S. cerevisiae

Microbiologia, 1992 Nov, 8(2), 82 - 93
Identification of yeast cloned genes by genetic analysis; Martin-Rendon E et al.; Gene cloning in yeast is usually carried out by complementation of recessive mutations . However, the fact that a DNA fragment is able to complement a mutation in a certain gene does not necessarily mean that it contains that gene . The identification of a cloned gene can involve the use of Molecular and/or Classical Genetics techniques . In this paper we describe the strategy to be followed in order to establish the identity of a cloned gene, by using genetic crosses and tetrad analysis . As a practical example of the use of this strategy, we describe the cloning of the THR1 gene which codes for the homoserine kinase in S . cerevisiae . This gene has been isolated from a yeast genomic library by complementation of a thr1 mutation . The complementing DNA fragment has been subcloned and integrated into the yeast genome . By genetic crosses and tetrad analysis it has been demonstrated that integration has occurred at the THR1 locus . Since in this organism integration takes place mainly by homologous recombination, it can be inferred that we have, in fact, cloned the THR1 gene . Biochemical analysis of the transformant that carries multiple copies of the cloned gene confirms this result . It shows that this strain presents a homoserine kinase activity about 60 times higher than that of the wild type.

Mol Cell Biol, 1992 Nov, 12(11), 4988 - 93
Genome rearrangement in top3 mutants of Saccharomyces cerevisiae requires a functional RAD1 excision repair gene; Bailis AM et al.; Saccharomyces cerevisiae cells that are mutated at TOP3, a gene that encodes a protein homologous to bacterial type I topoisomerases, have a variety of defects, including reduced growth rate, altered gene expression, blocked sporulation, and elevated rates of mitotic recombination at several loci . The rate of ectopic recombination between two unlinked, homologous loci, SAM1 and SAM2, is sixfold higher in cells containing a top3 null mutation than in wild-type cells . Mutations in either of the two other known topoisomerase genes in S . cerevisiae, TOP1 and TOP2, do not affect the rate of recombination between the SAM genes . The top3 mutation also changes the distribution of recombination events between the SAM genes, leading to the appearance of novel deletion-insertion events in which conversion tracts extend beyond the coding sequence, replacing the DNA flanking the 3' end of one SAM gene with nonhomologous DNA flanking the 3' end of the other . The effects of the top3 null mutation on recombination are dependent on the presence of an intact RAD1 excision repair gene, because both the rate of SAM ectopic gene conversion and the conversion tract length were reduced in rad1 top3 mutant cells compared with top3 mutants . These results suggest that a RAD1-dependent function is involved in the processing of damaged DNA that results from the loss of Top3 activity, targeting such DNA for repair by recombination.

Cell, 1992 Oct 30, 71(3), 489 - 503
A mammalian homolog of SEC61p and SECYp is associated with ribosomes and nascent polypeptides during translocation; Gorlich D et al.; SEC61p is essential for protein translocation across the endoplasmic reticulum membrane of S . cerevisiae . We have found a mammalian homolog that shows more than 50% sequence identity with the yeast protein . Moreover, several regions of SEC61p have significant similarities with corresponding ones of SecYp of bacteria, indicating a strong evolutionary conservation of the mechanism of protein translocation . Mammalian Sec61p, like the yeast protein, is located in the immediate vicinity of nascent polypeptides during their membrane passage . It is tightly associated with membrane-bound ribosomes, suggesting that the nascent chain passes directly from the ribosome into a protein-conducting channel . These results define Sec61p as a ubiquitous key component of the protein translocation apparatus.

Nucleic Acids Res, 1992 Oct 25, 20(20), 5297 - 303
Characterization of cDNA encoding the mouse DNA topoisomerase II that can complement the budding yeast top2 mutation; Adachi N et al.; Several cDNA clones encoding mouse DNA topoisomerase II were obtained from a mouse spermatocyte cDNA library and the entire coding sequence of the gene was determined . The mouse DNA topoisomerase II consists of 1528 amino acids with a molecular weight of 173 kDa . It shares significant homologies with the other eucaryotic enzymes, although species-specific sequences are observed in their highly charged C-terminal regions . The complete mouse TOP2 cDNA was put under yeast GAL1 promoter and examined for complementation of top2ts mutation in S.cerevisiae . We found that the cloned mouse gene could rescue the temperature-sensitive top2ts mutation, depending on its induction by galactose . The functional expression of the mouse DNA topoisomerase II in yeast was further confirmed by enzymatic assays and by immunological methods with antibodies specific for the mouse enzyme.

Cell, 1992 Oct 16, 71(2), 267 - 76
The arrest of replication forks in the rDNA of yeast occurs independently of transcription; Brewer BJ et al.; Replication forks, moving opposite to the direction of transcription, are arrested at the 3' ends of the 35S transcription units in the rDNA locus of S . cerevisiae . Because of its position and polarity, we tested the hypothesis that this replication fork barrier (RFB) results from the act of transcription . Three results contradict this hypothesis . First, the RFB persists in a strain containing a disruption of the gene for the 135 kd subunit of RNA polymerase I . Second, the RFB causes a polar arrest of replication forks when transplanted to a plasmid . Third, transcription by RNA polymerase II of a plasmid copy of the 35S transcription unit lacking the RFB does not generate a barrier . We propose that replication forks are arrested in a directional manner through the binding of one or more proteins to two closely spaced sites in the RFB.

Cell, 1992 Oct 16, 71(2), 221 - 30
A suppressor of TBP mutations encodes an RNA polymerase III transcription factor with homology to TFIIB; Buratowski S et al.; The TDS4 gene of S . cerevisiae was isolated as an allele-specific high copy suppressor of mutations within the basic region of the TATA-binding protein (TBP) . The gene is essential for viability and encodes a 596 aa protein . The first 300 aa of the TDS4 protein exhibit significant sequence similarity to the RNA polymerase II transcription factor TFIIB . However, TDS4 is required for RNA polymerase III transcription in vivo and in vitro . Antibodies specific for TDS4 or TBP react with the TFIIIB complex, indicating that both proteins are components of the RNA polymerase III initiation complex . These findings suggest that the RNA polymerase II and III initiation mechanisms are extremely similar, and they explain how the TATA-binding protein can function in both systems.

Cell, 1992 Oct 16, 71(2), 211 - 20
PCF4 encodes an RNA polymerase III transcription factor with homology to TFIIB; Lopez-De-Leon A et al.; A dominant mutation in the PCF4 gene of S . cerevisiae was isolated as a suppressor of a tRNA gene A block promoter mutation . In vitro studies indicate that PCF4 is a stoichiometrically-required RNA polymerase III (pol III) transcription initiation factor . We show that the PCF4-1 mutation increases the number of transcriptionally competent preinitiation complexes by affecting a limiting activity in yeast cell extracts that is squelched by excess TFIIIC . The PCF4 gene encodes a TFIIB homolog whose size, biochemical, and genetic properties are consistent with those of the 70 kd subunit of TFIIIB . The TFIIB homology of PCF4 suggests a means for determining the polymerase specificity of a gene.

J Biol Chem, 1992 Oct 15, 267(29), 20774 - 81
Regulation of iron uptake in Saccharomyces cerevisiae . The ferrireductase and Fe(II) transporter are regulated independently; Eide D et al.; Iron is required for the growth of Saccharomyces cerevisiae . High concentrations of iron, however, are toxic, forcing this yeast to tightly regulate its concentration of intracellular free iron . We demonstrate that S . cerevisiae accumulates iron through the combined action of a plasma membrane ferrireductase and an Fe(II) transporter . This transporter is highly selective for Fe(II) . Several other transition metals did not inhibit iron uptake when these metals were present at a concentration 100-fold higher than the Km (0.15 microM) for iron transport . Pt(II) inhibited ferrireductase activity but not the ability of cells to transport iron that was chemically reduced to Fe(II) . Incubation of cells in a synthetic iron-limited media resulted in the induction of both ferrireductase and Fe(II) transporter activities . In complex media, Fe(II) transport activity was regulated in response to media iron concentration, while the activity of the ferrireductase was not . When stationary phase cells were inoculated into fresh media, ferrireductase activity increased independent of the iron content of the media; in contrast, transporter activity varied inversely with iron levels . These results demonstrate that the ferrireductase and Fe(II) transporter are separately regulated and that iron accumulation may be limited by changes in either activity.

FEBS Lett, 1992 Oct 5, 310(3), 265 - 8
Identification of the mitochondrial receptor complex in Saccharomyces cerevisiae; Moczko M et al.; Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane . In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22 . We isolated a complex out of S . cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N . crassa MOM7, MOM8, MOM19, MOM22 and MOM30 . A receptor complex isolated out of yeast cells transformed with N . crassa MOM19 contained the N . crassa master receptor in addition to the yeast proteins . This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19.

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2029 - 33
The catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase in Saccharomyces cerevisiae; Dickinson JR et al.; Saccharomyces cerevisiae possesses 2-oxoacid dehydrogenase (EC 1.2.4.4) similar to that found in mammalian cells . The activity is readily detected in cells which have been cultured in a minimal medium containing a branched-chain amino acid . Mutants defective in lipoamide dehydrogenase also lack 2-oxoacid dehydrogenase and are thus unable to catabolize branched-chain amino acids: 2-oxoacids accumulate in the cultures of these cells . The 2-oxoacid dehydrogenase activity is distinct from both 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase, because it could not be detected in assay conditions which permitted the measurement of 2-oxoglutarate dehydrogenase and vice versa . In addition, a strain lacking 2-oxoglutarate dehydrogenase (kgd1::URA3) retained 2-oxoacid dehydrogenase as did a mutant specifically lacking pyruvate dehydrogenase (pda1::Tn5ble) . In complex media the specific activity of this enzyme is highest in YEP (yeast extract-peptone)-glycerol and lowest in YEP-acetate and YEP-fructose . 2-Oxoacid dehydrogenase could not be detected in cells which had been transferred to sporulation medium . These results suggest that in S . cerevisiae the catabolism of branched-chain amino acids occurs via 2-oxoacid dehydrogenase, not via the 'Ehrlich Pathway'.

J Gen Microbiol, 1992 Oct, 138 ( Pt 10), 2021 - 8
Physiological analysis of mutants of Saccharomyces cerevisiae impaired in sulphate assimilation; Thomas D et al.; The assimilation of sulphate in Saccharomyces cerevisiae, comprising the reduction of sulphate to sulphide and the incorporation of the sulphur atom into a four-carbon chain, requires the integrity of 13 different genes . To date, the functions of nine of these genes are still not clearly established . A set of strains, each bearing a mutation in one MET gene, was studied . Phenotypic studies and enzyme determinations showed that the products of at least five genes are needed for the synthesis of an enzymically active sulphite reductase . These genes are MET1, MET5, MET8, MET10 and MET20 . Wild-type strains of S . cerevisiae can use organic metabolites such as homocysteine, cysteine, methionine and S-adenosylmethionine as sulphur sources . They are also able to use inorganic sulphur sources such as sulphate, sulphite, sulphide or thiosulphate . Here we show that both of the two sulphur atoms of thiosulphate are used by S . cerevisiae . Thiosulphate is cleaved into sulphite and sulphide prior to utilization by the sulphate assimilation pathway, as the metabolism of one sulphur atom from thiosulphate requires the presence of an active sulphite reductase.

FEMS Microbiol Lett, 1992 Oct 1, 76(1-2), 135 - 9
Anthranilic acid release in adenosine-inhibited cultures of Saccharomyces cerevisiae and its inhibition by thiamin; Iwashima A et al.; Adenosine, at 1 mM concentrations or above, was found to have a fungistatic effect on Saccharomyces cerevisiae . A substance with amethyst fluorescence was detected in the medium of adenosine-inhibited cultures of S . cerevisiae . This compound was isolated and physicochemically identified as anthranilic acid . Both the inhibition of growth and release of anthranilic acid induced by adenosine were abrogated by thiamin or by the pyrimidine portion of thiamin, 2-methyl-4-amino-5-hdroxymethyl-pyrimidine (hydroxymethyl-pyrimidine); the latter was found to restore intracellular thiamin content that had been reduced by adenosine . It was demonstrated that effects of thiamin and hydroxymethylpyrimidine on S . cerevisiae cultured with adenosine resulted from their inhibition of adenosine uptake by growing yeast cells.

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9302 - 5
Characterization and mutagenesis of the gene encoding the A49 subunit of RNA polymerase A in Saccharomyces cerevisiae; Liljelund P et al.; The gene encoding the 49-kDa subunit of RNA polymerase A in Saccharomyces cerevisiae has been identified by formation of a hybrid enzyme between the S . cerevisiae A49 subunit and Saccharomyces douglasii subunits based on a polymorphism existing between the subunits of RNA polymerase A in these two species . The sequence of the gene reveals a basic protein with an unusually high lysine content, which may account for the affinity for DNA shown by the subunit . No appreciable homology with any polymerase subunits, enzymes, or transcription factors is found . Complete deletion of the single-copy RPA49 gene leads to viable but slowly growing colonies . Insertion of the HIS3 gene halfway into the RPA49 coding region results in synthesis of a truncated A49 subunit that is incorporated into the polymerase . The truncated and wild-type subunits compete equally for assembly in the heterozygous diploid, although the wild type is phenotypically dominant.

Curr Genet, 1992 Oct, 22(4), 337 - 9
High-level resistance to cycloheximide resulting from an interaction of the mutated pdr3 and cyh genes in yeast; Ruttkay-Nedecky B et al.; In addition to pdr3-1, the S . cerevisiae nuclear pleiotropic drug resistance mutant 2D was found to contain another recessive nuclear mutation, cyh, conferring specific resistance to cycloheximide only . The cycloheximide resistance level due to either the pdr3-1 or the cyh mutation alone was low and was not altered by the ogd1 mutation which increased the physiological acidification of the culture . When pdr3-1 and cyh mutations occurred simultaneously in the haploid yeast strain their interaction was synergistic and resulted in high-level resistance to cycloheximide.

Curr Genet, 1992 Oct, 22(4), 283 - 8
Genetic mapping of 1,3-beta-glucanase-encoding genes in Saccharomyces cerevisiae; Correa J et al.; The map position of three 1,3-beta-glucanase-encoding genes in S . cerevisiae has been determined following conventional meiotic and mitotic mapping combined with recombinant DNA techniques . EXG1, EXG2 and SSG1 were localized to chromosomes XII, IV and XV, respectively, by hybridizing the cloned genes to Southern blots of chromosomes separated by pulsed-field gel electrophoresis, in conjunction with the rad52-1-dependent chromosome-loss mapping technique . Meiotic tetrad analyses further localized the EXG1 gene 6.1 centimorgans centromere-proximal to CDC25 on the right arm of chromosome XII . EXG2 was positioned between LYS4 and GCN2 on the right arm of chromosome IV, at distances of 6.2 centimorgans from LYS4 and 4.9 centimorgans from GCN2 . Finally, the SSG1 locus mapped on the right arm of chromosome XV, about 8.2 centimorgans to the centromere-proximal side of HIS3.

Cell, 1992 Sep 18, 70(6), 961 - 73
Translation initiation requires the PAB-dependent poly(A) ribonuclease in yeast; Sachs AB et al.; Messenger RNA translation initiation and cytoplasmic poly(A) tail shortening require the poly(A)-binding protein (PAB) in yeast . The PAB-dependent poly(A) ribonuclease (PAN) has been purified to near homogeneity from S . cerevisiae based upon its PAB requirement, and its gene has been cloned . The essential PAN1 gene encodes a 161 kd protein organized into distinct domains containing repeated sequence elements . Deletion analysis of the gene revealed that only one-third of the protein is needed to maintain cell viability . Conditional mutations in PAN1 lead to an arrest of translation initiation and alterations in mRNA poly(A) tail lengths . These data suggest that PAN could mediate each of the PAB-dependent reactions within the cell, and they provide evidence for a direct relationship between translation initiation and mRNA metabolism.

J Biol Chem, 1992 Sep 15, 267(26), 18890 - 5
Farnesylation of YDJ1p is required for function at elevated growth temperatures in Saccharomyces cerevisiae; Caplan AJ et al.; The Saccharomyces cerevisiae YDJ1 protein (YDJ1p) contains a C-terminal "CaaX box" motif common to proteins that are modified by prenylation . In the present study we show that YDJ1p is a specific substrate for both yeast and mammalian protein farnesyltransferase enzymes in vitro . A mutant form of YDJ1p, in which the conserved cysteine of the CaaX box is mutated to a serine (ydj1-S406p), cannot be farnesylated in vitro . After expression in S . cerevisiae, ydj1-S406p displays a reduced electrophoretic mobility and an increased cytosolic localization in subcellular fractionation experiments when compared to wild type YDJ1p . Expression of ydj1-S406 in cells lacking YDJ1 results in a temperature-sensitive growth phenotype in S . cerevisiae . These data indicate that farnesylation of YDJ1p is required for its function at elevated temperatures.

