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