doses . Visible light plus 8-MOP was also genetically active in strain D5 . The haploid strain was more sensitive to the lethal effects of UV-C, UV-B, UV-A, and impure visible light plus 8-MOP than the diploid strain.

Antonie Van Leeuwenhoek, 1992 May, 61(4), 269 - 76
Alterations in the cell wall of Saccharomyces cerevisiae induced by the alpha sex factor or a mutation in the cell cycle; Diaz S et al.; We performed experiments in parallel to study the rate of synthesis of cell wall polysaccharides and the activity of glycosyl transferases in Saccharomyces cerevisiae after arrest of a cdc 28 mutant in G1 phase by either addition of alpha-factor or transfer to the non-permissive temperature . Both effectors brought about similar time-dependent increases in the rate of synthesis and deposition of the cell wall polysaccharides chitin, glucan and mannan . These changes in cell wall composition were accompanied by an increase in the specific activities of glucan and chitin synthetases . This increase was inhibited by cycloheximide suggesting that it represented de novo enzyme biosynthesis and not enzyme activation . Our data are consistent with the notion that both alpha-factor and the cdc 28 mutation affect the same stage-specific function that controls the temporal expression of glycosyl transferases.

Mutat Res, 1992 May, 273(3), 281 - 8
Evidence for excision repair in promitochondrial DNA of anaerobic cells of Saccharomyces cerevisiae; Pasupathy K et al.; The respiratory adaptation (i.e., essentially mitochondrial biogenesis) in the excision repair-defective rad3-type mutants of Saccharomyces cerevisiae undergoing transition from the anaerobic to the aerobic state is found to be far more sensitive to 254-nm ultraviolet radiation (UV) than that of the RAD wild-type strain . We confirm that mitochondria of aerobic cells of a RAD strain lack the excision repair capacity of UV-induced pyrimidine dimers at all doses tested (1-15 J/m2) . In contrast, in promitochondria of anaerobic cells of the wild-type strain excision repair appears to take place . This process is very efficient at low doses (at 0.5-5 J/m2 100% of the UV endonuclease-sensitive sites disappear), whereas at high doses its efficiency is reduced by about 50% . The promitochondrial excision repair of pyrimidine dimers appears to be under nuclear control since it is blocked in the rad2 mutant . Finally photoreactivation is found to be operating in nuclei, mitochondria and promitochondria.

Mutat Res, 1992 May 1, 279(1), 41 - 8
Comparison of chemically induced chromosome loss in a diploid, triploid, and tetraploid strain of Saccharomyces cerevisiae; Mayer VW et al.; Triploid and tetraploid strains of Saccharomyces cerevisiae were constructed and the spontaneous loss during mitosis of one, two or three copies of chromosome VII was determined . In one strain, a triploid (VM2) in which expression of the recessive alleles can be observed only after loss of two copies of chromosome VII (3N-2), the spontaneous frequency of chromosome loss was lower than in the diploid D61.M . In another strain, a tetraploid (VM4) that also requires the loss of two copies of chromosome VII for observation (4N-2) of the recessive alleles, the spontaneous frequency was slightly higher than in the diploid D61.M . The spontaneous frequency of other genetic events (that is, mutation, recombination or chromosome breakage) were lower by 2-3 orders of magnitude than in the diploid strain D61.M . Induction of chromosome loss and other genetic events by nocodazole, ethyl acetate, hydroxyurea and ethyl methanesulfonate was determined in D61.M, VM2, and VM4, and the results were compared . Nocodazole and ethyl acetate induced chromosome loss in both the triploid and the tetraploid strains at lower concentrations than required in the diploid . These compounds also induced elevated frequencies of other genetic events in both the triploid and the tetraploid strains but not in the diploid . Hydroxyurea induced elevated frequencies of chromosome loss in the diploid and the tetraploid . Frequencies of chromosome loss in the triploid treated with hydroxyurea, although elevated, are based on observation of very few colonies of the correct phenotype . Ethyl methanesulfonate failed to induce chromosome loss in any of the three strains . Hydroxyurea and ethyl methanesulfonate did, however, induce very high frequencies of other genetic events.

J Bacteriol, 1992 May, 174(10), 3125 - 32
Effects of bleomycin on growth kinetics and survival of Saccharomyces cerevisiae: a model of repair pathways; Keszenman DJ et al.; In order to analyze the roles of some repair genes in the processing of bleomycin-induced DNA damage and, especially, the interrelationships among the involved repair pathways, we investigated the potentially lethal effect of bleomycin on radiosensitive mutants of Saccharomyces cerevisiae defective in recombination, excision, and RAD6-dependent DNA repair . Using single, double, and triple rad mutants, we analyzed growth kinetics and survival curves as a function of bleomycin concentration . Our results indicate that genes belonging to the three epistasis groups interact in the repair of bleomycin-induced DNA damage to different degrees depending on the concentration of bleomycin . The most important mechanisms involved are recombination and postreplication repair . The initial action of a potentially inducible excision repair gene could provide intermediate substrates for the RAD6- and RAD52-dependent repair processes . Interaction between RAD6 and RAD52 genes was epistatic for low bleomycin concentrations . RAD3 and RAD52 genes act independently in processing DNA damage induced by high concentrations of bleomycin . The synergistic interaction observed at high concentrations in the triple mutant rad2-6 rad6-1 rad52-1 indicates partial independence of the involved repair pathways, with possible common substrates . On the basis of the present results, we propose a heuristic model of bleomycin-induced DNA damage repair.

Mol Cell Biol, 1992 May, 12(5), 2322 - 30
Different sequence elements are required for function of the cauliflower mosaic virus polyadenylation site in Saccharomyces cerevisiae compared with in plants; Irniger S et al.; We show that the polyadenylation site derived from the plant cauliflower mosaic virus (CaMV) is specifically functional in the yeast Saccharomyces cerevisiae . The mRNA 3' endpoints were mapped at the same position in yeast cells as in plants, and the CaMV polyadenylation site was recognized in an orientation-dependent manner . Mutational analysis of the CaMV 3'-end-formation signal revealed that multiple elements are essential for proper activity in yeast cells, including two upstream elements that are situated more than 100 and 43 to 51 nucleotides upstream of the poly(A) addition site and the sequences at or near the poly(A) addition site . A comparison of the sequence elements that are essential for proper function of the CaMV signal in yeast cells and plants showed that both organisms require a distal and a proximal upstream element but that these sequence elements are not identical in yeast cells and plants . The key element for functioning of the CaMV signal in yeast cells is the sequence TAGTATGTA, which is similar to a sequence previously proposed to act in yeast cells as a bipartite signal, namely, TAG .. . TATGTA . Deletion of this sequence in the CaMV polyadenylation signal abolished 3'-end formation in yeast cells, and a single point mutation in this motif reduced the activity of the CaMV signal to below 15% . These results indicate that the bipartite sequence element acts as a signal for 3'-end formation in yeast cells but only together with other cis-acting elements.

Genetics, 1992 May, 131(1), 31 - 42
Fitness effects of Ty transposition in Saccharomyces cerevisiae; Wilke CM et al.; It has been suggested that the primary evolutionary role of transposable elements is negative and parasitic . Alternatively, the target specificity and gene regulatory capabilities of many transposable elements raise the possibility that transposable element-induced mutations are more likely to be adaptively favorable than other types of mutations . Populations of Saccharomyces cerevisiae containing large amounts of variation for Ty1 genomic insertions were constructed, and the effects of Ty1 copy number on two components of fitness, yield and growth rate were determined . Although mean stationary phase density decreased with increased Ty1 copy number, the variance and range increased . The distributions of stationary phase densities indicate that many Ty1 insertions have negative effects on fitness, but also that some may have positive effects . To test directly for adaptively favorable Ty1 insertions, populations containing large amounts of variability for Ty1 copy number were grown in continuous culture . After 98-112 generations the frequency of clones containing zero Ty1 elements had decreased to approximately 0.0, and specific Ty1-containing clone families had predominated . Considering that most of the genetic variation in the populations was due to Ty1 transposition, and that Ty1 insertions had, on average, a negative effect on fitness, we conclude that Ty1 transposition events were directly responsible for the production of adaptive mutations in the clones that predominated in the populations.

Gene, 1992 May 1, 114(1), 51 - 8
A novel, highly regulated, rapidly inducible system for the expression of chicken progesterone receptor, cPRA, in Saccharomyces cerevisiae; Poletti A et al.; A rapidly inducible and tightly regulated system for the expression of protein in yeast is based on a chimeric promoter constructed of two copies of a vitellogenin-estrogen-response element (ERE) which are inserted upstream from the promoter of the yeast gene encoding iso-1-cytochrome c . The chimeric promoter was inserted in a yeast expression plasmid upstream from the coding sequence of ubiquitin fused in frame to a cDNA encoding the full-length chicken progesterone receptor A (cPRA) . The resultant plasmid (YEpA2) was co-transformed in Saccharomyces cerevisiae with a plasmid which encodes the human estrogen receptor . Estradiol (E2)-induced transactivation of the chimeric promoter results in transcription of the cPRA gene from YEpA2, and synthesis of cPRA . The fusion protein, ubiquitin-cPRA, is rapidly cleaved in vivo to produce cPRA . Analysis of samples by Western immunoblot shows that cPRA is almost undetectable in the absence of E2, and that treatment with 50 nM E2 results in a 500-1000-fold induction of cPRA (0.06-0.3% of the total protein) after 1 h . The plasmid-expressed soluble receptor is stable and demonstrates the correct affinity for its ligand . We have prepared yeast extracts using enzymatic digestion of the cell wall with oxalyticase followed by hypotonic shock . This has resulted in a dramatic increase in the % of receptor which binds hormone compared to previous studies which used mechanical disruption techniques . The cPRA is biologically active since it activates transcription of a co-transformed reporter gene containing its response element.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1992 May, 174(9), 3065 - 9
The low-affinity component of Saccharomyces cerevisiae maltose transport is an artifact; Benito B et al.; It has been reported by several laboratories that maltose transport in Saccharomyces cerevisiae consists of two components with high- and low-affinity constants for maltose . We have investigated the characteristics of the low-affinity component and have found that it shows an abnormal behavior without similarity to any transport mechanism described in this organism . The results strongly indicate that this apparent transport activity is due not to a genuine transport process but to nonspecific binding of maltose to the cell wall and plasma membrane.