J Biol Chem, 1992 Sep 15, 267(26), 18790 - 6
Purification and characterization of casein kinase II (CKII) from delta cka1 delta cka2 Saccharomyces cerevisiae rescued by Drosophila CKII subunits . The free catalytic subunit of casein kinase II is not toxic in vivo; Bidwai AP et al.; Casein kinase II (CKII) is composed of a catalytic (alpha) and a regulatory (beta) subunit which unite to form an alpha 2 beta 2 holoenzyme . Saccharomyces cerevisiae CKII consists of two distinct catalytic (Sc alpha and Sc alpha') and regulatory (Sc beta and Sc beta') subunits . Simultaneous disruption of the CKA1 and CKA2 genes (encoding the alpha and alpha' subunits, respectively) is lethal . Such double disruptions can be rescued by GAL1, 10-induced expression of the Drosophila alpha and beta subunits (Dm alpha+beta) together or by GAL10-induced expression of the Drosophila alpha subunit (Dm alpha) alone (Padmanabha, R., Chen-Wu, J . L.-P., Hanna, D . E., and Glover, C . V . C . (1990) Mol . Cell . Biol . 10, 4089-4099) . Here we report quantitation, purification, and characterization of casein kinase II activity from such rescued strains . Casein kinase II activity from a strain rescued by Dm alpha alone purifies as a free, catalytically active alpha subunit monomer, whereas that from a strain rescued by Dm alpha/beta purifies as a mixture of tetrameric holoenzyme and monomeric alpha subunit . Interestingly, neither Sc beta nor Sc beta' is present at detectable levels in the enzyme obtained from either strain, raising the possibility that rescue by Dm alpha alone may be mediated via the free, monomeric catalytic subunit . Overexpression of total casein kinase II activity from 6- to 18-fold is not toxic and indeed has no overt phenotypic consequences . Production of large amounts of free catalytic subunit also appears to be without effect, even though free catalytic subunit is normally undetectable in S . cerevisiae.

Nucleic Acids Res, 1992 Sep 11, 20(17), 4649 - 55
Cloning of human and bovine homologs of SNF2/SWI2: a global activator of transcription in yeast S . cerevisiae; Okabe I et al.; We performed positional cloning of genes carried on yeast artificial chromosomes that span a human translocation breakpoint associated with a human disease and isolated by chance human and bovine genes with strong homology to the S . cerevisiae genes, SNF2/SWI2 and STH1, and the D . melanogaster gene brahma . We report here sequence analysis, expression data, and functional studies for this human SNF2-like gene (hSNF2L) and its bovine homolog (bovSNF2L) . Despite strong homology at the amino acid level, hSNF2L is not capable of complementing the yeast mutations snf2 or sth1 in S . cerevisiae . Furthermore, in contrast to SNF2 itself, a fusion protein consisting of the DNA binding domain of LexA and hSNF2L did not transactivate a reporter gene downstream of LexA binding sites in a yeast expression system . The strong similarity between hSNF2L and these yeast and drosophila genes suggest that the mammalian genes are part of an evolutionarily conserved family that has been implicated as global activators of transcription in yeast and fruitflies but whose function in mammals remains unknown.

Gene, 1992 Sep 10, 118(2), 247 - 53
Overproduction of a human snRNP-associated Sm-D autoantigen in Escherichia coli and Saccharomyces cerevisiae; Rokeach LA et al.; To conduct functional and autoimmunity studies, we overproduced human Sm-D1 (hSm-D1), a small nuclear ribonucleoprotein 'core' protein and autoantigen, in Escherichia coli and Saccharomyces cerevisiae . Optimal expression in these organisms was achieved by designing vectors that synthesized abundant hSm-D1 mRNA under the control of the strong, regulatable promoters: T7 phi 10 (E . coli) and GAL1 (yeast) . In addition, efficient translation initiation of the hSm-D1 coding sequence was effected in E . coli by utilizing a two-cistron approach; for expression in yeast, we created a 5' untranslated leader whose sequence was based on the consensus of highly expressed genes in S . cerevisiae . The hSm-D1 protein accumulated at high levels in both bacteria and yeast, representing, respectively, approx . 10% and 7% of the total protein . However, in comparison with the authentic protein, the recombinant hSm-D1 displayed different immunoreactive determinants as assessed by Western blot . We thus conclude that certain hSm-D1 immunologic properties are most likely dependent on posttranslational modifications that take place in the cells of higher eukaryotes.

J Mol Biol, 1992 Sep 5, 227(1), 54 - 71
Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein; Alani E et al.; Single-stranded DNA binding proteins (SSBs) have been isolated from many organisms, including Escherichia coli, Saccharomyces cerevisiae and humans . Characterization of these proteins suggests they are required for DNA replication and are active in homologous recombination . As an initial step towards understanding the role of the eukaryotic SSBs in DNA replication and recombination, we examined the DNA binding and strand exchange stimulation properties of the S . cerevisiae single-strand binding protein y-RPA (yeast replication protein A) . y-RPA was found to bind to single-stranded DNA (ssDNA) as a 115,000 M(r) heterotrimer containing 70,000, 36,000 and 14,000 M(r) subunits . It saturated ssDNA at a stoichiometry of one heterotrimer per 90 to 100 nucleotides and binding occurred with high affinity (K omega greater than 10(9) M-1) and co-operativity (omega = 10,000 to 100,000) . Electron microscopic analysis revealed that y-RPA binding was highly co-operative and that the ssDNA present in y-RPA-ssDNA complexes was compacted fourfold, arranged into nucleosome-like structures, and was free of secondary structure . y-RPA was also tested for its ability to stimulate the yeast Sepl and E . coli RecA strand-exchange proteins . In an assay that measures the pairing of circular ssDNA with homologous linear duplex DNA, y-RPA stimulated the strand-exchange activity of Sepl approximately threefold and the activity of RecA protein to the same extent as did E . coli SSB . Maximal stimulation of Sepl occurred at a stoichiometry of one y-RPA heterotrimer per 95 nucleotides of ssDNA . y-RPA stimulated RecA and Sepl mediated strand exchange reactions in a manner similar to that observed for the stimulation of RecA by E . coli SSB; in both of these reactions, y-RPA inhibited the aggregation of ssDNA and promoted the co-aggregation of single-stranded and double-stranded linear DNA . These results demonstrate that the E . coli and yeast SSBs display similar DNA-binding properties and support a model in which y-RPA functions as an E . coli SSB-like protein in yeast.

J Bacteriol, 1992 Sep, 174(17), 5702 - 10
The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae; Racenis PV et al.; The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae . In the total membrane fraction of S . cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein) . The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide . Total membrane fraction of S . cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 1.1.1.101) activity, which has not been reported previously . The reductase has a Vmax of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHAP (Km = 15 microM) by NADPH (Km = 20 microM) . Both acyl DHAP and alkyl DHAP acted as substrates . NADPH was the specific cofactor . Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction . Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells . Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells . The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHAP synthase) in S . cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.

Mol Cell Biol, 1992 Sep, 12(9), 4142 - 52
Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II; Archambault J et al.; Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors . One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro . In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene . Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU) . Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S . cerevisiae RNAPII (RPO21) . A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis . All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII . The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII . We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS . These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites . In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.

Mol Cell Biol, 1992 Sep, 12(9), 3948 - 58
Early meiotic transcripts are highly unstable in Saccharomyces cerevisiae; Surosky RT et al.; Meiosis in Saccharomyces cerevisiae requires the induction of a large number of genes whose mRNAs accumulate at specific times during meiotic development . This study addresses the role of mRNA stability in the regulation of meiosis-specific gene expression . Evidence is provided below demonstrating that the levels of meiotic mRNAs are exquisitely regulated by both transcriptional control and RNA turnover . The data show that (i) early meiotic transcripts are extremely unstable when expressed during either vegetative growth or sporulation, and (ii) transcriptional induction, rather than RNA turnover, is the predominant mechanism responsible for meiosis-specific transcript accumulation . When genes encoding the early meiotic mRNAs are fused to other promoters and expressed during vegetative growth, their mRNA half-lives, of under 3 min, are among the shortest known in S . cerevisiae . Since these mRNAs are only twofold more stable when expressed during sporulation, we conclude that developmental regulation of mRNA turnover can be eliminated as a major contributor to meiosis-specific mRNA accumulation . The rapid degradation of the early mRNAs at all stages of the yeast life cycle, however, suggests that a specific RNA degradation system operates to maintain very low basal levels of these transcripts during vegetative growth and after their transient transcriptional induction in meiosis . Studies to identify specific cis-acting elements required for the rapid degradation of early meiotic transcripts support this idea . A series of deletion derivatives of one early meiosis-specific gene, SPO13, indicate that its mRNA contains determinants, located within the coding region, which contribute to the high instability of this transcript . Translation is another component of the degradation mechanism since frameshift and nonsense mutations within the SPO13 mRNA stabilize the transcript.

J Gen Microbiol, 1992 Sep, 138 ( Pt 9), 1865 - 73
Induction of specific enzymes of the oxidative pentose phosphate pathway by glucono-delta-lactone in Saccharomyces cerevisiae; Sinha A et al.; Growth of Saccharomyces cerevisiae on D-glucono-delta-lactone (delta gl) was found to be associated with a specific coordinate induction of the synthesis of two enzymes of the oxidative pentose phosphate pathway--6-phosphogluconate dehydrogenase and 6-phosphogluconolactonase--together with that of a third enzyme, gluconokinase . The gnd1 mutation, responsible for an approximately 80% loss of 6-phosphogluconate dehydrogenase activity and the inability of the cells to grow on delta gl, completely abolished the induction of all three enzymes, while the gnd2 mutation affected this only partially . One class of gnd1 revertants, selected for growth on delta gl, was found to have recovered normal dehydrogenase activity and the ability to synthesize the three enzymes when induced by delta gl . Another class of delta gl-positive revertants possessed constitutively elevated levels of gluconokinase . In contrast, glucose-positive revertants of gnd1, with restored constitutive dehydrogenase activity, continued to remain deficient in induction of the three enzymes and also failed to grow on delta gl . Induction of 6-phosphogluconate dehydrogenase activity was associated with increased transcription of the gene coding for the major isoenzyme; the transcript remained undetectable in the gnd1 mutant . Induction of these specific enzymes thus appears to be essential for growth of S . cerevisiae on delta gl.

Eur J Biochem, 1992 Sep 1, 208(2), 487 - 91
Activation by ATP of a proton-conducting pathway in yeast mitochondria; Prieto S et al.; The growth of Saccharomyces cerevisiae cells under aerobic conditions, in the presence of an energy-rich source, leads to production of an excess of NAD(P)H . Since the redox balance must be maintained, it has been postulated that NAD(P)H reoxidation is accelerated by the activation of energy-dissipating reactions, which would, in turn, explain the low growth efficiencies observed . It has been demonstrated already in S . cerevisiae cultures that these putative energy-dissipating reactions are stimulated both by oxygen and high cytosolic ATP levels . In this paper, we show that ATP induces a proton-permeability pathway in mitochondria at concentrations which are within the physiological range, as revealed both from the ATP stimulation of respiration and from the induction of H(+)-dependent swelling . We also demonstrate that phosphate acts as a competitive inhibitor of the nucleotide, and since activation is observed even in the presence of atractylate, we postulate that the ATP-binding site is located in the outer face of the mitochondrial inner membrane.

EMBO J, 1992 Sep, 11(9), 3441 - 7
Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae; Zenvirth D et al.; We present a scheme for locating double-strand breaks (DSBs) in meiotic chromosomes of Saccharomyces cerevisiae, based on the separation of large DNA molecules by pulsed field gel electrophoresis . Using a rad50S mutant, in which DSBs are not processed, we show that DSBs are widely induced in S . cerevisiae chromosomes during meiosis . Some of the DSBs accumulate at certain preferred sites . We present general profiles of DSBs in chromosomes III, V, VI and VII . A map of the 12 preferred sites on chromosome III is presented . At least some of these sites correlate with known 'hot spots' for meiotic recombination . The data are discussed in view of current models of meiotic recombination and chromosome segregation.

EMBO J, 1992 Sep, 11(9), 3421 - 30
A single-stranded DNA binding protein required for mitochondrial DNA replication in S . cerevisiae is homologous to E . coli SSB; Van Dyck E et al.; It has previously been shown that the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae becomes thermosensitive due to the inactivation of the mitochondrial DNA helicase gene, PIF1 . A suppressor of this thermosensitive phenotype was isolated from a wild-type plasmid library by transforming a pif1 null strain to growth on glycerol at the non-permissive temperature . This suppressor is a nuclear gene encoding a 135 amino acid protein that is itself essential for mtDNA replication; cells lacking this gene are totally devoid of mtDNA . We therefore named this gene RIM1 for replication in mitochondria . The primary structure of the RIM1 protein is homologous to the single-stranded DNA binding protein (SSB) from Escherichia coli and to the mitochondrial SSB from Xenopus laevis . The mature RIM1 gene product has been purified from yeast extracts using a DNA unwinding assay dependent upon the DNA helicase activity of SV40 T-antigen . Direct amino acid sequencing of the protein reveals that RIM1 is a previously uncharacterized SSB . Antibodies against this purified protein localize RIM1 to mitochondria . The SSB encoded by RIM1 is therefore an essential component of the yeast mtDNA replication apparatus.

FEBS Lett, 1992 Aug 31, 309(1), 103 - 6
Ca2+/calmodulin-activated protein phosphatase (PP2B) of Saccharomyces cerevisiae . PP2B activity is not essential for growth; Nakamura T et al.; Protein phosphatase (PP2B) whose activity is stimulated 12-20-fold by Ca2+/calmodulin (CaM) was partially purified by CaM-Sepharose and heparin-agarose chromatographies from cell extract of the yeast Saccharomyces cerevisiae . PP2B activity was not detectable in a mutant in which two genes (CMP1 and CMP2) encoding homologs of mammalian PP2B catalytic subunit were disrupted . We have previously shown that the double gene disruption has no significant effect on the growth of yeast {1991, Mol . Gen . Genet . 227, 52-59} . The results indicated that CMP1 and CMP2 are the only genes that encode the PP2B catalytic polypeptide in S . cerevisiae, and PP2B activity is not essential for the growth of the yeast under normal conditions.

J Biol Chem, 1992 Aug 15, 267(23), 16297 - 304
Purification, characterization, and kinetic analysis of a 55-kDa form of phosphatidylinositol 4-kinase from Saccharomyces cerevisiae; Nickels JT Jr et al.; A 55-kDa form of membrane-associated phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 10,166-fold from Saccharomyces cerevisiae . The purification procedure included solubilization of microsome membranes with 1% Triton X-100 followed by chromatography with DE52, hydroxylapatite I, Q-Sepharose, Mono Q, and hydroxylapatite II . The procedure resulted in a nearly homogeneous 55-kDa phosphatidylinositol 4-kinase preparation . The 55-kDa phosphatidylinositol 4-kinase and the previously purified 45-kDa phosphatidylinositol 4-kinase differed with respect to their amino acid composition, isoelectric points, and peptide maps . Furthermore, the two forms of phosphatidylinositol 4-kinase did not show an immunological relationship . Maximum 55-kDa phosphatidylinositol 4-kinase activity was dependent on magnesium (10 mM) or manganese (0.5 mM) ions and Triton X-100 at the pH optimum of 7.0 . The activation energy for the reaction was 12 kcal/mol, and the enzyme was labile above 30 degrees C . The enzyme was inhibited by thioreactive agents, MgADP, and calcium ions . A detailed kinetic analysis of the purified enzyme was performed using Triton X-100/phosphatidylinositol-mixed micelles . 55-kDa phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol and followed surface dilution kinetics . The interfacial Michaelis constant (Km) and the dissociation constant (Ks) for phosphatidylinositol in the Triton X-100 micelle surface were 1.3 mol % and 0.035 mM, respectively . The Km for MgATP was 0.36 mM . 55-kDa phosphatidylinositol 4-kinase catalyzed a sequential reaction mechanism as indicated by the results of kinetic and isotopic exchange reactions . The enzyme bound to phosphatidylinositol before ATP and released phosphatidylinositol 4-phosphate before ADP . The enzymological and kinetic properties of the 55-kDa phosphatidylinositol 4-kinase differed significantly from those of the 45-kDa phosphatidylinositol 4-kinase . This may suggest that the two forms of phosphatidylinositol 4-kinase from S . cerevisiae are regulated differentially in vivo.

J Biol Chem, 1992 Aug 25, 267(24), 16801 - 5
Effects of amino-terminal extensions and specific mutations on the activity of restrictocin; Yang R et al.; The cytotoxic activities of restrictocin with aminoterminal extensions and specific mutations were investigated using in vivo and in vitro systems . Genes were constructed from the cDNA clone of restrictocin which encode: the native form of restrictocin (including the leader sequence); Met-prorestrictocin, in which a codon for methionine was placed before a putative pro region; Met-mature restrictocin, with a methionine codon prior to the mature form of restrictocin; and three mutated forms of Met-mature restrictocin, E95G, E115G/H136L, and H136L . These constructions were placed under the control of the GAL1 promoter and were transformed into Saccharomyces cerevisiae . Transformants were killed, and a new RNA band formed when any of these genes except those containing the H136L mutation were expressed . Restrictocin protein was detected by immunoblot only in cells expressing the native form of restrictocin and the forms containing the H136L mutation . Native restrictocin, Met-prorestrictocin, and Met-mature restrictocin mRNA were translated in an in vitro system resulting in proteins of the expected molecular weight and inactivation of the translation system . Restrictocin was not inactivated by the presence of the leader sequence and the putative prosequence . Amino acid His136 is putatively in the active site of restrictocin by analogy to ribonuclease U2 and the elimination of toxic effects in the S . cerevisiae expression and in vitro translation systems.