FEMS Microbiol Lett, 1992 Apr 15, 71(2), 125 - 7
Mapping of the PCK1 gene encoding phosphoenolpyruvate carboxykinase on chromosome XI of Saccharomyces cerevisiae; Delgado MA et al.; The gene PCK1 encoding phosphoenolpyruvate carboxykinase of Saccharomyces cerevisiae has been mapped on the right arm of chromosome XI, 12.7 centimorgans proximal to MAL4 and 20.1 centimorgans distal to MET1 . In order to map the gene, hybridization of PCK1 DNA with separated yeast chromosomes and tetrad analysis of diploids with adequate markers were carried out.

J Biol Chem, 1992 Apr 15, 267(11), 7386 - 94
Characterization of ATP11 and detection of the encoded protein in mitochondria of Saccharomyces cerevisiae; Ackerman SH et al.; In Saccharomyces cerevisiae, expression of functional F1-ATPase requires two proteins encoded by the ATP11 and ATP12 genes . Mutations in either gene block some crucial late step in assembly of F1, causing the alpha and beta subunits to accumulate in mitochondria as inactive aggregates (Ackerman, S . H., and Tzagoloff, A . (1991) Proc . Natl . Acad . Sci . U.S.A . 87, 4986-4990) . In the present study we have cloned and determined the sequence of ATP11 . The encoded product is protein of 37 kDa with no obvious homology to any known protein . In vitro import assays of ATP11 precursor and immunochemical evidence indicate that the protein is located in mitochondria . A fusion was made between ATP11 and a short sequence coding for 78 amino acids with the biotination signal of bacterial transcarboxylase . The protein expressed from this construct complements atp11 mutants, indicating that the addition of the extra 78 amino acids at the carboxyl terminus of the ATP11 protein does not compromise its function . The hybrid protein is detected in mitochondria with antibodies and with peroxidase-conjugated avidin . Biotinated ATP11 protein can be partially purified by affinity chromatography on monomeric or tetrameric avidin coupled to Sepharose . A fraction eluted from the avidin column and enriched for the biotinated ATP11 protein also contains the alpha and beta subunits of F1-ATPase.

J Biol Chem, 1992 Apr 15, 267(11), 7839 - 44
Substrate specificity of the Rad3 ATPase/DNA helicase of Saccharomyces cerevisiae and binding of Rad3 protein to nucleic acids; Naegeli H et al.; Rad3 protein from the yeast Saccharomyces cerevisiae is a single-stranded DNA-dependent ATPase which catalyzes the unwinding of DNA.DNA duplexes . In the present studies we have demonstrated that the purified enzyme additionally catalyzes the displacement of RNA fragments annealed to complementary DNA . Quantitative comparisons using otherwise identical partially duplex DNA.DNA and DNA.RNA substrates indicate a significant preference for the latter . Competition for ATPase or DNA helicase activity by various homopolymers suggests that Rad3 protein does not discriminate between ribonucleotide and deoxyribonucleotide homopolymers with respect to binding . However, neither single-stranded RNA nor various ribonucleotide homopolymers supported the hydrolysis of nucleoside 5'-triphosphates . Additionally, Rad3 protein was unable to catalyze the displacement of oligo(dA) annealed to poly(U), suggesting that the catalytic domain of the enzyme is exquisitely sensitive to chemical and/or or conformational differences between DNA and RNA . Hence, it appears that Rad3 protein is not an RNA helicase.

Biochemistry, 1992 Apr 14, 31(14), 3694 - 702
Excision repair of DNA in nuclear extracts from the yeast Saccharomyces cerevisiae; Wang Z et al.; Excision repair of DNA is an important cellular response to DNA damage caused by a broad spectrum of physical and chemical agents . We have established a cell-free system in which damage-specific DNA repair synthesis can be demonstrated in vitro with nuclear extracts from the yeast Saccharomyces cerevisiae . Repair synthesis of UV-irradiated plasmid DNA was observed in a radiation dose-dependent manner and was unaffected by mutations in the RAD1, RAD2, RAD3, RAD4, RAD10, or APN1 genes . DNA damaged with cis-platin was not recognized as a substrate for repair synthesis . Further examination of the repair synthesis observed with UV-irradiated DNA revealed that it is dependent on the presence of endonuclease III-sensitive lesions in DNA, but not pyrimidine dimers . These observations suggest that the repair synthesis observed in yeast nuclear extracts reflects base excision repair of DNA . Our data indicate that the patch size of this repair synthesis is at least seven nucleotides . This system is expected to facilitate the identification of specific gene products which participate in base excision repair in yeast.

FEBS Lett, 1992 Apr 13, 301(1), 29 - 33
A gene family homologous to the S-phase specific gene in higher plants is essential for cell proliferation in Saccharomyces cerevisiae; Ito M et al.; Previously we reported the isolation and characterization of the gene, cyc07, which was specifically expressed in the S phase during the cell cycle in synchronous cell division cultures of the higher plant, Catharanthus roseus . We found that the yeast Saccharomyces cerevisiae contains two closely related genes which show a high degree of similarity (about 64% at the amino acid level) to cyc07 of C . roseus . Site-directed disruption mutations demonstrated that the two yeast genes, homologous to cyc07, constitute an essential gene family for cell proliferation in yeast cells . Furthermore, the rate of cell proliferation varied with the gene copy number.

J Mol Biol, 1992 Apr 5, 224(3), 575 - 87
NAM7 nuclear gene encodes a novel member of a family of helicases with a Zn-ligand motif and is involved in mitochondrial functions in Saccharomyces cerevisiae; Altamura N et al.; The yeast nuclear gene NAM7 was previously isolated within a genomic fragment of 7.7 kb (1 kb = 10(3) bases or base-pairs), having the ability to suppress mitochondrial intronic mutations defective in RNA splicing . We have identified and sequenced the region on the insert corresponding to the NAM7 gene . A long open reading frame has been revealed which could code for a protein of 971 amino acids . Comparison of the NAM7 putative protein with data libraries did not reveal any strong similarity with known proteins . However, the NAM7 protein contains several motifs typical for proteins interacting with nucleic acids: (1) five motifs diagnostic for a superfamily of helicases appear in the same order and with similar distances; (2) the N-terminal portion possesses potential Zn-ligand structures belonging to the C chi superfamily . Deletion of the chromosomal copy of NAM7 gene leads to a partial impairment in respiratory growth that is particularly striking at low temperature . Southern blot analysis of DNA extracted from a nam7 :: URA3 deleted strain revealed the presence of a second gene whose sequence is related to that of the NAM7 gene and which could participate in the same process.

FEMS Microbiol Lett, 1992 Apr 1, 71(1), 57 - 62
The promoter element GTACAAG of the SGA and STA2 genes is a possible target site for repression by the STA10 gene product from Saccharomyces cerevisiae; Claros MG et al.; The SGA and STA2 genes that, respectively, encode the intra- and extracellular glucoamylases of Saccharomyces cerevisiae are coregulated negatively, at the level of transcription, by the STA10 gene . This finding was re-examined by determining the effects of STA10 on the expression of gene constructs containing different fragments from the SGA and STA2 promoter regions fused to the lacZ gene . Repression was observed only for promoter fragments carrying the sequence GTACAAG indicating that this element is responsible for the coregulation of SGA and STA2 by STA10.

Mol Gen Genet, 1992 Apr, 232(3), 408 - 14
Isolation and phenotypic analysis of conditional-lethal, linker-insertion mutations in the gene encoding the largest subunit of RNA polymerase II in Saccharomyces cerevisiae; Archambault J et al.; Linker-insertion mutagenesis was used to isolate mutations in the Saccharomyces cerevisiae gene encoding the largest subunit of RNA polymerase II (RPO21, also called RPB1) . The mutant rpo21 alleles carried on a plamid were introduced into a haploid yeast strain that conditionally expresses RPO21 from the inducible promoter pGAL10 . Growth of this strain on medium containing glucose is sustained only if the plasmid-borne rpo21 allele encodes a functional protein . Of nineteen linker-insertion alleles tested, five (rpo21-4 to -8) were found that impose a temperature-sensitive (ts) lethal phenotype on yeast cells . Four of these five ts alleles encode mutant proteins in which the site of insertion lies near one of the regions of the largest subunit that have been conserved during evolution . Two of the ts mutants (rpo21-4 and rpo21-7) display pleiotropic phenotypes, including an auxotrophy for inositol and a decreased proliferation rate at the permissive temperature . The functional relationship between RPO21 and RPO26, the gene encoding the 17.9 kDa subunit shared by RNA polymerases I, II, and III was investigated by determining the ability of increased dosage of RPO26 to suppress the ts phenotype imposed by rpo21-4 to -8 . Suppression of the ts defect was specific for the rpo21-4 allele and was accompanied by co-suppression of the inositol auxotrophy . These results suggest that mutations in the largest subunit of RNA polymerase II can have profound effects on the expression of specific subsets of genes, such as those involved in the metabolism of inositol . In the rpo21-4 mutant, these pleiotropic phenotypes can be attributed to a defective interaction between the largest subunit and the RPO26 subunit of RNA polymerase II.

Genetics, 1992 Apr, 130(4), 703 - 16
Commitment to meiosis in Saccharomyces cerevisiae: involvement of the SPO14 gene; Honigberg SM et al.; This paper describes the identification, cloning and phenotypic analysis of SPO14, a new gene required for meiosis and spore formation . Studies of strains carrying a temperature-sensitive mutation or a disruption/duplication allele indicate that spo14 mutants have the unusual property of being able to return to mitotic division, even from the late stages of meiotic development . Early meiotic events, such as DNA replication and intragenic and intergenic recombination, occur normally . In contrast, later meiotic processes are defective in spo14 mutants: the meiosis I division appears to be executed at slightly depressed levels, the meiosis II division is reduced more severely, and no spores are formed . Epistasis tests using mutants defective in recombination or reductional division support these findings . Based on these data, we suggest that the SPO14 gene product is involved in the coordinate induction of late meiotic events and that this induction is responsible for the phenomenon of commitment.

Cell Mol Biol, 1992 Apr, 38(2), 135 - 43
Cryoprotection provided by heat shock treatment in Saccharomyces cerevisiae; Kaul SC et al.; Saccharomyces cerevisiae cells exposed to 43 degrees C (normal being 30 degrees C) exhibit the synthesis of heat shock proteins (hsps) . Time course studies indicated that the major hsps (97 kDa, 85 kDa and 70 kDa family) are induced within 10 min . of heat shock and attain maximum amount with two hours of treatment . The viability of cells decreased by 99% when directly frozen into liquid nitrogen . However, a prior heat shock (2 hours) increased the cell survival by 20-30 fold . Such an effect of prior heat shock treatment could be supported by light and electron microscopical studies . Differential scanning calorimetric analysis of whole cells revealed that heat shock treatment decreases the denaturation (delta H) of total cellular proteins . A direct correlation between the degree of hsp inducibility and protection against freezing and thawing injury was observed . Cycloheximide treatment curtailed the synthesis of hsps as well as protection against subsequent freezing . This suggests that prior heat shock treatment protects the cells from freezing injury and, furthermore, that hsps can act as biological cryoprotectants.

Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 3098 - 102
Addition of extra origins of replication to a minichromosome suppresses its mitotic loss in cdc6 and cdc14 mutants of Saccharomyces cerevisiae; Hogan E et al.; Many cell division cycle (cdc) mutants of Saccharomyces cerevisiae exhibit elevated mitotic loss of pDK243, a 14-kilobase minichromosome with a centromere and one autonomous replicating sequence (ARS) . Tandem copies of different ARSs were added to pDK243 . The addition of these ARS clusters to pDK243 had no effect on its mitotic loss in cdc7 (protein kinase), cdc9 (DNA ligase), or cdc16 or cdc17 (DNA polymerase) mutants . However, in cdc6 and cdc14 mutants, the mitotic loss of pDK243 with an ARS cluster was suppressed by a factor of 6-8 compared to pDK243 without the cluster . This suppression was dependent upon the number of ARSs in the cluster and the integrity of the ARS consensus sequence in each ARS of the cluster . ARSs are known to be DNA replication origins . Therefore, the suppression of mini-chromosome loss by ARSs in cdc6 and cdc14 mutants suggests that these mutants are defective in the initiation of DNA replication . Since the CDC6 protein appears to act at the G1/S phase transition, the CDC6 protein may be a factor required at the beginning of S phase to initiate DNA replication at origins . In contrast, the CDC14 protein acts after mitosis . We suggest that the CDC14 protein performs a function late in the cell cycle that may be required for efficient initiation of DNA replication during S phase of the next cell cycle.

Eur J Biochem, 1992 Apr 1, 205(1), 311 - 9
Isolation and characterization of two biologically active O-glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae . Identification of a new motif for O-glycosylation; Olstad OK et al.; Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha . Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses . The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone . In both the O-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79 . In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66 . Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for O-glycosylation in yeast . The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone-sensitive adenylate cyclase assay in bone sarcoma cells . Thus, a C-terminal O-glycosylation of hPTH with up to seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.

Mol Cell Biol, 1992 Apr, 12(4), 1893 - 902
An essential Saccharomyces cerevisiae gene homologous to SNF2 encodes a helicase-related protein in a new family; Laurent BC et al.; The Saccharomyces cerevisiae SNF2 gene affects the expression of many diversely regulated genes and has been implicated in transcriptional activation . We report here the cloning and characterization of STH1, a gene that is homologous to SNF2 . STH1 is essential for mitotic growth and is functionally distinct from SNF2 . A bifunctional STH1-beta-galactosidase protein is located in the nucleus . The predicted 155,914-Da STH1 protein is 72% identical to SNF2 over 661 amino acids and 46% identical over another stretch of 66 amino acids . Both STH1 and SNF2 contain a putative nucleoside triphosphate-binding site and sequences resembling the consensus helicase motifs . The large region of homology shared by STH1 and SNF2 is conserved among other eukaryotic proteins, and STH1 and SNF2 appear to define a novel family of proteins related to helicases.

Mol Cell Biol, 1992 Apr, 12(4), 1805 - 14
Measurements of excision repair tracts formed during meiotic recombination in Saccharomyces cerevisiae; Detloff P et al.; During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplexes are formed at a high frequency between HIS4 genes located on homologous chromosomes . Using mutant alleles of the HIS4 gene that result in poorly repaired mismatches in heteroduplex DNA, we find that heteroduplexes often span a distance of 1.8 kb . In addition, we show that about one-third of the repair tracts initiated at well-repaired mismatches extend 900 bp.

Mol Cell Biol, 1992 Apr, 12(4), 1719 - 27
MET4, a leucine zipper protein, and centromere-binding factor 1 are both required for transcriptional activation of sulfur metabolism in Saccharomyces cerevisiae; Thomas D et al.; Inactivation of the centromere-binding factor 1 (CBF1) gene results in yeast strains that require methionine for growth . This auxotrophy is due to the inability of such strains to concentrate and assimilate sulfate from the medium . Northern (RNA) blot experiments reveal that the CBF1 protein is required for full induction of MET25 and MET16 gene transcription . However, we show that induction of the sulfate assimilation pathway is not achieved solely by CBF1 . This induction also requires the integrity of a positive trans-acting factor, encoded by the MET4 gene . The MET4 gene was cloned, and its sequence reveals that it encodes a protein related to the family of the bZIP transcriptional activators . Evidence that MET4 is a transcriptional activator was provided by demonstrating that DNA-bound LexA-MET4 fusion proteins stimulate expression of a nearby promoter . The use of LexA-MET4 fusion proteins also reveals that the leucine zipper of MET4 is required for the recognition of the MET25 promoter . Moreover, an 18-bp fragment of the MET25 5' upstream region was found to confer S-adenosylmethionine-dependent regulation of a fusion gene . This regulation was shown to depend on both MET4 and CBF1 . The obtained results suggest that the binding of CBF1 to its cognate sequences increases the ability of MET4 to stimulate transcription of the MET genes.

Mol Cell Biol, 1992 Apr, 12(4), 1621 - 9
Nucleosome loss activates CUP1 and HIS3 promoters to fully induced levels in the yeast Saccharomyces cerevisiae; Durrin LK et al.; We have previously shown that nucleosome loss, obtained by repressing histone H4 mRNA synthesis, activates otherwise inactive PHO5, GAL1, and CYC1 gene promoters (fused to the bacterial beta-galactosidase {lacZ} reporter gene) to moderate levels of activity (approximately 2 to 15% of fully induced levels) . We now report that nucleosome loss activates the expression of two additional promoters that are normally induced by independent mechanisms: CUP1 (induced by heavy-metal toxicity) and HIS3 (induced by amino acid starvation) . Surprisingly, the level of CUP1-lacZ and HIS3-lacZ activation by nucleosome loss approximates fully induced levels of transcription . These CUP1 and HIS3 promoter activities are increased similarly from either episomal or genomic constructs . Our results emphasize the universality of the mechanism by which nucleosome loss activates yeast promoters . Moreover, a comparison of absolute levels of activation for different promoters suggests that activation by nucleosome loss results in a relatively constant level of activation, while levels obtained by normal induction vary considerably . These data argue that nucleosome loss may play a uniquely dominant role in the regulation of certain promoters.

Mol Cell Biol, 1992 Apr, 12(4), 1432 - 42
Mutational analysis of conserved positions potentially important for initiator tRNA function in Saccharomyces cerevisiae; von Pawel-Rammingen U et al.; The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis . However, the role of these positions is not known . We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs . Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed . Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene . We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo . Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process . Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain . Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth . In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro . From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.