Nucleic Acids Res, 1992 Aug 25, 20(16), 4325 - 30
Cloning of the gene for the 73 kD subunit of the DNA polymerase alpha primase of Drosophila melanogaster; Cotterill S et al.; We have isolated both cDNA and genomic clones for the 73 kDa subunit of the DNA polymerase alpha primase of Drosophila melanogaster . Analysis of these clones has identified an open reading frame of 1959 bases coding for a protein of 72.5 kDa . Northern analysis has shown the mRNA for the gene to be approximately 2.5 kb, and comparison of the cDNA and the genomic clones shows that the coding region of the gene lacks introns . The 5' end of the transcript has been mapped by primer extension, and the position of the gene in the genome mapped using in situ analysis . Computer analysis has been carried out on both coding and non coding regions of the gene . The protein sequence shows some homology to the analogous subunit in the S . cerevisiae DNA polymerase alpha, however a search of the data banks failed to reveal other homologies, or provide any clues as to the function of the protein . Analysis of the non-coding regions indicates some potential control regions for the gene . The 73 kDa protein has been overproduced, but a preliminary analysis failed to reveal any enzymatic activities.

J Biol Chem, 1992 Aug 25, 267(24), 17178 - 85
Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase; Kim RA et al.; The TOP3 gene of the yeast Saccharomyces cerevisiae was postulated to encode a DNA topoisomerase, based on its sequence homology to Escherichia coli DNA topoisomerase I and the suppression of the poor growth phenotype of top3 mutants by the expression of the E . coli enzyme (Wallis, J.W., Chrebet, G., Brodsky, G., Golfe, M., and Rothstein, R . (1989) Cell 58, 409-419) . We have purified the yeast TOP3 gene product to near homogeneity as a 74-kDA protein from yeast cells lacking DNA topoisomerase I and overexpressing a plasmid-borne TOP3 gene linked to a phosphate-regulated yeast PHO5 gene promoter . The purified protein possesses a distinct DNA topoisomerase activity: similar to E . coli DNA topoisomerases I and III, it partially relaxes negatively but not positively supercoiled DNA . Several experiments, including the use of a negatively supercoiled heteroduplex DNA containing a 29-nucleotide single-stranded loop, indicate that the activity has a strong preference for single-stranded DNA . A protein-DNA covalent complex in which the 74-kDa protein is linked to a 5' DNA phosphoryl group has been identified, and the nucleotide sequences of 30 sites of DNA-protein covalent complex formation have been determined . These sequences differ from those recognized by E . coli DNA topoisomerase I but resemble those recognized by E . coli DNA topoisomerase III . Based on these results, the yeast TOP3 gene product can formally be termed S . cerevisiae DNA topoisomerase III . Analysis of supercoiling of intracellular yeast plasmids in various DNA topoisomerase mutants indicates that yeast DNA topoisomerase III has at most a weak activity in relaxing negatively supercoiled double-stranded DNA in vivo, in accordance with the characteristics of the purified enzyme.

Cell, 1992 Aug 21, 70(4), 659 - 70
Recombination between similar but not identical DNA sequences during yeast transformation occurs within short stretches of identity; Mezard C et al.; Interactions between similar but not identical (homeologous) DNA sequences play an important biological role in the evolution of genes and genomes . To gain insight into the underlying molecular mechanism(s) of genetic recombination, we have studied inter- and intramolecular homeologous recombination in S . cerevisiae during transformation . We found that homeologous DNAs recombine efficiently . Hybrid sequences were obtained between two mammalian cytochrome P450 cDNAs, sharing 73% identity, and between the yeast ARG4 gene and its human homeologous cDNA, sharing 52% identity . Sequencing data showed that the preferred recombination events are those corresponding to the overall alignment of the DNA sequences and that the junctions are within stretches of identity of variable length (2-21 nt) . We suggest that these events occur by a conventional homologous recombination mechanism.

Cell, 1992 Aug 21, 70(4), 647 - 57
Autoregulation of the yeast lysyl-tRNA synthetase gene GCD5/KRS1 by translational and transcriptional control mechanisms; Lanker S et al.; We cloned the GCD5 gene of S . cerevisiae and found it to be identical to KRS1, which encodes lysyl-tRNA synthetase (LysRS) . The mutation gcd5-1 changes a conserved residue in the putative lysine-binding domain of LysRS . This leads to a defect in lysine binding and, consequently, to reduced charging of tRNA(Lys) . Mutant gcd5-1 cells compensate for the defect in LysRS by increasing GCN4 expression at the translational level . GCN4 protein in turn stimulates transcription of GCD5, leading to increased LysRS activity . We propose an autoregulatory model in which uncharged tRNA(Lys) stimulates the protein kinase GCN2, a translational activator of GCN4, and thereby increases transcription of GCD5 and other genes regulated by GCN4.

J Inorg Biochem, 1992 Aug 15-Sep, 47(3-4), 249 - 55
Ferric iron reduction and iron assimilation in Saccharomyces cerevisiae; Anderson GJ et al.; We have used the yeast Saccharomyces cerevisiae as a model organism to study the role of ferric iron reduction in eucaryotic iron uptake . S . cerevisiae is able to utilize ferric chelates as an iron source by reducing the ferric iron to the ferrous form, which is subsequently internalized by the cells . A gene (FRE1) was identified which encodes a protein required for both ferric iron reduction and efficient ferric iron assimilation, thus linking these two activities . The predicted FRE1 protein appears to be a membrane protein and shows homology to the beta-subunit of the human respiratory burst oxidase . These data suggest that FRE1 is a structural component of the ferric reductase . Subcellular fractionation studies showed that the ferric reductase activity of isolated plasma membranes did not reflect the activity of the intact cells, implying that cellular integrity was necessary for function of the major S . cerevisiae ferric reductase . An NADPH-dependent plasma membrane ferric reductase was partially purified from plasma membranes . Preliminary evidence suggests that the cell surface ferric reductase may, in addition to mediating cellular iron uptake, help modulate the intracellular redox potential of the yeast cell.

Biochem Biophys Res Commun, 1992 Aug 14, 186(3), 1567 - 74
The binding of bridged bis-pyridinium oximes to DNA and its relevance to the induction of mitochondrial dysfunction in yeast; Dodin G et al.; Bis-pyridium oximes and methoximes from a newly synthesized series are weak DNA binders (K = 3.10(4) M-1 under physiological conditions) . From the number of binding sites per phosphate, 0.25, the ionic strength dependence of the binding constant and the negative electric dichroism, it is concluded that monointercalation is the mode of association . In contrast to methoxy compounds, the oxime derivatives are able both to induce the mutated "petite" phenotype in yeast S . cerevisiae and to cause "in vitro" extensive condensation of single stranded DNA . This reaction is postulated to be relevant to the mutational process that leads to "peptide" cells . The absence of nuclear mutation is interpreted in terms of sequestration of the drug in mitochondria under the effect of the organelle inner membrane electrochemical potential.

FEBS Lett, 1992 Aug 10, 308(1), 62 - 4
Tyrosine-89 is important for enzymatic activity of S . cerevisiae inorganic pyrophosphatase; Raznikov AV et al.; 7-Chloro-4-nitro-benzofurazan selectively modifies one PPase Tyr residue per subunit and lowers the enzyme activity . Hydrolysis of the modified protein by trypsin and then by chymotrypsin produces the 82-89 peptide which possesses modified Tyr-89 . Substrate analog (CaPPi) and the product of the enzyme reaction, MgPi, protect the enzyme against inactivation . Ions of metal-activators (Mg2+, Zn2+) exert no influence on the inactivation rate . On the contrary, the Ca(2+)-inhibitor of the enzyme accelerates the reaction by binding to the high-affinity site, and effectively decreases it when Ca2+ binds to both sites . Mg2+ competes with Ca2+ for one binding site, which is the low affinity site for Mg2+ and the high-affinity site for Ca2+ . The Ca2+ saturation of the high-affinity site decreases the pK2 of Tyr-89, probably due to direct coordination between Tyr and Ca2+ . The observed properties of Tyr-89 modification enable us to propose that Tyr-89 serves as a proton donor for phosphate releasing during enzymatic hydrolysis of pyrophosphate . The Ca2+ inhibitory effect on the enzyme activity may be due to the existence of a Tyr-89 bond in the Ca2+ pyrophosphatase complex.

Cell, 1992 Aug 7, 70(3), 451 - 8
Kinesin-related proteins required for structural integrity of the mitotic spindle; Saunders WS et al.; For S . cerevisiae cells, the assembly of a bipolar mitotic spindle requires the action of either Cin8p or Kip1p, gene products related to the mechanochemical enzyme kinesin . In this paper we demonstrate that the activity of either one of these proteins is also required following spindle assembly . When their function was eliminated, preanaphase bipolar spindles rapidly collapsed, with previously separated poles being drawn together . In contrast, anaphase spindles were apparently resistant to collapse . Deletion of kinesin-related KAR3 partially suppressed the phenotypes associated with loss of Cin8p/Kip1p function . Our findings suggest that the structure of the preanaphase bipolar spindle is maintained by counteracting forces produced by kinesin-related proteins.

J Med Chem, 1992 Aug 7, 35(16), 3050 - 8
2,3-Epoxy-10-aza-10,11-dihydrosqualene, a high-energy intermediate analogue inhibitor of 2,3-oxidosqualene cyclase; Ceruti M et al.; 2,3-Epoxy-10-aza-10,11-dihydrosqualene, a high-energy intermediate analogue inhibitor of 2,3-oxidosqualene (SO) cyclase was obtained by total synthesis . This involved the preparation of three main building blocks: (1) C17 squalenoid N-methylamine, (2) 3-(diphenylphosphinoyl)propanal, and (3) 5,6-epoxy-6-methylheptan-2-one . The final stages of the reconstruction of the 6E double bond were obtained by a Wittig-Horner reaction which was modified for poorly reactive systems . This compound was designed to mimic the C-8 carbonium ion formed during SO cyclization . Its inhibitory activity on various SO cyclases was evaluated and compared with the 6 Z isomer which has an unfavorable geometry . Only isomer 6 E, the carbocation analogue, was active on SO cyclases from rat liver, pig liver, S . cerevisiae, and C . albicans microsomes, with an I50 varying from 3 to 5 microM . Both E and Z isomers were inactive on squalene epoxidase at the higher concentrations tested.

Cell, 1992 Aug 7, 70(3), 419 - 29
Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit; Hiles ID et al.; Purified bovine brain phosphatidylinositol 3-kinase (Pl3-kinase) is composed of 85 kd and 110 kd subunits . The 85 kd subunit (p85 alpha) lacks Pl3-kinase activity and acts as an adaptor, coupling the 110 kd subunit (p110) to activated protein tyrosine kinases . Here the characterization of the p110 subunit is presented . cDNA cloning reveals p110 to be a 1068 aa protein related to Vps34p, a S . cerevisiae protein involved in the sorting of proteins to the vacuole . p110 expressed in insect cells possesses Pl3-kinase activity and associates with p85 alpha into an active p85 alpha-p110 complex that binds the activated colony-stimulating factor 1 receptor . p110 expressed in COS-1 cells is catalytically active only when complexed with p85 alpha.

FEBS Lett, 1992 Aug 3, 307(3), 318 - 23
Structural determination of Saccharomyces cerevisiae rig gene and identification of its product as ribosomal protein S21; Takasawa S et al.; rig was originally isolated from a rat insulinoma-derived cDNA library . The 145 amino acid sequence of the rig protein is invariant in mammalian cDNAs . In this paper, we have isolated the cDNA and genomic clones for yeast (Saccharomyces cerevisiae) rig, determined their nucleotide sequences, and identified the gene product . The gene and the mRNA encode a basic protein of 142 amino acids which has 61.3% amino acid identity with mammalian rig protein . On two-dimensional gel electrophoresis, the in vitro transcription/translation product of yeast rig cDNA co-migrated with yeast ribosomal protein S21 . These results led to the conclusion that yeast rig ribosomal protein S21 and to the determination of the previously unknown primary structure of yeast S21 protein . Unlike most ribosomal protein genes of S . cerevisiae, the gene exists as a single copy in a haploid set of the yeast genome and has no intron, locating at chromosome VII or XV.

Gene, 1992 Aug 1, 117(1), 137 - 40
Cloning of chromosome I DNA from Saccharomyces cerevisiae: analysis of the FUN52 gene, whose product has homology to protein kinases; Barton AB et al.; A gene whose product has homology to protein kinases and is closely related to the Aspergillus nidulans nimA cell-cycle gene was identified on chromosome I of the yeast, Saccharomyces cerevisiae . This gene has been temporarily designated FUN52, where FUN is the acronym for 'function unknown now' . In A . nidulans, nimA is required to enter mitosis . In addition, overexpression of nimA causes premature onset of mitosis and cell cycle arrest . In contrast, S . cerevisiae cells that were either deleted for FUN52 or were overexpressing it had no detectable growth phenotypes . FUN52 proved to be the same as the previously identified KIN3 gene {Jones and Rosamond, Gene 90 (1990) 87-92} that was reported to map on chromosome VI.

J Virol, 1992 Aug, 66(8), 5157 - 60
Genetic assay for multimerization of retroviral gag polyproteins; Luban J et al.; We have established a genetic assay for the multimerization of retroviral gag polyproteins . This assay is based on the GAL4 two-hybrid system for studying protein-protein interactions (S . Fields and O . Song, Nature (London) 340:245-246, 1989) . In our initial experiments, we generated Saccharomyces cerevisiae plasmids that separately express the GAL4 DNA-binding and GAL4 activation domains fused to the human immunodeficiency virus type 1 (HIV-1) gag polyprotein, Pr55gag . The coexpression of these two hybrid proteins in S . cerevisiae results in the association of the GAL4 domains and the potent activation of an integrated GAL4-responsive lacZ indicator gene . Similar results were obtained with plasmids encoding GAL4-Moloney murine leukemia virus (M-MuLV) gag polyprotein hybrid proteins . In contrast, the heterologous GAL4-HIV-1 gag and GAL4-M-MuLV gag fusion proteins were unable to interact with each other to induce lacZ expression . The results suggest that this yeast system provides a rapid and specific assay for the interactions of retroviral gag proteins that occur during virion assembly.

Mol Gen Genet, 1992 Aug, 234(2), 193 - 200
ore2, a mutation affecting proline biosynthesis in the yeast Saccharomyces cerevisiae, leads to a cdc phenotype; Neuville P et al.; We report here the isolation of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae which exhibit cdc phenotypes . The recessive mutations defined four complementation groups, named ore1, ore2, ore3 and ore4 . At the non-permissive temperature, strains bearing these mutations arrested in the G1 phase of the cell cycle . The wild-type allele of the gene altered in ore2 mutants was cloned . The nucleotide sequence of a fragment which can complement the mutation showed the presence of an open reading frame capable of encoding a protein with 286 amino acid residues . The deduced amino acid sequence showed 25% identity with that of the Escherichia coli delta 1-pyrroline-5-carboxylate reductase, an enzyme of the pathway for the biosynthesis of proline . The ore2 mutants, correspondingly, were found to be capable of growing at the non-permissive temperature on a synthetic medium supplemented with proline . In addition, the chromosomal location of the gene and its restriction map were compatible with those previously reported for the PRO3 gene which encodes the S . cerevisiae delta 1-pyrroline-5-carboxylate reductase.

Eur J Biochem, 1992 Aug 1, 207(3), 1093 - 100
Molecular cloning and characterization of the Saccharomyces cerevisiae CYT2 gene encoding cytochrome-c1-heme lyase; Zollner A et al.; Cytochrome c1, a subunit of the mitochondrial ubiquinol--cytochrome-c reductase, is synthesized on cytosolic ribosomes as a precursor protein of 37 kDa . Maturation to the mature 31-kDa form involves two proteolytic processing steps of the amino-terminal presequence . After removal of the amino-terminal part by the matrix-localized processing peptidase, the carboxy-terminal part of the presequence is cleaved off by an unknown intermembrane space protease . This step depends on covalent linkage of heme to the apoprotein . At least two complementation groups (I and II) can be distinguished among mutants of the yeast Saccharomyces cerevisiae, which are defective in this second proteolytic processing, i.e . they accumulate the intermediate-sized form of cytochrome c1 instead of the mature form . Recently, it was shown that complementation group II defines the structural gene for cytochrome c1 {Sadler, I., Suda, K., Schatz, G., Kaudewitz, F . & Haid, A., (1984) EMBO J . 3, 2137-2143} . We report on the molecular cloning and characterization of the CYT2 gene representing complementation group I . It maps on chromosome XI and encodes a mitochondrial protein of about 26 kDa . Extensive similarity to Neurospora crassa and S . cerevisiae cytochrome-c--heme lyase, as well as the phenotype of cyt2 mutants, strongly suggest that we have identified the gene for cytochrome-c1--heme lyase.

Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 6846 - 50
Yeast glycoprotein biosynthesis: MNT1 encodes an alpha-1,2-mannosyltransferase involved in O-glycosylation; Hausler A et al.; The Saccharomyces cerevisiae MNT1 gene encodes a Golgi mannosyltransferase . Gene disruption of the MNT1 locus leads to a greater than 90% reduction of specific alpha-1,2-mannosyltransferase activity with alpha-methylmannoside as acceptor . Null mutants of MNT1 are viable, have no apparent growth defect, and are blocked in the elongation of protein O-linked mannobiose . Structural analysis of the N-linked outer chain isolated from an mnn1 mnn10 mnt1 strain revealed no alteration in carbohydrate structure compared to the parental mnn1 mnn10 strain . The MNT1 gene is identical to KRE2, and mutations in the gene render cells resistant to the killer toxin K1 of S . cerevisiae, which suggests a role for O-mannosylated proteins in the resistance mechanism . In addition, MNT1 is part of a multigene family whose members are presumed to be yeast Golgi mannosyltransferases.

Yeast, 1992 Aug, 8(8), 599 - 612
Genetic homology between Saccharomyces cerevisiae and its sibling species S . paradoxus and S . bayanus: electrophoretic karyotypes; Naumov GI et al.; Chromosomal DNAs of many monosporic strains of the biological species Saccharomyces cerevisiae, S . paradoxus and S . bayanus were analysed using contour-clamped homogeneous electric field electrophoresis . Southern blot hybridization with eight cloned S . cerevisiae genes (ADC1, CUP1, GAL4, LEU2, rDNA, SUC2, TRP1 and URA3) assigned to different chromosomes was used to study homology and chromosomal location of the genes in the three sibling species . A comparative study of Ty1, Ty2 and telomere-associated Y' sequences having multiple chromosomal location was also done . Chromosome length polymorphism was found in cultured strains of S . cerevisiae . Wild S . cerevisiae and S . paradoxus strains yielded chromosome banding patterns very similar to each other . The karyotype pattern of S . bayanus was readily distinguishable from that of S . cerevisiae and S . paradoxus . Southern blot analysis revealed a low degree of homology between the S . cerevisiae genes studied and the corresponding S . paradoxus and S . bayanus genes . The number of chromosomes appears to be 16 in all three species.

Curr Opin Cell Biol, 1992 Aug, 4(4), 573 - 80
Protein targeting to and translocation across the membrane of the endoplasmic reticulum; Nunnari J et al.; Several approaches are currently being taken to elucidate the mechanisms and the molecular components responsible for protein targeting to and translocation across the membrane of the endoplasmic reticulum . Two experimental systems dominate the field: a biochemical system derived from mammalian exocrine pancreas, and a combined genetic and biochemical system employing the yeast, Saccharomyces cerevisiae . Results obtained in each of these systems have contributed novel, mostly non-overlapping information . Recently, much effort in the field has been dedicated to identifying membrane proteins that comprise the translocon . Membrane proteins involved in translocation have been identified both in the mammalian system, using a combination of crosslinking and reconstitution approaches, and in S . cerevisiae, by selecting for mutants in the translocation pathway . None of the membrane proteins isolated, however, appears to be homologous between the two experimental systems . In the case of the signal recognition particle, the two systems have converged, which has led to a better understanding of how proteins are targeted to the endoplasmic reticulum membrane.

Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7100 - 4
Identification of a mammalian gene structurally and functionally related to the CDC25 gene of Saccharomyces cerevisiae; Wei W et al.; The yeast Saccharomyces cerevisiae CDC25 gene encodes a nucleotide-exchange-factor (NEF) that can convert the inactive GDP-bound state of RAS proteins to an active RAS-GTP complex . CDC25 can activate the yeast RAS proteins as well as the human H-ras protein . CDC25 is a member of a family of yeast genes that likely encode NEFs capable of regulating the RAS-related proteins found in yeast . By aligning the amino acid sequence of CDC25-related gene products we found a number of conserved motifs . Using degenerate oligonucleotides that encode these conserved sequences, we have used polymerase chain reactions to amplify fragments of mouse and human cDNAs related to the yeast CDC25 gene . We show that a chimeric molecule, part mouse and part yeast CDC25, can suppress the loss of CDC25 function in the yeast S . cerevisiae.

Mol Biol Cell, 1992 Aug, 3(8), 895 - 911
Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae; Ogg SC et al.; In mammalian cells, the signal recognition particle (SRP) receptor is required for the targeting of nascent secretory proteins to the endoplasmic reticulum (ER) membrane . We have identified the Saccharomyces cerevisiae homologue of the alpha-subunit of the SRP receptor (SR alpha) and characterized its function in vivo . S . cerevisiae SR alpha is a 69-kDa peripheral membrane protein that is 32% identical (54% chemically similar) to its mammalian homologue and, like mammalian SR alpha, is predicted to contain a GTP binding domain . Yeast cells that contain the SR alpha gene (SRP101) under control of the GAL1 promoter show impaired translocation of soluble and membrane proteins across the ER membrane after depletion of SR alpha . The degree of the translocation defect varies for different proteins . The defects are similar to those observed in SRP deficient cells . Disruption of the SRP101 gene results in an approximately sixfold reduction in the growth rate of the cells . Disruption of the gene encoding SRP RNA (SCR1) or both SCR1 and SRP101 resulted in an indistinguishable growth phenotype, indicating that SRP receptor and SRP function in the same pathway . Taken together, these results suggest that the components and the mechanism of the SRP-dependent protein targeting pathway are evolutionarily conserved yet not essential for cell growth . Surprisingly, cells that are grown for a prolonged time in the absence of SRP or SRP receptor no longer show pronounced protein translocation defects . This adaptation is a physiological process and is not due to the accumulation of a suppressor mutation . The degree of this adaptation is strain dependent.

Biokhimiia, 1992 Aug, 57(8), 1263 - 70
{A functionally important Tyr-89 residue in Saccharomyces cerevisiae pyrophosphatase . II . A possible role in the mechanism of enzyme action}; Raznikov AV et al.; Earlier it has been demonstrated that inactivation of inorganic pyrophosphatase (PPase) of S . cerevisiae by 7-chloro-4-nitronbenzofurasane is due to modification of Tyr89 . The effect of pH and active center ligands on this reaction has been studied . It was found that pK for Tyr89 does not exceed 8.5; the phosphate-metal complex binding to the high affinity center protects Tyr89 from inactivation . Activating ions (Mg2+ and Zn2+) do not influence the inactivation, whereas the PPase inhibitor, Ca2+, enhances this process after saturation of the high affinity binding site . Saturation of two binding sites with Ca2+ has a protective effect on the enzyme . An increase in the rate of Tyr89 binding to the inhibitor in the presence of low concentrations of Ca2+ is due to the decrease of Tyr89 pK . The data obtained suggest that Tyr89 is located near the high affinity binding site for phosphate . The high reactivity of Tyr89 and its tight binding in the active center point to the presence of a hydrogen bondage with the substrate and suggest a role of a proton donor whose acceptor is the product of the enzymatic reaction, i.e., phosphate.

Biokhimiia, 1992 Aug, 57(8), 1255 - 62
{Functionally important tyrosine residues in Saccharomyces cerevisiae pyrophosphatase . I . Chemical modification and localization in the primary structure}; Raznikov AV et al.; Inorganic pyrophosphatase (PPase) of S . cerevisiae is effectively inactivated by 7-chloro-4-nitrobenzofuran; the CaPP1 substrate analog has a protective effect . The modified enzyme separated from low molecular weight contaminants has an adsorption maximum at 345 nm . Preliminary modification of PPase SH-groups does not influence the enzyme binding to the inhibitor . The PPase activity is reconstituted by beta-mercapto-ethanol; hence, the inhibiting effect of the reagent is due to modification of tyrosine residues . A single reagent-containing peptide was isolated by specific adsorption from the tryptic hydrolysate of modified PPase . Within the primary structure of PPase, this peptide occupies positions 82-111 and contains two tyrosine residues . Hydrolysis of the isolated peptide by chymotrypsin and determination of the structure of fragments obtained by mass spectrometry and automated sequencing revealed that inactivation of PPase is due to selective modification of Tyr89.

Exp Cell Res, 1992 Aug, 201(2), 299 - 306
cAMP-mediated increase in the critical cell size required for the G1 to S transition in Saccharomyces cerevisiae; Baroni MD et al.; In Saccharomyces cerevisiae, cyclic AMP is required for cellular growth . In this study we show that cAMP also specifically inhibits the G1-S transition of the S . cerevisiae cell cycle by increasing the critical cell size required at start, the major yeast cell cycle control step . In fact: (a) addition of cAMP delays the time of entering into the S budded phase of small G1 cells, while it is ineffective on large fast-growing cells . (b) If cell growth is strongly depressed, cAMP permanently inhibits cell cycle commitment of cells arrested at the alpha-factor-sensitive step . The cell fraction inhibited by cAMP is inversely correlated with the average cell size of treated populations . (c) The critical protein content (Ps) and the critical cell volume (VB) required for budding in unperturbed exponentially growing yeast populations are largely increased by cAMP . On these bases, we propose a new cAMP role at start.

Nucleic Acids Res, 1992 Jul 25, 20(14), 3599 - 606
The Saccharomyces cerevisiae MGT1 DNA repair methyltransferase gene: its promoter and entire coding sequence, regulation and in vivo biological functions; Xiao W et al.; We previously cloned a yeast DNA fragment that, when fused with the bacterial lacZ promoter, produced O6-methylguanine DNA repair methyltransferase (MGT1) activity and alkylation resistance in Escherichia coli (Xiao et al., EMBO J . 10,2179) . Here we describe the isolation of the entire MGT1 gene and its promoter by sequence directed chromosome integration and walking . The MGT1 promoter was fused to a lacZ reporter gene to study how MGT1 expression is controlled . MGT1 is not induced by alkylating agents, nor is it induced by other DNA damaging agents such as UV light . However, deletion analysis defined an upstream repression sequence, whose removal dramatically increased basal level gene expression . The polypeptide deduced from the complete MGT1 sequence contained 18 more N-terminal amino acids than that previously determined; the role of these 18 amino acids, which harbored a potential nuclear localization signal, was explored . The MGT1 gene was also cloned under the GAL1 promoter, so that MTase levels could be manipulated, and we examined MGT1 function in a MTase deficient yeast strain (mgt1) . The extent of resistance to both alkylation-induced mutation and cell killing directly correlated with MTase levels . Finally we show that mgt1 S.cerevisiae has a higher rate of spontaneous mutation than wild type cells, indicating that there is an endogenous source of DNA alkylation damage in these eukaryotic cells and that one of the in vivo roles of MGT1 is to limit spontaneous mutations.

Mech Ageing Dev, 1992 Jul 15, 64(3), 235 - 45
The effect of aging on protein synthesis in the yeast Saccharomyces cerevisiae; Motizuki M et al.; The protein synthetic rate in the yeast S . cerevisiae, measured by the incorporation of radioactive amino acids per unit amount of proteins, decreased linearly with age reaching 50% of the rate of 2nd generation cells (young cells) in 20th generation cells (old cells), whereas the RNA content of the old cells was increased three times . Using a cell-free system for poly(U)-directed poly-phenylalanine synthesis, the activity of run-off ribosomes from old cells was shown to be about 40% less than the activity of ribosomes from young cells and the polysome level in old cells was much decreased compared to that in young cells . However, as protein content was increased twice in 20 generations, the cell is considered to maintain a constant level of protein synthesis during the process of aging compensating the decrease in the activity of ribosomes . Thus, it is likely that the decrease in the synthesis of certain proteins whose requirement was raised by the increase in cell volume, which is twice the increase in protein content, causes prolongation of the unbudded phase in old cells.

J Biol Chem, 1992 Jul 15, 267(20), 13791 - 4
Polypeptide translocation across the endoplasmic reticulum membrane; Sanders SL et al.; Many polypeptides have been postulated to play direct roles in secretory protein translocation based on genetic criteria, cross-linking, and antibody inhibition . Much of the excitement in the next few years will come from the resolution of current controversies . What is the nature of the ribosome receptor, and is it essential for translocation? Is BiP required for translocation in mammalian cells? Are all of the polypeptides of signal peptidase and oligosaccharyltransferase required for catalytic function, or do some of them mediate steps of protein translocation? One of the best ways to resolve these problems will be to determine the importance of each in reconstituted translocation reactions by fractionation or immunodepletion, or by analysis in a purified reaction . Another approach is to identify homologues of these molecules in S . cerevisiae and to assess their importance in in vivo translocation . Several mechanistic questions remain to be addressed as well . Does the protein translocation apparatus consist of protein, or lipid, or both? How are integral membrane proteins inserted? How is the translocon gated to admit only unfolded or partially folded secretory polypeptides and to exclude cytoplasmic molecules? The answers to these questions will illuminate a basic enigma in cell biology that has remained unanswered for many years.

Nucleic Acids Res, 1992 Jul 11, 20(13), 3443 - 51
An experimental study of Saccharomyces cerevisiae U3 snRNA conformation in solution; Segault V et al.; The conformation of Saccharomyces cerevisiae U3 snRNA (snR17A RNA) in solution was studied using enzymatic and chemical probes . In vitro synthesized and authentic snR17A RNAs have a similar conformation in solution . The S . cerevisiae U3 snRNA is folded in two distinct domains . The 5'-domain has a low degree of compactness; it is constituted of two stem-loop structures separated by a single-stranded segment, which has recently been proposed to basepair with the 5'-ETS of pre-ribosomal RNA . We demonstrate that, as previously proposed, the 5'-terminal region of U3 snRNA has a different structure in higher and lower eukaryotes and that this may be related to pre-rRNA 5'-ETS evolution . The S . cerevisiae U3 snRNA 3'-domain has a cruciform secondary structure and a compact conformation resulting from an higher order structure involving the single-stranded segments at the center of the cross and the bottom parts of helices . Compared to tRNA, where long range interactions take place between terminal loops, this represents another kind of tertiary folding of RNA molecules that will deserve further investigation, especially since the implicated single-strands have highly evolutionarily conserved primary structures that are involved in snRNP protein binding.

Gene, 1992 Jul 1, 116(1), 105 - 8
Cloning and sequence analysis of a Candida maltosa gene which confers resistance to cycloheximide; Sasnauskas K et al.; A CYHR gene from Candida maltosa, which confers resistance to cycloheximide, was cloned in Saccharomyces cerevisiae . A 2.3-kb DNA fragment carrying this gene was sequenced, and an open reading frame able to encode 553 amino acids (aa) was found in the sequence . Computer searches of the GenBank, EMBL, SWIS-PROT and Gen-Pept databases using the FASTA program failed to detect any proteins with extensive similarities to the deduced aa sequence for CYHR . The cloned gene transforms S . cerevisiae at a frequency similar to auxotrophic markers and can be used as a dominant selectable marker for introducing recombinant plasmids into wild-type strains of S . cerevisiae, as well as for gene disruption experiments.

J Bacteriol, 1992 Jul, 174(14), 4701 - 6
A positive regulatory gene, THI3, is required for thiamine metabolism in Saccharomyces cerevisiae; Nishimura H et al.; We have isolated a thiamine auxotrophic mutant carrying a recessive mutation which lacks the positive regulatory gene, THI3, which differs in the regulation of thiamine transport from the THI2 (PHO6) gene described previously (Y . Kawasaki, K . Nosaka, Y . Kaneko, H . Nishimura, and A . Iwashima, J . Bacteriol . 172:6145-6147, 1990) for expression of thiamine metabolism in Saccharomyces cerevisiae . The mutant (thi3) had a markedly reduced thiamine transport system as well as reduced activity of thiamine-repressible acid phosphatase and of several enzymes for thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-beta-hydroxyethylthiazole . These results suggest that thiamine metabolism in S . cerevisiae is subject to two positive regulatory genes, THI2 (PHO6) and THI3 . We have also isolated a hybrid plasmid, pTTR1, containing a 6.2-kb DNA fragment from an S . cerevisiae genomic library which complements thiamine auxotrophy in the thi3 mutant . This gene was localized on a 3.0-kb ClaI-BglII fragment in the subclone pTTR5 . Complementation of the activities for thiamine metabolism in the thi3 mutant transformed by some plasmids with the THI3 gene was also examined.