J Virol, 1992 Apr, 66(4), 2359 - 68
Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae; Leavitt AD et al.; Integration of retroviral DNA into the host cell genome requires the interaction of retroviral integrase (IN) protein with the outer ends of both viral long terminal repeats (LTRs) to remove two nucleotides from the 3' ends (3' processing) and to join the 3' ends to newly created 5' ends in target DNA (strand transfer) . We have purified the IN protein of human immunodeficiency virus type 1 (HIV-1) after production in Saccharomyces cerevisiae and found it to have many of the properties described for retroviral IN proteins . The protein performs both 3' processing and strand transfer reactions by using HIV-1 or HIV-2 attachment (att) site oligonucleotides . A highly conserved CA dinucleotide adjacent to the 3' processing site of HIV-1 is important for both the 3' processing and strand transfer reactions; however, it is not sufficient for full IN activity, since alteration of nucleotide sequences internal to the HIV-1 U5 CA also impairs IN function, and Moloney murine leukemia virus att site oligonucleotides are poor substrates for HIV-1 IN . When HIV-1 att sequences are positioned internally in an LTR-LTR circle junction substrate, HIV-1 IN fails to cleave the substrate preferentially at positions coinciding with correct 3' processing, implying a requirement for positioning att sites near DNA ends . The 2 bp normally located beyond the 3' CA in linear DNA are not essential for in vitro integration, since mutant oligonucleotides with single-stranded 3' or 5' extensions or with no residues beyond the CA dinucleotide are efficiently used . Selection of target sites is nonrandom when att site oligonucleotides are joined to each other in vitro . We modified an in vitro assay to distinguish oligonucleotides serving as the substrate for 3' processing and as the target for strand transfer . The modified assay demonstrates that nonrandom usage of target sites is dependent on the target oligonucleotide sequence and independent of the oligonucleotide used as the substrate for 3' processing.

Curr Genet, 1992 Apr, 21(4-5), 319 - 24
Isolation and characterization of additional genes influencing resistance to various mutagens in the yeast Saccharomyces cerevisiae; Haase E et al.; Screening of a multi-copy vector-based yeast genomic library in haploid cells of wild-type Saccharomyces cerevisiae yielded transformants hyper-resistant to various chemical mutagens . Genetical analysis of the yeast insert DNAs revealed three genes SNG1, SNQ2, and SNQ3 that confer the phenotype hyper-resistance to MNNG, to 4-NQO and triaziquone, and to mutagens 4-NQO, MNNG, and triaziquone, respectively . Integration of the gene disruption-constructs into the haploid yeast genome yielded viable null-mutants with a mutagen-sensitive phenotype . Thus, copy number of these non-essential yeast genes determines the relative resistance to certain chemical mutagens, with zero copies yielding a phenotype of mutagen sensitivity and multiple copies one of mutagen hyper-resistance, respectively.

Curr Genet, 1992 Apr, 21(4-5), 309 - 18
Mitotic hyperploidy for chromosomes VIII and III in Saccharomyces cerevisiae; Spector LM et al.; The arg4-8 and cup1s markers comprise a copy-number-dependent signal device in the yeast Saccharomyces cerevisiae . These alleles permit reliable discrimination between euploid and disomic haploids as well as between euploid and trisomic diploids . To investigate and compare inherent inter-chromosomal differences as regards propensity for hyperploidy, we transplaced arg4-8 and cup1s by deleting them from chromosome VIII and then re-introducing them at the leu2 locus on chromosome III . The rate of chromosome gain was significantly greater for the chromosome III construct compared to the native chromosome VIII, in both diploid and haploid strains . In addition, more coincident aneuploidy for other chromosomes was found among chromosome VIII hyperploids compared to chromosome III hyperploids.

Curr Genet, 1992 Apr, 21(4-5), 301 - 7
The UGA43 negative regulatory gene of Saccharomyces cerevisiae contains both a GATA-1 type zinc finger and a putative leucine zipper; Coornaert D et al.; The UGA43 gene of Saccharomyces cerevisiae is required for repression of inducible genes involved in the utilization of 4-aminobutyric acid (GABA) or urea as nitrogen sources . The UGA43 gene has been cloned by complementation of a uga43 mutation . The N-terminal region of the UGA43 protein is very similar to the DNA-binding zinc-finger region typical of the GATA regulatory factor family in vertebrates . UGA43 is the first reported instance of a GATA protein with a negative regulatory function . The C-terminal region of the predicted UGA43 protein contains a putative leucine zipper . Sequencing of three uga43 mutant alleles suggests that the GATA and putative leucine-zipper regions are both required for the repressive activity of UGA43 . UGA43 appears to be a highly regulated gene . On "poor" nitrogen sources, UGA43 transcripts are measured at high levels whereas they are nearly undetectable in conditions of nitrogen catabolite repression . The levels measured on "poor" nitrogen sources are further increased in uga43 mutant cells, suggesting that UGA43 exerts negative autoregulation.

Curr Genet, 1992 Apr, 21(4-5), 291 - 3
Phenotype traits associated with different alleles at the RPS5 locus in Saccharomyces cerevisiae; Valentin E et al.; The RPS5 gene has been characterised through its ability to reduce invertase production by the SUC5 gene . In this paper we show that RPS5 acts by maintaining low levels of SUC5 mRNA . We also show that RPS5 acts on the SUC1 and SUC4 genes but not on SUC2 and SUC3, which are members of the SUC family . RPS5 also shows a pleiotropic effect on the amount of mitochondrial cytochromes.

Curr Genet, 1992 Apr, 21(4-5), 285 - 9
Cloning and mapping of the CYS4 gene of Saccharomyces cerevisiae; Ono B et al.; A DNA fragment containing the CYS4 gene of Saccharomyces cerevisiae was isolated from a genomic library . The cloned fragment hybridized to the transverse-alternating-field-electrophoresis band corresponding to chromosomes VII and XV . According to the 2 microns DNA chromosome-loss procedure, the cys2 and cys4 mutations, which are linked together and co-operatively confer cysteine dependence, were assigned to chromosome VII . By further mapping involving tetrad analysis, the cys2-cys4 pair was localized between SUP77 (SUP166) and ade3 on the right arm of chromosome VII.

Yeast, 1992 Apr, 8(4), 273 - 89
Genetic and molecular analysis of DNA43 and DNA52: two new cell-cycle genes in Saccharomyces cerevisiae; Solomon NA et al.; Two Saccharomyces cerevisiae genes previously unknown to be required for DNA synthesis have been identified by screening a collection of temperature-sensitive mutants . The effects of mutations in DNA43 and DNA52 on the rate of S phase DNA synthesis were detected by monitoring DNA synthesis in synchronous populations that were obtained by isopycnic density centrifugation . dna43-1 and dna52-1 cells undergo cell-cycle arrest at the restrictive temperature (37 degrees C), exhibiting a large-budded terminal phenotype; the nuclei of arrested cells are located at the neck of the bud and have failed to undergo DNA replication . These phenotypes suggest that DNA43 and DNA52 are required for entry into or completion of S phase . DNA43 and DNA52 were cloned by their abilities to suppress the temperature-sensitive lethal phenotypes of dna43-1 and dna52-1 cells, respectively . DNA sequence analysis suggested that DNA43 and DNA52 encode proteins of 59.6 and 80.6 kDa, respectively . Both DNA43 and DNA52 are essential for viability and genetic mapping experiments indicate that they represent previously unidentified genes: DNA43 is located on chromosome IX, 32 cM distal from his5 and DNA52 is located on chromosome IV, 0.9 cM from cdc34.

Mol Biol Cell, 1992 Apr, 3(4), 429 - 44
Actin- and tubulin-dependent functions during Saccharomyces cerevisiae mating projection formation; Read EB et al.; Several conditional-lethal mutant alleles of the single-copy Saccharomyces cerevisiae beta-tubulin and actin genes were used to evaluate the roles of microtubules and actin filaments in the pheromone-induced extension of mating projections . Mutants defective in tubulin assembly form projections indistinguishable in appearance from those formed by wild-type cells . However, the tubulin mutants are unable to move their nuclei into the projections and to orient the spindle pole body associated with each nucleus toward the projection tip . Actin mutants are defective in spatial orientation of cell-surface growth required for formation of normal mating projections . Migration of nuclei into mating projections and Spa2p segregation to projection tips are also defective in actin mutants . Studies with abp1 null mutants showed that the function of the Abp1p actin-binding protein is either not required for projection formation or there are other proteins in yeast with similar functions . Our findings demonstrate that actin is required to restrict cell-surface growth to a defined region for pheromone-induced morphogenesis and suggest that nuclear position and orientation in mating projections depend on direct or indirect interaction of microtubules with actin filaments.

Mol Cell Biol, 1992 Apr, 12(4), 1613 - 20
Involvement of cDNA in homologous recombination between Ty elements in Saccharomyces cerevisiae; Melamed C et al.; Strains carrying a marked Ty element (TyUra) in the LYS2 locus were transformed with plasmids bearing a differently marked Ty1 element (Ty1Neo) under the control of the GAL promoter . When these strains were grown in glucose, a low level of gene conversion events involving TyUra was detected . Upon growth on galactose an increase in the rate of gene conversion was seen . This homologous recombination is not the consequence of increased levels of transposition . When an intron-containing fragment was inserted into Ty1Neo, some of the convertants had the intron removed, implying an RNA intermediate . Mutations that affect reverse transcriptase or reverse transcription of Ty1Neo greatly reduce the induction of recombination in galactose . Thus, Ty cDNA is involved in homologous gene conversion with chromosomal copies of Ty elements . Our results have implications about the way families of repeated sequences retain homogeneity throughout evolution.

Yeast, 1992 Apr, 8(4), 303 - 9
Control of peroxisome proliferation in Saccharomyces cerevisiae by ADR1, SNF1 (CAT1, CCR1) and SNF4 (CAT3); Simon M et al.; The Saccharomyces cerevisiae ADR1 gene has recently been demonstrated to control transcription of several genes encoding peroxisomal proteins or proteins necessary for peroxisome formation . Therefore, the effect of two other genes (SNF1 (CAT1, CCR1) and SNF4 (CAT3)) known to control derepression of glucose-repressible genes was studied . Levels of transcripts of genes encoding catalase A, fatty acid beta-oxidation enzymes and of the PAS1 gene are reduced in snf1 and snf4 mutants on ethanol as well as on oleic acid medium . By immunogold labelling with an antibody directed against peroxisomal thiolase, clusters of peroxisomes were detected in wild-type cells, whereas smaller single peroxisomes were observed in adr1 mutant cells . Results of immunofluorescence experiments are consistent with these observations . No peroxisomes were detected in snf1 and snf4 mutants by immunogold labelling as well as by immunofluorescence.

Gene, 1992 Apr 1, 113(1), 67 - 74
Cloning, primary structure and regulation of the ARO4 gene, encoding the tyrosine-inhibited 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae; Kunzler M et al.; In Saccharomyces cerevisiae, two differently regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (DAHPS; EC 4.1.2.15) isoenzymes carry out the first step in the shikimate pathway . Mutations in both genes are necessary to cause aromatic amino acid (aa) auxotrophy, since one isoenzyme alone is sufficient to produce enough DAHP for normal growth of the cells . The phenylalanine-inhibited DAHPS is encoded by the previously isolated and characterized ARO3 gene . Here, we report the cloning and characterization of the ARO4 gene, encoding the second DAHPS, which is inhibited by tyrosine . The aa sequence of the ARO4 gene product reveals 76% similarity to the ARO3-encoded isoenzyme and 66 and 73% to the three DAHPS isoenzymes from Escherichia coli . ARO4 gene expression is regulated by the general control system of aa biosynthesis . As in the case of the ARO3 gene, a single GCN4-recognition element in the promoter is responsible for derepression of the ARO4 gene under aa starvation conditions . However, in contrast to the situation in the isogene, ARO3, GCN4 does not contribute to the basal level of ARO4 transcription under nonderepressing conditions.

Curr Genet, 1992 Apr, 21(4-5), 325 - 9
The CCS1 gene from Saccharomyces cerevisiae which is involved in mitochondrial functions is identified as IRA2 an attenuator of RAS1 and RAS2 gene products; Bussereau F et al.; The ccs1-1 mutation of Saccharomyces cerevisiae, which has been previously described, is associated with an increase in cytochrome content, in respiration, and in ATP synthesis . In addition, this mutation leads to the same phenotype as cells de-regulated in the cAMP pathway . From a yeast genomic library, we have isolated a DNA fragment in a recombinant plasmid pCD1 which complements the ccs1-1 mutation . Homologous integration of this DNA in the genome occurs at the CCS1 locus . An 11 kb of the DNA insert is necessary for complementation . Sequencing part of the fragment identifies CCS1 as the IRA2 gene . The IRA2 gene is known to encode an attenuator of RAS gene product activity which stimulates the GTPase activity of the RAS proteins . This result underlines the involvement of cAMP-dependent phosphorylation in mitochondrial function . We present the sequence of 1 kb DNA upstream of the putative ATG of the IRA2/CCS1 gene product which is devoid of an ORF and could contain several regulatory sites.

J Bacteriol, 1992 Apr, 174(8), 2548 - 59
Ty insertions upstream and downstream of native DUR1,2 promoter elements generate different patterns of DUR1,2 expression in Saccharomyces cerevisiae; Chisholm GE et al.; Expression of allantoin pathway genes is subject to induction and nitrogen catabolite repression . Two classes of cis-dominant mutations (DUR80 and DUR1,2-Oh) result in overproduction of DUR1,2 mRNA . In DUR80 mutants, DUR1,2 expression remained inducible, nitrogen catabolite repression sensitive, and unresponsive to cell ploidy, i.e., overproduction was superimposed on normal gene regulation . DUR1,2-Oh mutations, in contrast, generated a pattern of DUR1,2 expression similar to that often reported when a Ty element inserts upstream of a gene, the ROAM phenotype . We analyzed four independent DUR80 and DUR1,2-Oh alleles . The DUR1,2-Oh mutation was, as expected, a Ty insertion at -445 3' of the native DUR1,2 upstream activation sequences (UASs) . All three DUR80 alleles were also Ty insertions between -644 and -653 immediately 5' of the native DUR1,2 USASs . We suggest that the difference in DUR1,2-Oh and DUR80 phenotypes depends on whether the native cis-acting elements and transcription factors associated with them can operate . If they can, enhancement of normally regulated DUR1,2 expression is observed . This is a novel phenotype for Ty insertions . If the native DUR1,2 cis-acting elements are not present, the case when Ty insertion occurs 3' of them, a ROAM phenotype is generated . Nitrogen-regulated upstream activation sequence (UASNTR)-homologous sequences present in the Ty delta elements rather than cis-acting elements required for Ty transcription are the most likely candidates to serve as the cis-acting elements mediating the DUR80 phenotype.

Mol Cell Biol, 1992 Apr, 12(4), 1879 - 92
A presumptive helicase (MOT1 gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae; Davis JL et al.; Exposure of a haploid yeast cell to mating pheromone induces transcription of a set of genes . Induction is mediated through a cis-acting DNA sequence found upstream of all pheromone-responsive genes . Although the STE12 gene product binds specifically to this sequence element and is required for maximum levels of both basal and induced transcription, not all pheromone-responsive genes are regulated in an identical manner . To investigate whether additional factors may play a role in transcription of these genes, a genetic screen was used to identify mutants able to express pheromone-responsive genes constitutively in the absence of Ste12 . In this way, we identified a recessive, single gene mutation (mot1, for modifier of transcription) which increases the basal level of expression of several, but not all, pheromone-responsive genes . The mot1-1 allele also relaxes the requirement for at least one other class of upstream activating sequence and enhances the expression of another gene not previously thought to be involved in the mating pathway . Cells carrying mot1-1 grow slowly at 30 degrees C and are inviable at 38 degrees C . The MOT1 gene was cloned by complementation of this temperature-sensitive lethality . Construction of a null allele confirmed that MOT1 is an essential gene . MOT1 residues on chromosome XVI and encodes a large protein of 1,867 amino acids which contains all seven of the conserved domains found in known and putative helicases . The product of MOT1 is strikingly homologous to the Saccharomyces cerevisiae SNF2/SW12 and RAD54 gene products over the entire helicase region.

Biochem Biophys Res Commun, 1992 Mar 31, 183(3), 1191 - 6
Effect of 17 beta-estradiol on the generation time of old cells of the yeast Saccharomyces cerevisiae; Motizuki M et al.; The duration of unbudded period of the yeast Saccharomyces cerevisiae is known to be extended with age from unresolved causes; in this experiment the unbudded period of 20-generation-old cells was extended to be 1.6 times that of 6-generation-old cells . We found that the addition of 17 beta-estradiol into the culture medium reduced the age-related extension of the unbudded period reaching 1.35 times that of the young cells which was unaffected by the hormone . This effect of 17 beta-estradiol was not observed when the old cells were cultured in a glycerol-based medium instead of a glucose-based medium suggesting that the action of 17 beta-estradiol was mediated by facilitation of glycolysis . The administration of 17 beta-estradiol equally elevated the cAMP level of the old cells in either medium up to the level of the young cells but elevated the ATP level of only those in the glucose-based medium . Furthermore, the administration of cAMP shortens the unbudded period of the old cells cultured in the glucose-based medium . Therefore, it was suggested that 17 beta-estradiol causes the shortening of the unbudded period of the old cells by stimulating the energy metabolism through elevation of the cAMP level.

Nucleic Acids Res, 1992 Mar 25, 20(6), 1277 - 81
Mitochondrially-imported cytoplasmic tRNA(Lys)(CUU) of Saccharomyces cerevisiae: in vivo and in vitro targetting systems; Tarassov IA et al.; The cytoplasmic tRNA(Lys)(CUU) (tRNA(1Lys)) is the single yeast tRNA species to be traffiked from the cytoplasm into the mitochondrial compartment of the cell . To study mechanisms of this targetting we worked out two test systems . The in vivo system based on the electroporation of intact yeast cells was used to introduce labelled tRNAs into the cytoplasm . All tRNA species tested were effectively introduced into the cytoplasm, but only the cytoplasmic tRNA(1Lys) was found in the mitochondrial compartment within 1-2 hours after the electroporation procedure . The in vitro system permits specific transfer of the tRNA(1Lys) into isolated mitochondria . Contrary to the known systems for protein transport into isolated mitochondria, mitochondrial import of tRNA(1Lys) in vitro requires the presence of soluble cellular proteins in the reaction mixture . The translocation proved to be ATP-dependent and to require the presence of an ATP-generation system in the reaction . Preincubation of the tRNA with the total cellular extract of the cell markedly increases the rate of the translocation . Two protein fractions are necessary to direct the import in vitro . The first one has high heparin-binding affinity, while the other protein fraction is not retained by heparin-Sepharose.

FEBS Lett, 1992 Mar 16, 299(3), 283 - 6
The pro-region of the Kex2 endoprotease of Saccharomyces cerevisiae is removed by self-processing; Germain D et al.; We have produced in the baculovirus/insect cells expression system a soluble secreted form of the Saccharomyces cerevisiae Kex2 endoprotease . This secreted enzyme was purified and its NH2-terminal sequence determined . The NH2-terminal sequence started at residue Leu109 of the sequence deduced from the KEX2 gene nucleotide sequence, showing that the Kex2 enzyme is produced as a proenzyme . Residue Leu109 is preceded by a pair of basic amino acid residues (Lys107-Arg108) which is a potential processing site for the Kex2 endopeptidase . Furthermore, expression of an inactive form of this truncated enzyme resulted in the production of a protein with a higher molecular weight . These observations suggest that the pro-region of Kex2 endoprotease is removed by a self-processing event.