Mol Cell Biol, 1992 Jul, 12(7), 3297 - 304
Conditional defect in mRNA 3' end processing caused by a mutation in the gene for poly(A) polymerase; Patel D et al.; Maturation of most eukaryotic mRNA 3' ends requires endonucleolytic cleavage and polyadenylation of precursor mRNAs . To further understand the mechanism and function of mRNA 3' end processing, we identified a temperature-sensitive mutant of Saccharomyces cerevisiae defective for polyadenylation . Genetic analysis showed that the polyadenylation defect and the temperature sensitivity for growth result from a single mutation . Biochemical analysis of extracts from this mutant shows that the polyadenylation defect occurs at a step following normal site-specific cleavage of a pre-mRNA at its polyadenylation site . Molecular cloning and characterization of the wild-type allele of the mutated gene revealed that it (PAP1) encodes a previously characterized poly(A) polymerase with unknown RNA substrate specificity . Analysis of mRNA levels and structure in vivo indicate that shift of growing, mutant cells to the nonpermissive temperature results in the production of poly(A)-deficient mRNAs which appear to end at their normal cleavage sites . Interestingly, measurement of the rate of protein synthesis after the temperature shift shows that translation continues long after the apparent loss of polyadenylated mRNA . Our characterization of the pap1-1 defect implicates this gene as essential for mRNA 3' end formation in S . cerevisiae.

Curr Genet, 1992 Jul, 22(1), 69 - 74
In vitro mutagenesis of the mitochondrial leucyl-tRNA synthetase of S . cerevisiae reveals residues critical for its in vivo activities; Li GY et al.; The mitochondrial leucyl-tRNA synthetase (mLRS) of Saccharomyces cerevisiae is involved in both mitochondrial protein synthesis and pre-mRNA splicing . We have created mutations in the regions HIGH, GWD and KMSKS, which are involved in ATP-, amino acid- and tRNA-binding respectively, and which have been conserved in the evolution of group I tRNA synthetases . The mutants GRD and NMSKS have no discernible phenotype . The mutants AWD and ARD act as null alleles and lead to the production of 100% cytoplasmic petites . The mutants HIGN, NIGH and KMSNS are unable to grow on glycerol even in the presence of an intronless mitochondrial genome and accumulate petites to a greater extent than the wild-type but less than 40% . Experiments with an imported bI4 maturase indicate that the lesion in these mutations primarily affects the synthetase and not the splicing functions.

Yeast, 1992 Jul, 8(7), 519 - 33
Factors affecting homologous overexpression of the Saccharomyces cerevisiae lanosterol 14 alpha-demethylase gene; Weber JM et al.; The Saccharomyces cerevisiae Lanosterol 14 alpha-demethylase (14DM) gene was overexpressed in S . cerevisiae using promoter sequences of the highly expressed S . cerevisiae glyceraldehyde-3-phosphate dehydrogenase (TDH3) gene . To investigate factors affecting 14DM overproduction, the levels of 14DM-specific RNAs, apoprotein, and heme protein, respectively, were determined and the 14DM-specific RNA levels compared with the RNA levels originating from the endogenous TDH gene(s) . The quantitative measurements revealed that the 14DM steady-state RNA levels reached were some three- to five-fold below the theoretically expected values . With a view towards further improving expression of the 14DM gene, the spacing between the TDH3 promoter and the AUG was adjusted precisely and to rule out possible toxic effects exerted by the 14DM protein, the TDH3 promoter was placed under galactose regulation by introducing an UASG segment . Furthermore, the effects of the gene copy number on 14DM overproduction were investigated . From the analysis of the improved expression constructs five conclusions could be reached: (1) expression from the native 14DM gene is comparable to the expression driven by the TDH3 promoter-14DM fusion construct on single copy plasmid vectors; (2) expression from the TDH3 promoter-14DM construct on single-copy vectors is nearly as efficient as expression from the corresponding endogenous TDH3 gene; (3) the gene copy number has an effect on the relative expression levels of the TDH3 promoter-14DM constructs; (4) the steady-state amounts of protein produced are very nearly proportional to gene dosage; and (5) protein toxicity does not have a major impact on 14DM production . The maximum yield of 14DM was in the order of 7% of the total yeast protein and the maximum production of functional 14DM heme protein appears to be limited by the availability of heme.

FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 23 - 6
Molecular monitoring of the transcriptional activation of the yeast Saccharomyces kluyveri mating pheromone signal transduction by using FUS1-lacZ fusion gene; Fujimura H; To analyse the molecular mechanism by which a mating pheromone signal is transmitted in Saccharomyces kluyveri, the S . cerevisiae FUS1-lacZ fusion gene was introduced into S . kluyveri cells and its transcriptional activation was investigated . The expression of FUS1-lacZ fusion product was cell-type-specifically induced by pheromone treatment in S . kluyveri . The result suggests that S . kluyveri pheromone signal may be transduced by a mechanism similar to that in S . cerevisiae . This system may provide a model for analysing the molecular mechanism of the mating reaction in Saccharomyces kluyveri.

Mol Gen Genet, 1992 Jul, 234(1), 65 - 73
Recombinant repair of diverged DNAs: a study of homoeologous chromosomes and mammalian YACs in yeast; Resnick MA et al.; Recombinational repair is the means by which DNA double-strand breaks (DSBs) are repaired in yeast . DNA divergence between chromosomes was shown previously to inhibit repair in diploid G1 cells, resulting in chromosome loss at low nonlethal doses of ionizing radiation . Furthermore, 15-20% divergence prevents meiotic recombination between individual pairs of Saccharomyces cerevisiae and S . carlsbergensis chromosomes in an otherwise S . cerevisiae background . Based on analysis of the efficiency of DSB-induced chromosome loss and direct genetic detection of intragenic recombination, we conclude that limited DSB recombinational repair can occur between homoeologous chromosomes . There is no difference in loss between a repair-proficient Pms+ strain and a mismatch repair mutant, pms1 . Since DSB recombinational repair is tolerant of diverged DNAs, this type of repair could lead to novel genes and altered chromosomes . The sensitivity to DSB-induced loss of 11 individual yeast artificial chromosomes (YACs) containing mouse or human (chromosome 21 or HeLa) DNA was determined . Recombinational repair between a pair of homologous HeLa YACs appears as efficient as that between homologous yeast chromosomes in that there is no loss at low radiation doses . Single YACs exhibited considerable variation in response, although the response for individual YACs was highly reproducible . Based on the results with the yeast homoeologous chromosomes, we propose that the potential exists for intra- YAC recombinational repair between diverged repeat DNA and that the extent of repair is dependent upon the amount of repeat DNA and the degree of divergence . The sensitivity of YACs containing mammalian DNA to ionizing radiation-induced loss may thus be an indicator of the extent of repeat DNA.

Biokhimiia, 1992 Jul, 57(7), 1100 - 8
{Analysis of the quaternary structure of secreted repressible acid phosphatase from the yeast Saccharomyces cerevisiae}; Shnyreva MG et al.; The structural organization of extracellular repressible acid phosphatase from S . cerevisiae has been studied . The existence of multiple acid phosphatase forms with isoelectric points at pH 4.1-4.8 has been confirmed by isoelectrofocusing . The molecular masses of three acid phosphatase isoforms (56, 57-59, and 60 kDa) obtained after enzymatic deglycosylation correlate with the data obtained previously during the analysis of translation products in cell-free systems . Electron microscopic studies revealed that the acid phosphatase molecule has a square shape and is made up of four identical subunits with molecular masses of about 125 kDa.

Mol Cell Biol, 1992 Jul, 12(7), 2924 - 30
PMI40, an intron-containing gene required for early steps in yeast mannosylation; Smith DJ et al.; We have previously described a temperature-sensitive pmi40-1 mutant of Saccharomyces cerevisiae which is defective in glycosylation and secretion because of a thermolabile phosphomannose isomerase (PMI) activity . Inactivation of PMI at the restrictive temperature of 37 degrees C prevents synthesis of the GDP-mannose and dolichol-phosphate-mannose required for a number of critical mannosyl transfer reactions and results in cell death . Here, we report the isolation of the PMI40 gene by complementation of the corresponding mutation . The PMI40 gene contains an efficiently spliced intron which differs from the majority of those so far identified in S . cerevisiae in that it is short and the branch-forming structure has an AACTAAC motif replacing the highly conserved consensus TACTAAC . The 48.2-kDa protein predicted to be encoded by PMI40 contains amino acid sequences corresponding to those of internal peptides derived from purified S . cerevisiae PMI . Deletion of the PMI40 coding sequence results in a strain requiring D-mannose for growth . The PMI40 gene is located on chromosome V, and its transcription is increased 12-fold when cells are grown on D-mannose as sole carbon source instead of D-glucose . PMI enzyme activity, however, is not increased in D-mannose-grown cells, and PMI protein levels remain constant, suggesting that the PMI40 gene is subject to additional levels of regulation.

Nucleic Acids Res, 1992 Jun 25, 20(12), 3011 - 20
Physical dissection and characterization of chromosomes V and VIII of Saccharomyces cerevisiae; Tanaka S et al.; Chromosomes V and VIII of S . cerevisiae were dissected and ordered clone banks were constructed and characterized . Each bank contains almost the entire chromosome from the left to the right telomere except for a small gap in each case . The size of the banks constructed is in good agreement with the physical length of these chromosomes, 580 kb, estimated by pulsed-field gel electrophoresis . The remaining gap in the ordered clone bank of chromosome V was found to be only 1.6 kb in length and to contain a 1.5 kb-long portion of one of the two Ty elements located in tandem . The gap in the bank of chromosome VIII was 6.4 kb in length and contained four copies of the CUP1 gene . A genomic restriction map analysis of the corresponding region of chromosome VIII revealed that a unit of about 2 kb in length harbouring the CUP1 gene was repeated ten times in strain DC5 rho degrees which was used for the bank construction . A 588.5 kb-long high resolution physical map for chromosome V and a 585.6 kb-long one for chromosome VIII have thus been established.

J Mol Biol, 1992 Jun 20, 225(4), 1027 - 34
Purification and crystallization of glycogen phosphorylase from Saccharomyces cerevisiae; Rath VL et al.; Glycogen phosphorylase from Saccharomyces cerevisiae is activated by the covalent phosphorylation of a single threonine residue in the N terminus of the protein . We have hypothesized that the structural features that effect activation must be distinct from those characterized in rabbit muscle phosphorylase because the two enzymes have unrelated phosphorylation sites located in dissimilar protein contexts . To understand this potentially novel mechanism of activation by phosphorylation, we require information at atomic resolution of the phosphorylated and unphosphorylated forms of the enzyme . To this end, we have purified, characterized and crystallized glycogen phosphorylase from S . cerevisiae . The enzyme was isolated from a phosphorylase-deficient strain harboring a multicopy plasmid containing the phosphorylase gene under the control of its own promoter . One liter of cultured cells yields 12 mg of crystallizable material . The purified protein was not phosphorylated and had an activity of 4.7 units/mg in the presence of saturating amounts of substrate . Yeast phosphorylase was crystallized in four different crystal forms, only one of which is suitable for diffraction studies at high resolution . The latter belongs to space group P4(1)2(1)2 with unit cell constants of a = 161.1 A and c = 175.5 A Based on the density of the crystals, the solvent content is 49.7%, indicating that the asymmetric unit contains the functional dimer of yeast phosphorylase.

Biochem Biophys Res Commun, 1992 Jun 15, 185(2), 641 - 7
Functional co-expression of human oxidoreductase and cytochrome P450 1A1 in Saccharomyces cerevisiae results in increased EROD activity; Eugster HP et al.; A cDNA coding for human oxidoreductase (NADPH-cytochrome P450 reductase) was expressed in S . cerevisiae on a high copy plasmid under control of a constitutive promoter . Microsomes from a transformed strain lacking endogenous oxidoreductase exhibited cytochrome c reductase activity . An apparent Km of 7.3 microM for the substrate NADPH was determined . Expression of human oxidoreductase complemented a mutation in the yeast oxidoreductase gene CPR1 and fully reversed the ketoconazole sensitive phenotype of the respective strain . The 7-ethoxyresorufin-O-deethylase activity of yeast cells expressing human cytochrome P450 1A1 was increased by more than sixteen-fold upon coexpression of human oxidoreductase . These results strongly suggest that a more efficient coupling between the human enzymes might be responsible for the increase in enzyme activity.

Mol Microbiol, 1992 Jun, 6(11), 1413 - 9
Control of translation initiation in Saccharomyces cerevisiae; Yoon H et al.; The first observations regarding the control of translation initiation in the yeast Saccharomyces cerevisiae were made by Fred Sherman and his colleagues in 1971 . Elegant genetic studies of the CYC1 gene resulted in the formulation of 'Sherman's Rules' for translation initiation as follows: (i) AUG is the only initiator codon . (ii) the most proximal AUG from the 5' end of a message will serve as the start site of translation; and (iii) if the upstream AUG codon is mutated then initiation begins at the next available AUG in the message . Hidden within these rules is the mechanism of eukaryotic translation initiation, as these very same rules were later shown to apply to higher eukaryotic organisms and were formulated into the scanning model . However, only in the past five years has yeast been taken seriously as an organism for studying the mechanism of eukaryotic translation initiation . The basis for this is that the yeast genes for at least four mammalian translation initiation factor homologues have been identified and the number is growing . Similar factors suggest similar mechanisms for translation initiation between yeast and mammals . For some translation initiation factors, the genetics of yeast has provided new insights into their function . A mechanism for regulating translation initiation in mammalian cells is now evident in yeast . It seems clear that the molecular genetics of yeast coupled with the available in vitro translation system will provide a wealth of information in the future regarding translational control and regulatory mechanisms . The purpose of this review is to summarize what is known about translational control in S . cerevisiae.

Comp Biochem Physiol B, 1992 Jun, 102(2), 261 - 5
Study on fatty acid binding by proteins in yeast . Dissimilar results in Saccharomyces cerevisiae and Yarrowia lipolytica; Dell'Angelica EC et al.; 1 . The presence of soluble proteins with fatty acid binding activity was investigated in cell-free extracts from Saccharomyces cerevisiae and Yarrowia lipolytica cultures . 2 . No significant fatty acid binding by proteins was detected in S . cerevisiae, even when grown on a fatty acid-rich medium, thus indicating that such proteins are not essential to fatty acid metabolism . 3 . An inducible fatty acid binding protein (K0.5 = 3-4 microM) was found in Y . lipolytica which had grown on a minimal medium with palmitate as the sole source of carbon and energy . 4 . The relative molecular mass of this protein was 100,000 as inferred from Sephacryl S-200 gel filtration.

Mol Cell Biol, 1992 Jun, 12(6), 2653 - 61
Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein; Gross E et al.; The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein . The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25) . On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs . Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria . These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25 . The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S . cerevisiae . The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product . These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex . Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein . The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein . The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25 . Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals . These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

AIDS, 1992 Jun, 6(6), 541 - 6
Subcellular localization of recombinant truncated Gag precursor proteins of HIV expressed in Saccharomyces cerevisiae; Biemans R et al.; OBJECTIVE: To determine signals contained in the HIV-1 Gag precursor implicated in protein transport . DESIGN: To study the localization of truncated Gag proteins expressed in Saccharomyces cerevisiae . METHODS: Thin-section immunoelectron microscopy studies were performed on S . cerevisiae cells producing myristoylated or non-myristoylated Pr55gag, the core protein (p24) and several truncated Gag proteins . RESULTS: Pr55gag and the carboxy-terminal truncated Gag proteins were myristoylated and localized at the plasma membrane . p24 was localized in the nucleus or perinuclear membrane . However, addition of a myristoyl group to p24 targeted this molecule to the plasma membrane . CONCLUSIONS: The myristoylated amino-terminal 214 amino acids are sufficient to target Pr55gag to the plasma membrane . Subcellular signals implicated in protein transport are present in the core p24 polypeptide which may become dominant or accessible in the absence of the amino-myristoyl group.

J Biol Chem, 1992 May 25, 267(15), 10874 - 81
T double-stranded RNA (dsRNA) sequence reveals that T and W dsRNAs form a new RNA family in Saccharomyces cerevisiae . Identification of 23 S RNA as the single-stranded form of T dsRNA; Esteban LM et al.; Some strains of the yeast Saccharomyces cerevisiae harbor a double-stranded RNA (dsRNA) molecule, called T . We obtained T cDNA clones by random priming of denatured T dsRNA followed by reverse transcription . Sequence data of T show that only one strand ((+)-strand) has coding capacity for a protein with 940 amino acids which spans almost the entire length of the molecule (2.9 kilobases) . Within this protein we found a sequence pattern characteristic of RNA-dependent RNA polymerases of (+)-strand and double-stranded RNA viruses . Although T has no homology with other dsRNAs found in S . cerevisiae, such as L-A, L-BC, M1, or W, the T-encoded protein shows a high degree of conservation with the W-encoded protein . This conservation extends beyond a region that contains the consensus sequences for RNA-dependent RNA polymerases, suggesting that both T and W are evolutionarily related . With a (+)-strand-specific probe for T we identified 23 S RNA, a new single-stranded RNA (ssRNA) species with a sedimentation coefficient of 23 S . T and 23 S RNA have the same mobility under denaturing conditions with glyoxal, suggesting that 23 S RNA is, in fact, the (+)-single-stranded RNA form of T dsRNA . 23 S RNA synthesis is induced under stress conditions such as heat shock and starvation . The relationship between T and 23 S RNA clearly resembles the one between W and its single-stranded derivative form, 20 S RNA . Thus T and W dsRNAs (and their respective single-stranded species) constitute a new RNA family in S . cerevisiae.