Eur J Biochem, 1992 Mar 15, 204(3), 983 - 90
The ICL1 gene from Saccharomyces cerevisiae; Fernandez E et al.; The glyoxylate cycle is essential for the utilization of C2 compounds by the yeast Saccharomyces cerevisiae . Within this cycle, isocitrate lyase catalyzes one of the key reactions . We obtained mutants lacking detectable isocitrate lyase activity, screening for their inability to grow on ethanol . Genetic and biochemical analysis suggested that they carried a defect in the structural gene, ICL1 . The mutants were used for the isolation of this gene and it was located on a 3.1-kb BglII-SphI DNA fragment . We then constructed a deletion-substitution mutant in the haploid yeast genome . It did not have any isocitrate lyase activity and lacked the ability to grow on ethanol as the sole carbon source . Both strands of a DNA fragment carrying the gene and its flanking regions were sequenced . An open reading frame of 1671 bp was detected, encoding a protein of 557 amino acids with a calculated molecular mass of 62515 Da . The deduced amino acid sequence shows extensive similarities to genes encoding isocitrate lyases from various organisms . Two putative cAMP-dependent protein-kinase phosphorylation sites may explain the susceptibility of the enzyme to carbon catabolite inactivation.

J Biol Chem, 1992 Mar 15, 267(8), 5442 - 5
NH2-terminal acetylation of ribosomal proteins of Saccharomyces cerevisiae; Takakura H et al.; Using a mutant of Saccharomyces cerevisiae defective in the NAT1 gene, that encodes one of the NH2-terminal acetyltransferases, we have identified 14 ribosomal proteins whose electrophoretic mobility at pH 5.0 suggests they carry an additional charge, presumably due to the lack of NH2-terminal acetylation . At least 30 other ribosomal proteins from the mutant are electrophoretically normal . Attempted NH2-terminal analysis of most of the presumed acetylated proteins from wild type cells indicated that all were blocked . NH2-terminal analysis of the same proteins from the nat1 mutant strain yielded unique sequences . Each one carries an NH2-terminal serine . We conclude that these are normally acetylated due to the presence of the NAT1 gene product . It seems surprising that cells whose ribosomes have been altered to this degree grow rather well and synthesize the same spectrum of proteins as do wild type cells (Mullen, J . R., Kayne, P . S., Moerschell, R . P., Tsunasawa, S . Gribskov, M., Sherman, F., and Sternglanz, R . (1989) EMBO J . 8, 2067-2075) . Finally, this analysis has provided the first sequence information available for several of the acetylated ribosomal proteins and for one non-acetylated ribosomal protein, which is clearly the product of the MFT1 gene (Garrett, J . M., Singh, K . K., Vonder Haar, R . A., and Emr . S . D . (1991) Mol . Gen . Gen . 225, 483-491).

Proc Natl Acad Sci U S A, 1992 Mar 15, 89(6), 2302 - 6
Functional expression of human mdr1 in the yeast Saccharomyces cerevisiae; Kuchler K et al.; Development of multiple drug resistance in tumor cells involves amplification of the mdr1 gene product, a 170-kDa plasma membrane glycoprotein that is an ATP-driven pump that extrudes the drugs . Human mdr1 (also designated as PGY1) cDNA was expressed in yeast cells by using the promoter and translational initiation signal of a related yeast gene, STE6 . Immunoblotting of subcellular fractions showed that all of the Mdr1 (also known as P glycoprotein) was associated with the particulate material . Immunofluorescence microscopy revealed that the majority of the Mdr1 was localized to the plasma membrane (although a significant amount was also found in the endoplasmic reticulum) . In contrast to mammalian cells, Mdr1 was not glycosylated in yeast . Nevertheless, some, if not all, of the Mdr1 made in yeast was properly folded and functional because it could be photoaffinity labeled specifically with 8-azido-ATP and because cells overexpressing Mdr1 displayed increased resistance towards valinomycin, an ionophore known to interact with Mdr1 in animal cells . Hence, a human polytopic membrane protein was correctly inserted into the yeast plasma membrane, and glycosylation was not required for its function.

Nucleic Acids Res, 1992 Mar 11, 20(5), 1011 - 6
Yeast ribosomal proteins: XIII . Saccharomyces cerevisiae YL8A gene, interrupted with two introns, encodes a homolog of mammalian L7; Mizuta K et al.; We isolated and sequenced a gene, YL8A, encoding ribosomal protein YL8 of Saccharomyces cerevisiae . It is one of the two duplicated genes encoding YL8 and is located on chromosome VII while the other is on chromosome XVI . The haploid strains carrying disrupted YL8A grew more slowly than the parent strain . The open reading frame is interrupted with two introns . The predicted amino acid sequence reveals that yeast YL8 is a homolog of mammalian ribosomal protein L7, E.coli L30 and others.

Biochemistry, 1992 Mar 3, 31(8), 2359 - 67
Exon sequence and structure requirements for tRNA splicing in Saccharomyces cerevisiae; Shapero MH et al.; A survey of exon sequence and structure requirements for splicing was undertaken using labeled pre-tRNA substrates prepared by in vitro transcription of bacterial promoter-yeast tRNA(Tyr) gene fusions . Transcription templates were assembled from oligonucleotide cassettes allowing analysis of 22 derivatives affecting each of the potential secondary and certain tertiary interactions in the pre-tRNA . Effects on both excision of the intervening sequence by yeast endonuclease and joining of exons by ligase were examined . Replacements within the D- and T-stems and anticodon stems revealed that while the primary sequences of these segments were not essential for splicing, formation of base-paired structures was required . Replacements which altered the primary sequence while retaining the secondary structure of the aminoacyl stem allowed efficient excision by endonuclease but reduced joining by ligase . Potentially, the effects of changes within these stems may be indirect through effects on adjacent or overall structure . The presence of either structured or unstructured 5' leader and/or 3' trailer sequences had no effect on either splicing step . Alterations in the conserved Levitt tertiary pair (G15/C48), previously implicated in splicing of pre-tRNA(Phe), did not alter splicing of pre-tRNA(Tyr) . A precursor in which the small (type I) extra arm in pre-tRNA(Tyr) was replaced with the large (type II) extra arm sequence from tRNA(Ser) was efficiently spliced . These and previous results suggest that only limited features of exon sequence or structure are recognized by the splicing enzymes.

Mol Biol Cell, 1992 Mar, 3(3), 275 - 86
Two genes in Saccharomyces cerevisiae encode a membrane-bound form of casein kinase-1; Wang PC et al.; Two cDNAs encoding casein kinase-1 have been isolated from a yeast cDNA library and termed CKI1 and CKI2 . Each clone encodes a protein of approximately 62,000 Da containing a highly conserved protein kinase domain surrounded by variable amino- and carboxy-terminal domains . The proteins also contain two conserved carboxy-terminal cysteine residues that comprise a consensus sequence for prenylation . Consistent with this posttranslational modification, cell fractionation experiments demonstrate that intact CKI1 is found exclusively in yeast cell membranes . Gene disruption experiments reveal that, although neither of the two CKI genes is essential by itself, at least one CKI gene is required for yeast cell viability . Spores deficient in both CKI1 and CKI2 fail to grow and, therefore, either fail to germinate or arrest as small cells before bud emergence . These results suggest that casein kinase-1, which is distributed widely in nature, plays a pivotal role in eukaryotic cell regulation.

J Biochem (Tokyo), 1992 Mar, 111(3), 352 - 8
A dominant mutation that alters the regulation of INO1 expression in Saccharomyces cerevisiae; Hosaka K et al.; A dominant, single nuclear gene mutation, CSE1, caused inositol auxotrophy in yeast cells . The inositol requirement was marked when choline was present in the medium . Inositol-1-phosphate synthase, the regulatory enzyme of inositol synthesis, is repressed by inositol, or more profoundly by a combination of inositol and choline in the wild type . In CSE1, the level of inositol-1-phosphate synthase was low and was greatly repressed on the addition of choline alone . In accordance with this, INO1 mRNA encoding the enzyme was low even under the depressed conditions and was profoundly decreased by choline in CSE1 . But in the wild type, the addition of choline alone had little effect . An INO1-lacZ fusion was constructed and the control of the INO1 promoter in CSE1 was studied . lacZ expression was repressed not only by inositol, but also by choline in CSE1, whereas it was repressed by inositol, but only slightly by choline in the wild type . CSE1 was unlinked to the INO1 structural gene . Thus CSE1 was thought to be a regulatory mutation . Furthermore, when the CDP-choline pathway was mutationally blocked, choline did not affect INO1 expression, indicating that the metabolism of choline via the CDP-choline pathway is required for INO1 repression.

Microbiol Rev, 1992 Mar, 56(1), 1 - 11
Regulation of gene expression by oxygen in Saccharomyces cerevisiae; Zitomer RS et al.; The oxygen regulation of two broad categories of yeast genes is discussed in this review . The first is made up of genes regulated by heme, and the second is made up of genes whose regulation is heme independent . Heme-regulated genes fall into two classes: heme-activated and heme-repressed genes . Activation is achieved through one of two transcriptional activators, the heme-dependent HAP1 protein or the heme-activated, glucose-repressed HAP2/3/4 complex . Some of the properties and the DNA-binding sites of these activators are discussed . Heme repression is achieved through the action of the ROX1 repressor, the expression of which is transcriptionally activated by heme . Once ROX1 is synthesized, its function is heme independent . Evidence that ROX1 binds to DNA or is part of a DNA-binding complex is described . Factors which modulate the function of these regulatory proteins are discussed, and a schematic of heme activation and repression is presented . The mitochondrial subunits of cytochrome c oxidase are induced by oxygen in a heme-independent fashion . The translation of one, cytochrome c oxidase subunit III, is dependent upon three nucleus-encoded initiation factors . One of these, PET494, is itself translationally regulated by oxygen in a heme-independent fashion . The expression of at least four other mitochondrially encoded cytochrome subunits is dependent upon specific translation factors, raising the potential for translational regulation as a general mechanism . Finally, a number of anaerobic genes that show heme-independent, oxygen-repressed expression have been identified . These fall into two kinetic classes, suggesting that there are at least two different regulatory circuitries.