Carbohydr Res, 1992 May 22, 229(2), 347 - 53
New mannose-specific lectins from garlic (Allium sativum) and ramsons (Allium ursinum) bulbs; Kaku H et al.; Two new mannose-binding lectins were isolated from garlic (Allium sativum, ASA) and ramsons (Allium ursinum, AUA) bulbs, of the family Alliaceae, by affinity chromatography on immobilized mannose . The carbohydrate-binding specificity of these two lectins was studied by quantitative precipitation and hapten-inhibition assay . ASA reacted strongly with a synthetic linear (1----3)-alpha-D-mannan and S . cerevisiae mannan, weakly with a synthetic (1----6)-alpha-D-mannan, and failed to precipitate with galactomannans from T . gropengiesseri and T . lactis-condensi, a linear mannopentaose, and murine IgM . On the other hand, AUA gave a strong reaction of precipitation with murine IgM, and good reactions with S . cerevisiae mannan and both synthetic linear mannans, suggesting that the two lectins have somewhat different binding specificities for alpha-D-mannosyl units . Of the saccharides tested as inhibitors of precipitation, those with alpha-(1----3)-linked mannosyl units were the best inhibitors of ASA, the alpha-(1----2)-, alpha-(1----4)-, and alpha-(1----6)-linked mannobioses and biosides having less than one eighth the affinity of the alpha-(1----3)-linked compounds . The N-terminal amino acid sequence of ASA exhibits 79% homology with that of AUA, and moderately high homology (53%) with that of snowdrop bulb lectin, also an alpha-D-mannosyl-binding lectin.

Biochem Biophys Res Commun, 1992 May 15, 184(3), 1490 - 5
Two distinct sequences control the targeting and anchoring of the mouse P450 1A1 into the yeast endoplasmic reticulum membrane; Cullin C; We previously expressed mouse P450 1A1 in the yeast S . cerevisiae . In the present study, I describe experiments in which several deletions in the 5' end of the corresponding cDNA were created . The truncated forms were then expressed in yeast cells . Studies of microsomes obtained from transformed yeast show that the signal-sequence is not required in vivo for the integration of mouse P450 1A1 into the endoplasmic reticulum membrane . In addition, the cytochrome deleted for its hydrophobic signal-sequence appears to be enzymatically functional . These results strongly argue for the existence of a second determinant of membrane targeting and binding.

Proc Natl Acad Sci U S A, 1992 May 15, 89(10), 4338 - 42
SNC1, a yeast homolog of the synaptic vesicle-associated membrane protein/synaptobrevin gene family: genetic interactions with the RAS and CAP genes; Gerst JE et al.; SNC1, a gene from the yeast Saccharomyces cerevisiae, encodes a homolog of vertebrate synaptic vesicle-associated membrane proteins (VAMPs) or synaptobrevins . SNC1 was isolated by its ability to suppress the loss of CAP function in S . cerevisiae strains possessing an activated allele of RAS2 . CAP is a component of the RAS-responsive S . cerevisiae adenylyl cyclase complex . The N-terminal domain of CAP is required for full cellular responsiveness to activated RAS proteins . The C-terminal domain of CAP is required for normal cellular morphology and responsiveness to nutrient extremes . Multicopy plasmids expressing SNC1 suppress only the loss of the C-terminal functions of CAP and only in the presence of activated RAS2.

Nature, 1992 May 14, 357(6374), 128 - 34
ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex; Bell SP et al.; A multiprotein complex that specifically recognizes cellular origins of DNA replication has been identified and purified from the yeast Saccharomyces cerevisiae . We observe a strong correlation between origin function and origin recognition by this activity . Interestingly, specific DNA binding by the origin recognition complex is dependent upon the addition of ATP . We propose that the origin recognition complex acts as the initiator protein for S . cerevisiae origins of DNA replication.

Appl Environ Microbiol, 1992 May, 58(5), 1677 - 82
Overproduction of threonine by Saccharomyces cerevisiae mutants resistant to hydroxynorvaline; Ramos C et al.; In this work, we isolated and characterized mutants that overproduce threonine from Saccharomyces cerevisiae . The mutants were selected for resistance to the threonine analog alpha-amino-beta-hydroxynorvalerate (hydroxynorvaline), and, of these, the ones able to excrete threonine to the medium were chosen . The mutant strains produce between 15 and 30 times more threonine than the wild type does, and, to a lesser degree, they also accumulate isoleucine . Genetic and biochemical studies have revealed that the threonine overproduction is, in all cases studied, associated with the presence in the strain of a HOM3 allele coding for a mutant aspartate kinase that is totally or partially insensitive to feedback inhibition by threonine . This enzyme seems, therefore, to be crucial in the regulation of threonine biosynthesis in S . cerevisiae . The results obtained suggest that this strategy could be efficiently applied to the isolation of threonine-overproducing strains of yeasts other than S . cerevisiae, even those used industrially.

Proteins, 1992 May, 13(1), 41 - 56
Comparative analysis of the beta transducin family with identification of several new members including PWP1, a nonessential gene of Saccharomyces cerevisiae that is divergently transcribed from NMT1; Duronio RJ et al.; While investigating the expression of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT: E.C . 2.3.1.97) by Northern blot analysis, we observed another RNA transcript whose expression resembled that of NMT1 during meiosis and was derived from a gene located less than 1 kb immediately upstream of NMT1 . This new gene, designated PWP1 (for periodic tryptophan protein), is divergently transcribed from NMT1 and encodes a 576-residue protein . Null mutants of PWP1 are viable, but their growth is severely retarded and steady-state levels of several cellular proteins (including at least two proteins that label with exogenous {3H}myristic acid) are drastically reduced . New methods for database searching and assessing the statistical significance of sequence similarities identify PWP1 as a member of the beta-transducin protein superfamily . Two other previously unrecognized beta-transducin-like proteins (S . cerevisiae MAK11 and D . discoideum AAC3) were also identified, and an unexpectedly high degree of sequence homology was found between a Chlamydomonas beta-like polypeptide and the C12.3 gene of chickens . A systematic and quantitative comparative analysis resulted in classifying all beta-transducin-like sequences into 11 nonorthologous families . Based on specific sequence attributes, however, not all beta-transducin-like sequences are expected to be functionally similar, and quantitative criteria for inferring functional analogies are discussed . Possible roles of repetitive tryptophan residues in proteins are also considered.

Cell, 1992 May 1, 69(3), 457 - 70
Rad51 protein involved in repair and recombination in S . cerevisiae is a RecA-like protein; Shinohara A et al.; The RAD51 gene of S . cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis . We show that the rad51 null mutant accumulates meiosis-specific double-strand breaks (DSBs) at a recombination hotspot and reduces the formation of physical recombinants . Rad51 protein shows structural similarity to RecA protein, the bacterial strand exchange protein . Furthermore, we have found that Rad51 protein is similar to RecA in its DNA binding properties and binds directly to Rad52 protein, which also plays a crucial role in recombination . These results suggest that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination . Rad51 protein is also homologous to a meiosis-specific Dmc1 protein of S . cerevisiae.

J Bacteriol, 1992 May, 174(10), 3339 - 47
Cloning and characterization of the CYS3 (CYI1) gene of Saccharomyces cerevisiae; Ono B et al.; A DNA fragment containing the Saccharomyces cerevisiae CYS3 (CYI1) gene was cloned . The clone had a single open reading frame of 1,182 bp (394 amino acid residues) . By comparison of the deduced amino acid sequence with the N-terminal amino acid sequence of cystathionine gamma-lyase, CYS3 (CYI1) was concluded to be the structural gene for this enzyme . In addition, the deduced sequence showed homology with the following enzymes: rat cystathionine gamma-lyase (41%), Escherichia coli cystathionine gamma-synthase (36%), and cystathionine beta-lyase (25%) . The N-terminal half of it was homologous (39%) with the N-terminal half of S . cerevisiae O-acetylserine and O-acetylhomoserine sulfhydrylase . The cloned CYS3 (CYI1) gene marginally complemented the E . coli metB mutation (cystathionine gamma-synthase deficiency) and conferred cystathionine gamma-synthase activity as well as cystathionine gamma-lyase activity to E . coli; cystathionine gamma-synthase activity was detected when O-succinylhomoserine but not O-acetylhomoserine was used as substrate . We therefore conclude that S . cerevisiae cystathionine gamma-lyase and E . coli cystathionine gamma-synthase are homologous in both structure and in vitro function and propose that their different in vivo functions are due to the unavailability of O-succinylhomoserine in S . cerevisiae and the scarceness of cystathionine in E . coli.

J Bacteriol, 1992 May, 174(10), 3102 - 10
Adenine deaminase and adenine utilization in Saccharomyces cerevisiae; Deeley MC; Compared with other purine salvage and nitrogen catabolism enzymatic activities, adenine deaminase (adenine aminohydrolase {AAH}; EC 3.5.4.2) activity in Saccharomyces cerevisiae is uniquely regulated . AAH specific activity is not induced by adenine and is reduced sevenfold when cells are cultivated in medium containing proline in place of ammonium as the sole nitrogen source . Exogenous adenine enters metabolic pathways primarily via the function of either AAH or adenine phosphoribosyltransferase (APRT; EC 2.4.2.7) . Exogenous adenosine cannot normally be utilized as a purine source . Strains efficiently utilized adenosine or inosine when grown in pH 4.5 medium containing Triton X-100 . A recessive mutation permitting utilization of adenosine or inosine in standard media was isolated . In both situations, growth of purine auxotrophs required either AAH or APRT activity . With medium containing either ammonium or proline as a nitrogen source, minimum doubling times of purine auxotrophs deficient in either APRT or AAH were measured . In proline-based medium, AAH and APRT permitted equal utilization of exogenous adenine . In ammonium-based medium, the absence of APRT increased the minimum doubling time by 50% . Similar experiments using sufficient exogenous histidine to feedback inhibit histidine biosynthesis failed to affect the growth rates of adenine auxotrophs blocked in AAH or APRT, indicating that the histidine-biosynthetic pathway does not play a significant role in adenine utilization . The gene that encodes AAH in S . cerevisiae was isolated by complementation using yeast strain XD1-1, which is deficient in AAH, APRT, and purine synthesis . A 1.36-kb EcoRI-SphI fragment was demonstrated to contain the structural gene for AAH by expressing this DNA in Escherichia coli under control of the trp promoter-operator . Northern (RNA) studies using the AAH-, APRT-, and CDC3-coding regions indicated that AAH regulation was not mediated at the level of transcription or mRNA degradation.

J Cell Biol, 1992 May, 117(3), 515 - 29
Isolation of a Saccharomyces cerevisiae long chain fatty acyl:CoA synthetase gene (FAA1) and assessment of its role in protein N-myristoylation; Duronio RJ et al.; Regulation of myristoylCoA pools in Saccharomyces cerevisiae plays an important role in modulating the activity of myristoylCoA:protein N-myristoyltransferase (NMT), an essential enzyme with an ordered Bi Bi reaction that catalyzes the transfer of myristate from myristoylCoA to greater than or equal to 12 cellular proteins . At least two pathways are available for generating myristoylCoA: de novo synthesis by the multifunctional, multisubunit fatty acid synthetase complex (FAS) and activation of exogenous myristate by acylCoA synthetase . The FAA1 (fatty acid activation) gene has been isolated by genetic complementation of a faal mutant . This single copy gene, which maps to the right arm of chromosome XV, specifies a long chain acylCoA synthetase of 700 amino acids . Analyses of strains containing NMT1 and a faal null mutation indicated that FAA1 is not essential for vegetative growth when an active de novo pathway for fatty acid synthesis is present . The role of FAA1 in cellular lipid metabolism and protein N-myristoylation was therefore assessed in strains subjected to biochemical or genetic blockade of FAS . At 36 degrees C, FAA1 is required for the utilization of exogenous myristate by NMT and for the synthesis of several phospholipid species . This requirement is not apparent at 24 or 30 degrees C, suggesting that S . cerevisiae contains another acylCoA synthetase activity whose chain length and/or temperature optima may differ from Faalp.

J Cell Biol, 1992 May, 117(3), 473 - 82
A conserved phosphoprotein that specifically binds nuclear localization sequences is involved in nuclear import; Stochaj U et al.; We have purified proteins of 70 kD from Drosophila, HeLa cells, and Z . mays that specifically bind nuclear localization sequences (NLSs) . These proteins are recognized by antibodies raised against a previously identified NLS-binding protein (NBP) from the yeast S . cerevisiae . All NBPs are associated with nuclei and also present in the cytosol . NBPs are phosphorylated and phosphatase treatment abolished NLS binding . The requirement for NBPs in nuclear protein uptake is demonstrated in semipermeabilized Drosophila melanogaster tissue culture cells . Proper import of a fluorescent protein containing the large T antigen NLS requires cytosol and ATP . In the absence of cytosol and/or ATP, NLS-containing proteins are bound to cytosolic structures and the nuclear envelope . Addition of cytosol and ATP results in movement of this bound intermediate into the nucleus . Anti-NBP antibodies specifically inhibited the binding part of this import reaction . These results indicate that a phosphoprotein common to several eukaryotes acts as a receptor that recognizes NLSs before their uptake into the nucleus.

Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 4129 - 33
Mutations of human myristoyl-CoA:protein N-myristoyltransferase cause temperature-sensitive myristic acid auxotrophy in Saccharomyces cerevisiae; Duronio RJ et al.; We have isolated cDNAs encoding human myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) by complementing the nmtl-181 mutation of Saccharomyces cerevisiae, which causes temperature-sensitive myristic acid auxotrophy . Human NMT is derived from a single-copy gene, contains 416 amino acids, is 44% identical to S . cerevisiae NMT (yeast NMT), and can complement the lethal phenotype of an nmtl null mutation . Human and yeast NMTs have overlapping yet distinct protein substrate specificities as judged by a coexpression system that reconstitutes protein N-myristoylation in Escherichia coli . Both enzymes contain a glycine five residues from the C terminus . Gly----Asp or Lys mutagenesis in these orthologous NMTs produces marked reductions in their activities in E . coli as well as temperature-sensitive myristic acid auxotrophy in S . cerevisiae . These results indicate highly conserved structure-function relationships in vivo and underscore the usefulness of these functional assays for identifying factors that regulate protein N-myristoylation in mammalian systems.

Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 4062 - 5
Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo; Gottschling DE; Genes located near telomeres in Saccharomyces cerevisiae undergo position-effect variegation; their transcription is subject to reversible but mitotically heritable repression . This position effect and the finding that telomeric DNA is late replicating suggest that yeast telomeres exist in a heterochromatin-like state . Mutations in genes that suppress the telomeric position effect suggest that a special chromatin structure exists near chromosomal termini . Thus transcriptional repression may be explained by the inability of DNA binding proteins to access the DNA near telomeres . To test this hypothesis, the Escherichia coli Dam DNA methyltransferase, which modifies the sequence GATC, was introduced into S . cerevisiae cells . DNA sequences near the telomere were highly refractive to Dam methylation but were modified when located at positions more internal on the chromosome . Telomeric sequences were accessible to methyltransferase activity in strains that contained a mutation that suppressed the telomeric position effect . These data support the model that sequence-specific DNA binding proteins are excluded from telomere-proximal sequences in vivo and show that expression of DNA methyltransferase activity may serve as a useful tool for mapping chromosomal structural domains in vivo.

Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 3999 - 4003
Genes encoding transcription factor IIIA and the RNA polymerase common subunit RPB6 are divergently transcribed in Saccharomyces cerevisiae; Woychik NA et al.; The gene encoding Saccharomyces cerevisiae transcription factor TFIIIA has been found adjacent to RPB6, a gene that specifies a subunit shared by nuclear RNA polymerases . Analysis of DNA upstream of the RPB6 gene revealed an open reading frame that predicts a protein, designated PZF1, with nine C2H2 zinc fingers . The presence of nine C2H2 zinc fingers in PZF1 protein, a hallmark of amphibian TFIIIA proteins, suggested that PZF1 might be a TFIIIA homologue . We found that purified recombinant PZF1 specifically binds the internal control region (ICR) of the 5S rRNA gene in S . cerevisiae . The presence of nine C2H2 zinc fingers, the specific binding to ICR DNA, and the similarity of the predicted molecular mass of PZF1 with that determined for purified yeast TFIIIA, together indicate that PZF1 is TFIIIA . The yeast and amphibian TFIIIA proteins share only a limited number of residues outside of those normally conserved in C2H2 zinc fingers; these conserved residues may provide clues to the sequence specificity of these proteins . The PZF1 gene was found to be single copy, transcribed into a 1.5-kilobase mRNA, and essential for yeast cell viability . Interestingly, the yeast RPB6 and TFIIIA coding sequences are divergently transcribed and are separated by only 233 base pairs, providing the potential for coregulated expression of components of RNA polymerases and the 5S rRNA component of ribosomes.

EMBO J, 1992 May, 11(5), 1773 - 84
The Cln3-Cdc28 kinase complex of S . cerevisiae is regulated by proteolysis and phosphorylation; Tyers M et al.; In Saccharomyces cerevisiae, several of the proteins involved in the Start decision have been identified; these include the Cdc28 protein kinase and three cyclin-like proteins, Cln1, Cln2 and Cln3 . We find that Cln3 is a very unstable, low abundance protein . In contrast, the truncated Cln3-1 protein is stable, suggesting that the PEST-rich C-terminal third of Cln3 is necessary for rapid turnover . Cln3 associates with Cdc28 to form an active kinase complex that phosphorylates Cln3 itself and a co-precipitated substrate of 45 kDa . The cdc34-2 allele, which encodes a defective ubiquitin conjugating enzyme, dramatically increases the kinase activity associated with Cln3, but does not affect the half-life of Cln3 . The Cln--Cdc28 complex is inactivated by treatment with non-specific phosphatases; prolonged incubation with ATP restores kinase activity to the dephosphorylated kinase complex . It is thus possible that phosphate residues essential for Cln-Cdc28 kinase activity are added autocatalytically . The multiple post-translational controls on Cln3 activity may help Cln3 tether division to growth.

Arch Biochem Biophys, 1992 May 1, 294(2), 603 - 8
Expression of human ferredoxin in Saccharomyces cerevisiae: mitochondrial import of the protein and assembly of the {2Fe-2S} center; Seaton BL et al.; Vertebrate ferredoxins function in the transfer of reducing equivalents from NADPH:ferredoxin oxidoreductase to cytochrome P450 enzymes involved in steroid metabolism . We report here the expression of human mitochondrial ferredoxin in the yeast Saccharomyces cerevisiae . The full-length ferredoxin protein containing the ferredoxin mitochondrial leader sequence could not be stably expressed in S . cerevisiae, but a fusion protein consisting of the mature portion of ferredoxin linked to the mitochondrial leader sequence of the S . cerevisiae cytochrome c oxidase subunit Va protein (COX5a) could be stably expressed . The COX5a:ferredoxin fusion protein was targeted to the mitochondria as a preprotein and was cleaved at the normal processing site of the COX5a presequence during import into the matrix . Absorption spectra and electron transfer activity of the isolated fusion protein established that the {2Fe-2S} center was correctly assembled and incorporated into the recombinant ferredoxin in this heterologous system.

Plant J, 1992 May, 2(3), 417 - 22
Complementation of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNAs; Minet M et al.; An Arabidopsis thaliana cDNA bank has been constituted in a Saccharomyces cerevisiae expression vector based on the phosphoglycerate kinase (PGK) promoter and terminator . This bank was used to complement eight S . cerevisiae auxotrophic markers . All of them were corrected . These results confirm the quality of the bank and the feasibility of cloning plant genes by yeast mutant complementation . The cDNA complementing the ura1 yeast mutant was sequenced, analysed and shown to encode a dihydroorotic (DHO) dehydrogenase sequence.

Cell, 1992 Apr 17, 69(2), 343 - 52
Yeast Sec proteins interact with polypeptides traversing the endoplasmic reticulum membrane; Musch A et al.; We show by photocross-linking that nascent secretory proteins, during their passage through the endoplasmic reticulum membrane of S . cerevisiae, are in physical contact with Sec61p and Sec62p, two genetically identified membrane proteins that are essential for in vivo translocation . Sec61p seems to be in continuous contact, whereas Sec62p is involved only transiently . Translocation comprises both ATP-dependent and -independent phases of interaction with the Sec proteins . The results suggest a direct role of the Sec proteins in translocation.

Cell, 1992 Apr 17, 69(2), 317 - 27
Different G1 cyclins control the timing of cell cycle commitment in mother and daughter cells of the budding yeast S . cerevisiae; Lew DJ et al.; Growth of S . cerevisiae cells by budding gives rise to asymmetric progeny cells: a larger "mother" cell and a smaller "daughter" cell . The mother cell transits a brief G1 phase before forming a new bud and beginning DNA replication . The daughter cell stays in G1 for a longer period, growing in size before initiating a new cell cycle . We show that the timing of cell cycle initiation in mother and daughter cells is governed by different G1 cyclins . In daughter cells, transcription of CLN1 and CLN2 is induced in a size-dependent manner, and these cyclins are necessary for the normal timing of cell cycle initiation . CLN3 is not required in daughter cells, but is crucial for mother cells, in which the G1 phase is much longer in the absence of this cyclin.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1539 - 45
Mammalian p53 can function as a transcription factor in yeast; Scharer E et al.; p53 has previously been shown to contain a transactivation domain using GAL4 fusion proteins and to bind specifically to a 33 base pair DNA sequence in immunoprecipitation assays . We show here that mammalian p53 expressed in S . cerevisiae is able to activate transcription of a reporter gene placed under the control of a CYC1 hybrid promoter containing the 33 base pair p53-binding sequence . The activation is dependent on the orientation and number of copies of the binding site . Three p53 mutants commonly found in human tumours, 175H, 248W and 273H, are unable to activate transcription . A fourth human p53 mutant, 285K, is temperature-sensitive for transcriptional activation . Murine p53 activates transcription from the same sequence . The murine 135V mutant, which is temperature-sensitive for mammalian cell transformation, is also temperature-sensitive for transcriptional activation . There is a much better correlation between mutation and transcriptional competence than between mutation and the structure of p53 determined with conformation-sensitive antibodies . We have therefore developed a simple transcription assay for p53 mutation in which yeast are transfected with p53 PCR products and mutation is scored on X-gal plates.

Nature, 1992 Apr 9, 356(6369), 534 - 7
The S . cerevisiae SEC65 gene encodes a component of yeast signal recognition particle with homology to human SRP19; Stirling CJ et al.; Translocation of proteins across the endoplasmic reticulum (ER) membrane represents the first step in the eukaryotic secretory pathway . In mammalian cells, the targeting of secretory and membrane protein precursors to the ER is mediated by signal recognition particle (SRP), a cytosolic ribonucleoprotein complex comprising a molecule of 7SL RNA and six polypeptide subunits (relative molecular masses 9, 14, 19, 54, 68 and 72K) . In Saccharomyces cerevisiae, a homologue of the 54K subunit (SRP54) co-purifies with a small cytoplasmic RNA, scR1 (refs 4, 5) . Genetic data indicate that SRP54 and scR1 are involved in translocation in vivo, suggesting the existence of an SRP-like activity in yeast . Whether this activity requires additional components similar to those found in mammalian SRP is not known . We have recently reported a genetic selection that led to the isolation of a yeast mutant, sec65-1, which is conditionally defective in the insertion of integral membrane proteins into the ER . Here we report the cloning and sequencing of the SEC65 gene, which encodes a 31.2K protein with significant sequence similarity to the 19K subunit of human SRP (SRP19) . We also report the cloning of a multicopy suppressor of sec65-1, and its identification as the previously defined SRP54 gene, providing genetic evidence for an interaction between these gene products in vivo.

Nature, 1992 Apr 9, 356(6369), 532 - 3
SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity; Hann BC et al.; Protein targeting to the endoplasmic reticulum (ER) in mammalian cells is catalysed by the signal recognition particle (SRP), which consists of six protein subunits and an RNA subunit . Saccharomyces cerevisiae SRP is a 16S particle, of which only two subunits have been identified: a protein subunit, SRP54p, which is homologous to the mammalian SRP54 subunit, and an RNA subunit, scR1 (ref . 3) . The sec65-1 mutant yeast cells are temperature-sensitive for growth and defective in the translocation of several secreted and membrane-bound proteins . The DNA sequence of the SEC65 gene suggests that its product is related to mammalian SRP19 subunit and may have a similar function . Here we show that SEC65p is a subunit of the S . cerevisiae SRP and that it is required for the stable association of another subunit, SRP54p, with SRP . Overexpression of SRP54p suppresses both growth and protein translocation defects in sec65-1 mutant cells.

J Biol Chem, 1992 Apr 5, 267(10), 6646 - 53
Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae . Molecular analysis of the fox2 gene and gene product; Hiltunen JK et al.; The gene encoding the multifunctional protein (MFP) of peroxisomal beta-oxidation in Saccharomyces cerevisiae was isolated from a genomic library via functional complementation of a fox2 mutant strain . The open reading frame consists of 2700 base pairs encoding a protein of 900 amino acids . The predicted molecular weight (98,759) is in close agreement with that of the isolated polypeptide (96,000) . Analysis of the deduced amino acid sequence revealed similarity to the MFPs of two other fungi but not to that of rat peroxisomes or the multifunctional subunit of the Escherichia coli beta-oxidation complex . The FOX2 gene was overexpressed from a multicopy vector (YEp352) in S . cerevisiae and the gene product purified to apparent homogeneity . A truncated version of MFP lacking 271 carboxyl-terminal amino acids was also overexpressed and purified . Experiments to study the enzymatic properties of the wild-type MFP demonstrated an absence of activities originally assigned to an MFP of S . cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA) . The truncated form contained only the D-3-hydroxyacyl-CoA dehydrogenase activity . These results clearly demonstrate that the beta-oxidation of fatty acids in S . cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate.

Nature, 1992 Apr 2, 356(6368), 450 - 3
Solution structure of the DNA-binding domain of Cd2-GAL4 from S . cerevisiae; Baleja JD et al.; The GAL4 protein activates transcription of the genes required for galactose utilization in Saccharomyces cerevisiae . The protein, consisting of 881 amino acids, is dimeric when bound to one of the approximately twofold symmetrical DNA sites present in the galactose upstream activating sequence (UASG) . Here we use two-dimensional NMR spectroscopy to determine the structure of an amino-terminal fragment of GAL4 (residues 1-65) . This fragment, a monomer in solution, binds as a dimer specifically to UASG-containing DNA . Residues 9-40 form a well defined, compact globular cluster, whereas residues 1-8 and 41-66 show considerable conformational mobility in the absence of DNA . The compact domain contains a motif in which six cysteines, located on two symmetrically related helix/extended strand units connected by a long loop, coordinate two central zinc ions, forming a bimetal-thiolate cluster . The zincs were replaced by NMR-active 113Cd in most of our work and structural parameters are therefore derived from the Cd2-protein . The structure obtained for the GAL4 DNA-binding domain represents a novel DNA-binding motif . Essentially the same conformation is observed for the compact domain in solution using NMR techniques as was seen for the central core of the N-terminal fragment bound to DNA using crystallographic techniques . Thus, the core of the DNA-binding domain changes little upon binding DNA.

Gene, 1992 Apr 1, 113(1), 119 - 24
Cloning system for Candida glabrata using elements from the metallothionein-IIa-encoding gene that confer autonomous replication; Mehra RK et al.; The yeast Candida glabrata harbors two distinct gene families that encode metallothioneins (MTs) . One of these loci, the MT-IIa locus, exhibits selective and tandem amplification in many wild type strains of C . glabrata . The present paper demonstrates that the amplified MT-IIa gene contains autonomously replicating sequences (ARS) . These ARS elements have been used to construct vectors capable of replicating in C . glabrata . The ARS element(s) in the MT-IIa gene were localized to a 457-bp segment downstream from the MT-IIa coding sequence . Although plasmids containing this fragment transform C . glabrata with high frequency, the stability of the transformants and the copy number of the plasmid improve when the entire 1.25-kb MT-IIa gene is used . Transformation of C . glabrata with plasmids carrying the 2 microns circle ARS of Saccharomyces cerevisiae led to the formation of micro-colonies, indicating that the ARS elements of 2 microns plasmids replicate only to a limited extent in C . glabrata . Conversely, a C . glabrata plasmid carrying three copies of the MT-IIa gene was able to transform S . cerevisiae.

J Bacteriol, 1992 Apr, 174(8), 2575 - 81
Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity; Pinto WJ et al.; We have begun a biochemical-genetic analysis of the synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae and found evidence for the occurrence of serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase, enzymes that catalyze the initial steps of the pathway in other organisms . SPT activity was demonstrated in vitro with crude membrane preparations from S . cerevisiae as judged by the formation of radiolabeled 3-ketosphinganine from the condensation of palmitoyl-coenzyme A (CoA) with radiolabeled serine . Shorter (C12 and C14) and longer (C18) acyl-CoAs sustain significant SPT activity, a result consistent with the finding of both C18 and C20 long-chain bases in the organism . Three products of the long-chain-base synthetic pathway, 3-ketosphinganine, erythrosphinganine, and phytosphingosine, neither directly inhibited the reaction in vitro nor affected the specific activity of the enzyme when these bases were included in the culture medium of wild-type cells . Thus, no evidence for either feedback inhibition or repression of enzyme synthesis could be found with these putative effectors . Mutant strains of S . cerevisiae that require a sphingolipid long-chain base for growth fall into two genetic complementation groups, LCB1 and LCB2 . Membrane preparations from both lcb1 and lcb2 mutant strains exhibited negligible SPT activity when tested in vitro . Step 2 of the long-chain-base synthetic pathway was demonstrated by the stereospecific NADPH-dependent reduction of 3-ketosphinganine to erythrosphinganine . Membranes isolated from wild-type cells and from an lcb1 mutant exhibited substantial 3-ketosphinganine reductase activity . We conclude that the Lcb- phenotype of these mutants results from a missing or defective SPT, an activity controlled by both the LCB1 and LCB2 genes . These results and earlier work from this laboratory establish that SPT plays an essential role in sphingolipid synthesis in S . cerevisiae.

J Bacteriol, 1992 Apr, 174(8), 2565 - 74
Sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae: genetics, physiology, and a method for their selection; Pinto WJ et al.; A selection method for sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae was devised after observing that strains that require a long-chain base for growth become denser when starved for this substance . Genetic analysis of over 60 such strains indicated only two complementation classes, lcb1 and lcb2 . Mutant strains from each class grew equally well with 3-ketodihydrosphingosine, erythrodihydrosphingosine or threodihydrosphingosine, or phytosphingosine . Since these metabolites represent the first, second, and last components, respectively, of the long-chain-base biosynthetic pathway, it is likely that the LCB1 and LCB2 genes are involved in the first step of long-chain-base synthesis . The results of long-chain-base starvation in the Lcb- strains suggest that one or more sphingolipids have a vital role in S . cerevisiae . Immediate sequelae of long-chain-base starvation were loss of viability, exacerbated in the presence of alpha-cyclodextrin, and loss of phosphoinositol sphingolipid synthesis but not phosphatidylinositol synthesis . Loss of viability with long-chain-base starvation could be prevented by also blocking either protein or nucleic acid synthesis . Without a long-chain-base, cell division, dry mass accumulation, and protein synthesis continued at a diminished rate and were further inhibited by the detergent Tergitol . The cell density increase induced by long-chain-base starvation is thus explained as a differential loss of cell division and mass accumulation . Long-chain-base starvation in Lcb- S . cerevisiae and inositol starvation of Inos- S . cerevisiae share common features: an increase in cell density and a loss of cell viability overcome by blocking macromolecular synthesis.

Curr Genet, 1992 Apr, 21(4-5), 269 - 73
The PAR1 (YAP1/SNQ3) gene of Saccharomyces cerevisiae, a c-jun homologue, is involved in oxygen metabolism; Schnell N et al.; The PAR1/SNQ3 gene of S . cerevisiae, which increases resistance to iron chelators in multi-copy transformants, is identical to the YAP1 gene, a yeast activator protein isolated as a functional homologue of the human c-jun oncogene by binding specifically to the AP-1 consensus box . The observed H2O2-sensitivity of par1 mutants has been attributed to an increased sensitivity to reduced oxygen intermediates . Accordingly, par1 mutants did not survive an elevated oxygen pressure and were very sensitive to menadione and methylviologene, two chemicals enhancing the deleterious effects of oxygen . The specific activities of enzymes involved in oxygen detoxification, such as superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase, were decreased in par1 mutants and increased after PAR1 over-expression . As in the case of oxygen detoxification enzymes, the cellular levels of glutathione were similarly affected . These observations indicate that PAR1/YAP1/SNQ3 is involved in the gene regulation of certain oxygen detoxification enzymes . The finding that H2O2 promotes DNA-binding of human c-jun is consistent with a similar function for PAR1/YAP1/SNQ3 and c-jun in cellular metabolism.

Biotechniques, 1992 Apr, 12(4), 536 - 43
Use of a ligand-screening procedure to study the interaction of S . cerevisiae alpha 2 repressor with its operator sequence; Lorimer IA et al.; A simple and rapid screening procedure was developed to study the interaction of the S . cerevisiae alpha 2 repressor with its operator sequence . An E . coli expression vector was constructed in which the alpha 2 coding sequence was placed under control of the lac promoter . Bacterial colonies containing this vector could be lysed and assayed directly for binding of wild-type and mutant operator sequences when grown on nitrocellulose filters . alpha 2 assayed in this way showed the same sequence specificity as determined in vivo . Pools of mutant alpha 2 repressors in which the codons for Arg185 or Ser181 in the homeodomain region were randomized were created by cassette mutagenesis . These pools of mutants were screened with the wild-type operator sequence to determine allowed amino acid substitutions at each position . Results suggest that both Arg185 and Ser181 have a role in high affinity operator binding.