Yeast, 1992 Mar, 8(3), 227 - 38
Molecular cloning and physical analysis of an 8.2 kb segment of chromosome XI of Saccharomyces cerevisiae reveals five tightly linked genes; Abraham PR et al.; The nucleotide sequence of 6472 base pairs of an 8.2 kb segment of Saccharomyces cerevisiae chromosome XI has been determined . The sequence contains a cluster of four long open reading frames (ORF) designated YKL2, YKL3, YKL4 and TGL1 in the same orientation, flanked at the 5'-end by a divergent incomplete ORF (YKL1) . Transcription and Southern analysis of the four complete ORFs showed that all are expressed and are present in single copy on the haploid genome . The average codon adaptation index of the coding regions is approximately 0.2, suggesting that these genes are lowly expressed . The upstream regions of all four genes as well as the YKL1 ORF contain putative promoter elements previously found to be characteristic of nuclear genes encoding mitochondrial proteins . Significant sequence similarities were found between the YKL3 protein and Escherichia coli ribosomal protein S2 as well as between the TGL1 protein and triglyceride lipases from rat salivary gland and human gastric tissue . The 3'-end of the 6472 bp nucleotide sequence overlaps with the upstream region of the previously identified CTK1 gene, encoding the largest subunit of CTD kinase (Lee, J.M . and Greenleaf, A.L., 1991, Gene Expression 2, 149-167), thereby increasing the number of genes on the 8.2 kb fragment to at least five . The transcripts of these genes represent approximately 83% of the DNA fragment, making it one of the most highly transcribed regions of the yeast chromosome analysed to date.

Yeast, 1992 Mar, 8(3), 215 - 22
Sequence of the HMR region on chromosome III of Saccharomyces cerevisiae; Sor F et al.; A 10,095 base pair DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae has been sequenced and analysed . It encompasses the silent mating-type locus HMR . Both HMRa1 and HMRa2 genes, as well as their flanking regulatory regions, have been identified . Three new open reading frames longer than 80 amino acid residues were found in this fragment . One of them (YCR137) shows features compatible with a membranous localization and a transporter function . The other two do not show a similarity with any known gene . A new gene coding for tRNA(thr)al (ACU) has been identified . It is located in a region coding for several delta sequences.

Yeast, 1992 Mar, 8(3), 157 - 69
Identification of a novel secreted glycoprotein of the yeast Saccharomyces cerevisiae stimulated by heat shock; Lupashin VV et al.; A new secreted yeast glycoprotein with an Mr of about 400 kDa (gp400) has been found . The glycoprotein is an O-mannosylated oligomer, whose synthesis and export into culture medium are stimulated by heat shock . Intracellular transport of gp400 is carried out by membrane vesicles distinct from the known constitutive secretory vesicles . Immunological analysis revealed gp400 only in Saccharomyces species.

Curr Genet, 1992 Mar, 21(3), 183 - 9
Positive cis-acting regulatory sequences mediate proper control of POL1 transcription in Saccharomyces cerevisiae; Pizzagalli A et al.; The 5'ACGCGT3' MluI motif, which is found in the upstream region of several yeast DNA-synthesis genes which are periodically expressed during the mitotic cell-cycle, is present twice in the 5' non-coding region of the DNA-polymerase alpha gene (POL1) . Deletion of the most distal repeat does not affect POL1 transcription, while the adjacent 40 base-pair (bp) downstream sequence is necessary both for the proper level and the fluctuation of POL1 mRNA . This region contains the 5'ACGCGTCGCGT3' sequence, which is sufficient to control periodic transcription of a CYC1-lacZ reporter gene with the same kinetics observed for POL1 . The adjacent 29 bp AT-rich region does not show any activity by itself, but it acts synergistically in conjunction with at least one MluI hexamer to stimulate CYC1-lacZ expression . By further deletion analysis, DNA sequences necessary to initiate POL1 transcription at the proper sites have also been identified.

Mol Gen Genet, 1992 Mar, 232(2), 332 - 4
Temperature sensitivity of the cdc9-1 allele of Saccharomyces cerevisiae DNA ligase is dependent on specific combinations of amino acids in the primary structure of the expressed protein; Unternahrer S et al.; In this study we present the characterization of the temperature-sensitive mutant allele cdc9-1 encoding DNA ligase, of Saccharomyces cerevisiae strain A364A by DNA sequencing . Comparison with the published wild-type sequence from strain SK1 revealed 13 nucleotide exchanges between these two sequences, which are derived from non-isogenic genetic backgrounds . Only four of these changes, distributed over the whole coding region, lead to amino acid exchanges in the protein chain . Our analysis of the sequence of the wild-type CDC9 allele from strain A364A revealed differences from the isogenic cdc9-1 allele in only two nucleotides: one silent change and one leading to a single amino acid exchange . The latter is therefore responsible for the temperature-sensitive phenotype . A mosaic protein, in which a region carrying this amino acid exchange has been inserted in place of the corresponding part of CDC9 from the non-isogenic strain SK1, is not temperature sensitive . The exchange of a longer stretch of DNA leading to atteration of three amino acids of the protein compared with the original sequence of SK1 is required to obtain a temperature-sensitive DNA ligase in this strain, while in strain A364A a single amino acid change is sufficient for expression of a temperature-sensitive protein.

Mol Gen Genet, 1992 Mar, 232(2), 304 - 12
rna12+, a gene of Saccharomyces cerevisiae involved in pre-rRNA maturation . Characterization of a temperature-sensitive mutant, cloning and sequencing of the gene; Liang S et al.; RNA12-1 is a dominant temperature-sensitive (Ts) yeast mutant which has previously been reported to exhibit a defect in RNA accumulation at 37 degrees C . We further characterized this mutant through analyses of rRNA transcription rates and maturation . The results show that pre-rRNA is normally synthesized but that subsequent maturation is severely affected by a temperature upshift: the nascent rRNAs are under-methylated and little mature rRNA can be observed at 37 degrees C . Likewise, the accumulation of some mRNAs for ribosomal proteins is also prevented at 37 degrees C . The RNA12-1 mutation is recessive at 32 degrees C, which made it possible to clone the wild-type rna12+ gene by complementation of the Ts phenotype with plasmids from a multicopy yeast genomic library . The predicted gene product is a protein of 96,630 Da with no significant sequence similarity to any known proteins . Gene disruption is not lethal at either the permissive or the restrictive temperature . The gene is located on chromosome XIII, downstream of the ADH2 gene and 10 cM from the ADE4 gene . Furthermore, the mutant allele RNA12-1 was cloned and sequenced . A point mutation found in this allele leads to dominant thermosensitivity at 37 degrees C when the mutant gene is introduced into a wild-type strain . Taken together, these data suggest that the rna12+ gene product plays a dispensable role in early maturation of pre-rRNA but that its mutant gene product can interfere with the normal function of other proteins required for pre-rRNA maturation.

Mol Gen Genet, 1992 Mar, 232(2), 231 - 9
Inhibition of cell proliferation in Saccharomyces cerevisiae by expression of human NAD+ ADP-ribosyltransferase requires the DNA binding domain ("zinc fingers"); Kaiser P et al.; Constitutive expression of human nuclear NAD+: protein ADP-ribosyltransferase (polymerizing) {pADPRT; poly(ADP-ribose)polymerase; EC 2.4.2.30} as an active enzyme in Saccharomyces cerevisiae, under the control of the alcohol dehydrogenase promoter, was only possible with simultaneous inhibition of ADP-ribosylation by 3-methoxybenzamide . Induction of fully active pADPRT from the inducible galactose epimerase promoter resulted in inhibition of cell division and morphological changes reminiscent of cell cycle mutants . Expression of a pADPRT cDNA truncated at its 5' end had no influence on cell proliferation at all . Obviously the amino-terminal part of the DNA binding domain containing the first "zinc finger", which is essential for inducibility of pADPRT activity by DNA breaks, is also required for inhibition of cell growth on expression in yeast . Full-length as well as truncated pADPRT molecules were directed to the cell nucleus where the fully active enzyme produced large amounts of poly(ADP-ribose) by automodification . Since pADPRT turned out to be the only target for ADP-ribosylation in these cells, elevated levels of poly(ADP-ribose) were the most likely cause of inhibition of cell division, presumably resulting from interaction with chromosomal proteins.

Mol Cell Biol, 1992 Mar, 12(3), 1078 - 86
IME4, a gene that mediates MAT and nutritional control of meiosis in Saccharomyces cerevisiae; Shah JC et al.; In the yeast Saccharomyces cerevisiae, sporulation occurs in response to nutritional and genetic signals . The process is initiated when nutrient availability limits mitotic growth, but only in MATa/MAT alpha diploid cells . Under these conditions, the cells express an activator of meiosis (IME1), which is required for the expression of early sporulation-specific genes . We describe a new gene, IME4, whose activity is essential for IME1 transcript accumulation and sporulation . The IME4 transcript was induced in starved MATa/MAT alpha diploids but not in other cell types . In addition, excess IME4 promoted sporulation in mat-insufficient cells . Thus, IME4 appears to activate IME1 in response to cell type and nutritional signals . We have also explored the interactions between IME4 and two genes that are known to regulate IME1 expression . Normally, cells that lack complete MAT information cannot sporulate; when such strains lack RME1 activity or contain the semidominant RES1-1 mutation, however, they can express IME1 and sporulate to low levels . Our results show that mat-insufficient strains containing rme1::LEU2 or RES1-1 bypass mutations still retain MAT control of IME4 expression . Even though IME4 levels remained low, the rme1::LEU2 and RES1-1 mutations allowed IME1 accumulation, implying that these mutations do not require IME4 to exert their effects . In accord with this interpretation, the RES1-1 mutation allowed IME1 accumulation in MATa/MAT alpha strains that contain ime4::LEU2 alleles . These strains still sporulated poorly, suggesting that IME4 plays a role in sporulation in addition to promoting IME1 transcript accumulation . IME4 is located between ADE5 and LYS5 on chromosome VII.

Mol Cell Biol, 1992 Mar, 12(3), 1064 - 77
Role of multifunctional autonomously replicating sequence binding factor 1 in the initiation of DNA replication and transcriptional control in Saccharomyces cerevisiae; Rhode PR et al.; Autonomously replicating sequence (ARS) binding factor 1 (ABF1) is an abundant DNA-binding protein that specifically recognizes the motif RTCRYN5ACG at many sites in the yeast genome, including promoter elements, mating-type silencers, and ARSs . Mutational analysis of these sites suggests that ABF1 is involved in constitutive and carbon source-regulated transcriptional activation, transcriptional silencing, and ARS activity . To better assess the role of ABF1 in DNA replication and transcriptional control, temperature-sensitive lethal mutations in the ABF1 gene were isolated . Several of the abf1(Ts) strains show rapid growth arrest at the nonpermissive temperature . At the semipermissive temperature, these strains show an ARS-specific defect in the mitotic stability of ARS-CEN plasmids, such that the abf1 mutants show defects in ARS function identical to those of mutants bearing the mutations in the cis-acting ABF1 binding sites analyzed previously by numerous investigators . Flow cytometric analysis and in vivo DNA labeling experiments on an alpha-factor synchronized abf1(Ts) strain showed that at the nonpermissive temperature, these cells fail to progress efficiently from G1 through S phase and synthesize DNA at 25% of the level seen in the isogenic ABF1 strain . RNA synthesis is also reduced in the abf1(Ts) strains . In addition, transcriptional activation by an ABF1 binding site upstream activation sequence is completely defective in an abf1(Ts) strain at the semipermissive temperature . These phenotypes provide evidence that the same protein, ABF1, functions in the initiation of DNA replication and transcriptional activation.

J Cell Sci, 1992 Mar, 101 ( Pt 3), 709 - 19
Screening and identification of a gene, PSE-1, that affects protein secretion in Saccharomyces cerevisiae; Chow TY et al.; Utilizing a screening method designed for the identification of genes involved with enhanced protein secretion in Saccharomyces cerevisiae we identified a gene, which we named PSE-1 (Protein Secretion Enhancer) . Overexpression of PSE-1 in a multi-copy plasmid, as shown by Northern hybridization, gave a fourfold enhancement in total protein secretion . The repertoire of proteins that are found to be secreted in greater quantities include three known biologically active proteins: k1 killer toxin, alpha-factor, and acid phosphatase . The PSE-1 gene is located on chromosome XII of the yeast genome and codes for a hydrophobic protein containing 1089 amino acids . Haploid yeast cells that contained a LEU2 insertion mutation in PSE-1 grow very poorly, a phenotype similar to other conditional SEC mutants at restrictive temperature.

Appl Microbiol Biotechnol, 1992 Mar, 36(6), 754 - 8
The activity of a model heterologous protein in pep4-3 mutants of Saccharomyces cerevisiae; Wingfield JM et al.; The bacterial lacZ gene was introduced into two sibling strains of Saccharomyces cerevisiae, one a wild-type strain with normal proteinase activity and the other a pep4-3 mutant strain . The pep4-3 mutation resulted in 90% reduced activity of the four major vacuolar proteinases . By comparing the activity of the lacZ gene product (beta-galactosidase) in both strains the degradative effect of the major vacuolar proteinases on a heterologous protein was estimated . The mutant strain with reduced proteinase activity had higher beta-galactosidase activity under all the test conditions . In the most productive case the pep4-3 mutant had 55% higher beta-galactosidase activity than the wild-type . Batch cultures of the two strains were evaluated for growth characteristics . The strain with reduced proteinase activity grew to higher optical densities than the wild-type . Upon further examination it was found that not only were the optical densities of pep4-3 cultures greater but the cell numbers were much greater than expected due to the smaller size of pep4-3 cells . It is concluded that the strain lacking vacuolar proteinases maintained increased levels of beta-galactosidase and is physiologically as healthy as the wild-type.

J Bacteriol, 1992 Mar, 174(6), 1828 - 36
Sequence of the GLN1 gene of Saccharomyces cerevisiae: role of the upstream region in regulation of glutamine synthetase expression; Minehart PL et al.; The GLN1 gene, encoding glutamine synthetase in Saccharomyces cerevisiae, was sequenced, and its encoded polypeptide was shown to have significant homology to other eukaryotic glutamine synthetases . S1 analysis has defined the transcriptional start site of the gene . Upstream analysis of the gene using lacZ fusions has verified transcriptional control of the gene and has identified a nitrogen upstream activation sequence which is required for the increased transcription of GLN1 seen when glutamine is replaced by glutamate as the nitrogen source . cis-acting sites required for the increased transcription in response to purine starvation also have been localized.

Eur J Biochem, 1992 Mar 1, 204(2), 713 - 23
Identification and molecular characterization of the calmodulin-binding subunit gene (CMP1) of protein phosphatase 2B from Saccharomyces cerevisiae . An alpha-factor inducible gene; Ye RR et al.; A method has been developed for the rapid purification of yeast calmodulin in high yield . Using a 125I-labeled calmodulin SDS/PAGE gel overlay procedure with either yeast or bovine calmodulin, we show that the bovine and yeast proteins recognize the same proteins in total yeast extracts . However, yeast calmodulin does not bind to many of the proteins in vertebrate cells identified using bovine calmodulin . A lambda gt11 yeast genomic expression library was screened with yeast or bovine brain 125I-calmodulin to identify sequences derived from calmodulin binding proteins . Twelve clones were recovered, all containing a common DNA insert; all bound calmodulin in a Ca(2+)-dependent manner . The complete coding sequence was recovered and sequenced . The predicted protein sequence show greater than 50% identity to the A subunit of vertebrate protein phosphatase 2B . The gene was designated CMP1 and shown to reside on chromosome IV . Disruption or over-expression of CMP1 have no obvious phenotype; yeast appears to contain one or more CMP1-related genes . The protein product of the CMP1 gene is elevated by alpha-factor treatment, suggesting an involvement of protein phosphatase 2B in the mating response.

Eur J Biochem, 1992 Feb 15, 204(1), 121 - 6
Expression of the Saccharomyces cerevisiae Kex2p endoprotease in inset cells . Evidence for a carboxy-terminal autoprocessing event; Germain D et al.; The pheromone-processing Kex2p endoprotease of Saccharomyces cerevisiae has been difficult to characterize due to its low level of expression in yeast cells . To overcome this problem, we have overexpressed Kex2p using the baculovirus/insect cell expression system . Spodoptera frugiperda Sf9 insect cells infected with a recombinant baculovirus, containing the complete KEX2 gene which encodes the Kex2p protease (814 amino acids), accumulate an 120-kDa functional form of the enzyme . The inhibition profile of the insect-cell-derived endoprotease is similar to that of the yeast enzyme . The recombinant infected insect cells also secrete into the medium about half of the total Kex2p activity produced . Deleting the carboxyl-terminal tail and the transmembrane domain of Kex2p (Kex2 delta p, 666 amino acids) does not measurably interfere with the enzyme characteristics and results in the secretion of up to 90% of the total enzyme activity . The truncated form, Kex2 delta p, of the endoprotease accumulates in the cell supernatant to 6.7 x 10(5) U/l . The molecular mass of the secreted forms for both the wild-type Kex2p and Kex2 delta p is the same (70 kDa) and is 50-kDa lower than the intracellular form . This result implicates a processing event which gives rise to shorter extracellular forms of both the wild-type Kex2p and Kex2 delta p and which trims their carboxy termini upsteam of amino acid 666 . This processing event requires the integrity of the Ser385 of the Kex2p active site.

J Biol Chem, 1992 Feb 15, 267(5), 2894 - 901
Cloning of TFC1, the Saccharomyces cerevisiae gene encoding the 95-kDa subunit of transcription factor TFIIIC; Parsons MC et al.; The yeast gene encoding the 95-kDa subunit of the class III gene transcription factor TFIIIC was cloned . This gene, termed TFC1 (transcription factor C, gene 1), was isolated by screening a lambda gt11 yeast cDNA expression library using a polyclonal antiserum preparation which was previously shown to specifically recognize the 95-kDa subunit of yeast TFIIIC (Parsons, M . C., and Weil, P . A . (1990) J . Biol . Chem . 265, 5095-5103) . TFC1 was found to be a single copy gene which contained a continuous open reading frame about 2 kilobases in length . TFC1 was shown to encode the 95-kDa subunit of TFIIIC by several criteria . Like the authentic yeast protein, the protein encoded by TFC1 had an apparent molecular weight of 95,000 . In addition, the protein encoded by the TFC1 gene bound to the same antibody species as the yeast 95-kDa subunit of TFIIIC . Last, the sizes of the cleavage products of the Escherichia coli-expressed protein were indistinguishable from those of the cleavage products of the bona fide yeast 95-kDa protein.

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