Biophys J, 1992 Apr, 62(1), 123 - 31; discussion 131-5
Large scale rearrangement of protein domains is associated with voltage gating of the VDAC channel; Peng S et al.; The VDAC channel of the mitochondrial outer membrane is voltage-gated like the larger, more complex voltage-gated channels of the plasma membrane . However, VDAC is a low molecular weight (30 kDa), abundant protein, which is readily purified and reconstituted, making it an ideal system for analyzing the molecular basis for ion selectivity and voltage-gating . We have probed the VDAC channel by subjecting the cloned yeast (S . cerevisiae) VDAC gene to site-directed mutagenesis and introducing the resulting mutant channels into planar bilayers to detect the effects of specific sequence changes on channel properties . This approach has allowed us to formulate and test a model of the open state structure of the VDAC channel . Now we have applied the same approach to analyzing the structure of the channel's low-conducting "closed state" (essentially closed to important metabolites) . We have identified protein domains forming the wall of the closed conformation and domains that seem to be removed from the wall of the pore during channel closure . The latter can explain the reduction in pore diameter and volume and the dramatically altered channel selectivity resulting from the channel closure . This process would make a natural coupling between motion of the sensor and channel gating.

Mol Cell Biochem, 1992 Mar 25, 110(2), 135 - 43
Glutathione reductase from Saccharomyces cerevisiae undergoes redox interconversion in situ and in vivo; Peinado J et al.; Redox interconversion of glutathione reductase was studied in situ with S . cerevisiae . The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones . While 5 microM NADPH or 100 microM NADH caused 50% inactivation in normal cells in 30 min, 0.75 microM NADPH or 50 microM NADH promoted a similar effect in starved cells . GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion . Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process . Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases . Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP+ concentrations . The inactivation of yeast glutathione reductase has also been studied in vivo . The activity decreased to 75% after 2 hours of growth with glucono-delta-lactone as carbon source, while NADPH rose to 144% and NADPH+ fell to 86% of their initial values . Greater changes were observed in the presence of 1.5 microM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD+ and NADPH/NADP+ ratios rose to 171% and 262% of their initial values, respectively . Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-delta-lactone is oxidized promotes in vivo inactivation of glutathione reductase.

Cell, 1992 Mar 20, 68(6), 1077 - 90
Unipolar cell divisions in the yeast S . cerevisiae lead to filamentous growth: regulation by starvation and RAS; Gimeno CJ et al.; Diploid S . cerevisiae strains undergo a dimorphic transition that involves changes in cell shape and the pattern of cell division and results in invasive filamentous growth in response to starvation for nitrogen . Cells become long and thin and form pseudohyphae that grow away from the colony and invade the agar medium . Pseudohyphal growth allows yeast cells to forage for nutrients . Pseudohyphal growth requires the polar budding pattern of a/alpha diploid cells; haploid axially budding cells of identical genotype cannot undergo this dimorphic transition . Constitutive activation of RAS2 or mutation of SHR3, a gene required for amino acid uptake, enhance the pseudohyphal phenotype; a dominant mutation in RSR1/BUD1 that causes random budding suppresses pseudohyphal growth.

J Biol Chem, 1992 Mar 15, 267(8), 5366 - 73
Analysis of the compartmentalization of myristoyl-CoA:protein N-myristoyltransferase in Saccharomyces cerevisiae; Knoll LJ et al.; Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the cotranslational, covalent attachment of a rare fatty acid, myristic acid (C14:0), to the amino-terminal glycine residue of a number of eukaryotic proteins involved in cellular growth and signal transduction as well as several viral proteins necessary for assembly-replication . NMT has become a target for both anti-viral and anti-fungal therapy . Analysis of purified Saccharomyces cerevisiae NMT plus yeast strains with conditional lethal nmt1 mutations have provided insights about how this process is regulated in vivo . We have now defined the location of NMT in two strains of S . cerevisiae to better understand the functional and spatial relationships between this enzyme and cellular systems that generate its acyl-CoA and peptide ligands . Western blot studies using an affinity purified antibody raised in rabbits against purified S . cerevisiae NMT indicate that the acyltransferase represents 0.06% of total cellular proteins in an exponentially growing haploid strain with a wild type NMT1 allele . Another strain containing a single, integrated copy of a GAL1/NMT1 fusion gene and a nmt1 null allele had 12-fold higher levels of NMT when grown on galactose-containing media . This increase in NMT production had no detectable effects on growth or cellular morphology . Cell fractionation studies, confocal fluorescence immunocytochemical analysis, and immunogold electron microscopic surveys of fixed, gelatin-embedded cryosections of both strains revealed that NMT is a cytosolic protein that is not associated with cellular membranes (including the endoplasmic reticulum and plasma membrane), the nucleus, mitochondria, Golgi apparatus, or vacuoles . This finding is discussed in light of what is known about the location and activities of enzymes involved in de novo fatty acid biosynthesis and in amino-terminal processing of nascent proteins.

J Cell Biol, 1992 Mar, 116(5), 1221 - 9
Mutants in the S . cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect; Levin DE et al.; The PKC1 gene of Saccharomyces cerevisiae encodes a homologue of the Ca(2+)-dependent isozymes of mammalian protein kinase C (Levin, D.cE., F . O . Fields, R . Kunisawa, J . M . Bishop, and J . Thorner . 1990 . Cell . 62:213-224) . Cells depleted of the PKC1 gene product display a uniform phenotype, a behavior indicating a defect in the cell division cycle (cdc) . These cells arrest division after DNA replication, but before mitosis . Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrest growth with small buds . We created conditional alleles of PKC1 to explore the nature of this unusual cdc defect . In contrast to PKC1-depleted cells, all of the conditional pkc1 mutants isolated were suppressed by the addition of CaCl2 to the medium, suggesting that the mutant enzymes could be activated by Ca2+ . Arrest of growth and cell division in the conditional mutants was accompanied by cessation of protein synthesis, rapid loss of viability, and release of cellular material into the medium, suggesting cell lysis . This conclusion was supported by the observation that a pkc1 deletion mutant was capable of proliferation in osmotically stabilized medium, but underwent rapid cell lysis when shifted to hypo-osmotic medium . We have incorporated these observations into a model to explain the cdc-specific arrest of pkc1 mutants.

Biochem Int, 1992 Mar, 26(3), 521 - 30
Trehalase activity and its regulation during growth of Saccharomyces cerevisiae; Coutinho CC et al.; Trehalase activity decreased in 95% at the onset of the transition phase of growth of S . cerevisiae . The question which we raised was whether this phenomenon was due to proteolysis or to conversion of the enzyme to a less active form (dephosphorylation) . Immunological methods allowed to identify the presence of the trehalase protein during cell growth . At the same stage of growth, an increase in the non-phosphorylated enzyme was detected "in vitro" . Results utilizing mutant strains also indicated that regulation occurred by interconversion of forms . The same mechanism also seems to control trehalase activity in non proliferating conditions.

Microbiol Rev, 1992 Mar, 56(1), 180 - 94
Sexual agglutination in budding yeasts: structure, function, and regulation of adhesion glycoproteins; Lipke PN et al.; The sexual agglutinins of the budding yeasts are cell adhesion proteins that promote aggregation of cells during mating . In each yeast species, complementary agglutinins are expressed by cells of opposite mating type that interact to mediate aggregation . Saccharomyces cerevisiae alpha-agglutinin and its analogs from other yeasts are single-subunit glycoproteins that contain N-linked and O-linked oligosaccharides . The N-glycosidase-sensitive carbohydrate is not necessary for activity . The proposed binding domain of alpha-agglutinin has features characteristic of the immunoglobulin fold structures of cell adhesion proteins of higher eukaryotes . The C-terminal region of alpha-agglutinin plays a role in anchoring the glycoprotein to the cell surface . The S . cerevisiae alpha-agglutinin and its analogs from other species contain multiple subunits; one or more binding subunits, which interact with the opposite agglutinin, are disulfide bonded to a core subunit, which mediates cell wall anchorage . The core subunits are composed of 80 to 95% O-linked carbohydrate . The binding subunits have less carbohydrate, and both carbohydrate and peptide play roles in binding . The alpha-agglutinin and alpha-agglutinin genes from S . cerevisiae have been cloned and shown to be regulated by the mating-type locus, MAT, and by pheromone induction . The agglutinins are necessary for mating under conditions that do not promote cell-cell contact . The role of the agglutinins therefore is to promote close interactions between cells of opposite mating type and possibly to facilitate the response to phermone, thus increasing the efficiency of mating . We speculate that they mediate enhanced response to sex pheromones by providing a synapse at the point of cell-cell contact, at which both pheromone secretion and cell fusion occur.

Appl Environ Microbiol, 1992 Mar, 58(3), 948 - 52
Induction of ploidy level increments in an asporogenous industrial strain of the yeast Saccharomyces cerevisiae by UV irradiation; Sasaki T; Cells of an asporogenous industrial strain of the yeast Saccharomyces cerevisiae were irradiated with UV light by using a method that was developed previously (T . Sasaki and Y . Ohshima, Appl . Environ . Microbiol . 53:1504-1511, 1987) . This treatment gave rise to large-cell clones among the surviving cells, from which colonies consisting of cells with a normal morphology and a prototrophic property were obtained . The large-cell trait of these was stably inheritable, with the cell volumes being about twice that of the parent for 7 years on a slant agar medium at 4 degrees C with repeated transfers . The cellular DNA content of these clones, in comparison to those of two authentic haploid strains, was determined by chemical analysis . The ratio of the DNA contents showed that the parent and its large-cell derivatives were a diploid and tetraploids, respectively . No abnormality was found in the chromosomal DNA patterns of the large-cell clones, at least as determined by agarose gel electrophoresis with a CHEF-DR II pulsed-field electrophoresis system . These findings led to the conclusion that our UV light method is applicable for inducing ploidy level increments in the widely used yeast species S . cerevisiae.

Mol Cell Biol, 1992 Mar, 12(3), 1357 - 65
Human p53 and CDC2Hs genes combine to inhibit the proliferation of Saccharomyces cerevisiae; Nigro JM et al.; Human wild-type and mutant p53 genes were expressed under the control of a galactose-inducible promoter in Saccharomyces cerevisiae . The growth rate of the yeast was reduced in cells expressing wild-type p53, whereas cells transformed with mutant p53 genes derived from human tumors were less affected . Coexpression of the normal p53 protein with the human cell cycle-regulated protein kinase CDC2Hs resulted in much more pronounced growth inhibition that for p53 alone . Cells expressing p53 and CDC2Hs were partially arrested in G1, as determined by morphological analysis and flow cytometry . p53 was phosphorylated when expressed in the yeast, but differences in phosphorylation did not explain the growth inhibition attributable to coexpression of p53 and CDC2Hs . These results suggest that wild-type p53 has a growth-inhibitory activity in S . cerevisiae similar to that observed in mammalian cells and suggests that this yeast may provide a useful model for defining the pathways through which p53 acts.

Eur J Biochem, 1992 Mar 1, 204(2), 699 - 704
In vivo and in vitro studies of the purine-cytosine permease of Saccharomyces cerevisiae . Functional analysis of a mutant with an altered apparent transport constant of uptake; Brethes D et al.; The FCY2 gene of the purine-cytosine permease (PCP) of Saccharomyces cerevisiae and the allele fcy2-21 have been cloned on the yeast multicopy plasmid pJDB207 . The corresponding plasmids were introduced into a S . cerevisiae strain carrying a chromosomal deletion at the FCY2 locus . The resulting strains were designated pAB4 and pAB25 respectively . The pAB25 strain, which carries the fcy2-21 allele, contains four amino acid changes in the open reading frame of the PCP (Weber et al., 1989) . The influence of these mutations was studied on cells by determination of the uptake constants of purine bases and cytosine {apparent Michaelis constant of transport (Ktapp) and Vmax} and on plasma-membrane preparations, by measurements of binding parameters at equilibrium {(Kd and maximum amount of binding sites/Bmax)} . For strain pAB4, the Ktapp and Vmax of uptake were almost similar for all solutes considered {1.8-2.6 microM and 8.5-10.2 nmol.min-1.(10(7) cells)-1} . The main effect of the mutations in strain pAB25 was based on a large increase in Ktapp for all ligands except adenine . Plasma membranes of each strain displayed one class of specific binding sites . Variations in Kd of 0.4-1 microM were observed for pAB4 . These slight variations had no effect on the Ktapp of uptake measured for the corresponding solutes . In contrast, using pAB25 membranes, Kd increased dramatically; 2.6 microM, 40 microM and 96 microM for adenine, cytosine and hypoxanthine, respectively . These increments were correlated to variations in Ktapp of the uptake for cytosine and hypoxanthine . Therefore, we conclude that modification in the Ktapp of uptake in the strain carrying fcy2-21 allele is merely due to a modification of the binding ability of the permease for its ligands.

Mutat Res, 1992 Mar, 273(2), 231 - 6
Mitochondrial DNA repair by photolyase; Yasui A et al.; Photolyase genes of Saccharomyces cerevisiae and Escherichia coli were expressed in S . cerevisiae and photoreactivation in nuclei and mitochondria of the host cells was analyzed by determination of survival and petit rates . Yeast photolyase was able to repair mitochondrial DNA effectively, whereas E . coli photolyase could reduce only a small fraction of the petit rate produced by UV irradiation . Analysis using fusion between yeast photolyase and E . coli lacZ genes as well as a chimeric gene between yeast and E . coli photolyase genes suggests the importance of the protruding amino terminal region of the yeast photolyase for its transport into mitochondria . A significant similarity between the protruding amino termini of yeast photolyase and yeast uracil-DNA-glycosylase suggests a common functional importance of the terminal sequences for both DNA repair enzymes.

J Virol, 1992 Mar, 66(3), 1414 - 24
Ty3 GAG3 and POL3 genes encode the components of intracellular particles; Hansen LJ et al.; Ty3 is a Saccharomyces cerevisiae retrotransposon that integrates near the transcription initiation sites of polymerase III-transcribed genes . It is distinct from the copialike Ty1 and Ty2 retrotransposons of S . cerevisiae in both the sequences of encoded proteins and gene order . It is a member of the gypsylike family of retrotransposons which resemble animal retroviruses . This study was undertaken to investigate the nucleocapsid particle of a transpositionally active gypsylike retrotransposon . Characterization of extracts from cells in which Ty3 expression was induced showed the presence of Ty3 nucleoprotein complexes, or viruslike particles, that migrated on linear sucrose gradients with a size of 156S . These particles are composed of Ty3 RNA, full-length, linear DNA, and proteins . In this study, antibodies raised against peptides predicted from the Ty3 sequence were used to identify Ty3-encoded proteins . These include the capsid (26 kDa), nucleocapsid (9 kDa), and reverse transcriptase (55 kDa) proteins . Ty3 integrase proteins of 61 and 58 kDa were identified previously (L . J . Hansen and S . B . Sandmeyer, J . Virol . 64:2599-2607, 1990) . Reverse transcriptase activity associated with the particles was measured by using exogenous and endogenous primer-templates . Immunofluorescence studies of cells overexpressing Ty3 revealed cytoplasmic clusters of immunoreactive proteins . Transmission electron microscopy showed that Ty3 viruslike particles are about 50 nm in diameter . Thus, despite the unusual position specificity of Ty3 upstream of tRNA-coding regions, aspects of the Ty3 life cycle are fundamentally similar to those of retroviruses.

Nucleic Acids Res, 1992 Feb 25, 20(4), 791 - 6
Pleiotropic effect of a point mutation in the yeast SUP4-o tRNA gene: in vivo pre-tRNA processing in S . cerevisiae; Wilhelm ML et al.; The expression of mutant tyrosine-inserting ochre suppressor SUP4-o tRNA genes in vivo in S . cerevisiae was examined as a basis for further studies of tRNA transcription and processing . In vivo yeast precursor tRNAs have been identified by filter hybridization and primer extension analysis . We have previously shown that a mutant SUP4-o tRNA gene with a C52----A52 transversion at positive 52 (C52----A52(+IVS) allele) was transcribed but that the primary transcript was not processed correctly . We show here that 5' and 3' end processing as well as splicing are defective for this mutant but that the 5' end processing is restored when the intron is removed from the gene by oligonucleotide directed mutagenesis (C52----A52(-IVS) allele) . Our results imply that the C52----A52 transversion by itself cannot account for the lack of susceptibility to RNase P cleavage but that the overall tertiary structure of the mutant tRNA precursor is destabilized by the intron/anticodon stem . A second consequence of the C52----A52 transversion is to prevent complete maturation of the tRNA precursor at its 3' end since intermediates containing incompletely processed 3' trailers accumulate in the yeast cells transformed with the C52----A52(-IVS) allele . A correct structure of the T stem might therefore define a structural feature required for the recognition of the 3' processing activity.

J Mol Biol, 1992 Feb 20, 223(4), 899 - 910
Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA; van der Sande CA et al.; Using the previously described "tagged ribosome" (pORCS) system for in vivo mutational analysis of yeast rDNA, we show that small deletions in the 5'-terminal portion of ITS2 completely block maturation of 26 S rRNA at the level of the 29 SB precursor (5.8 S rRNA-ITS2-26 S rRNA) . Various deletions in the 3'-terminal part, although severely reducing the efficiency of processing, still allow some mature 26 S rRNA to be formed . On the other hand, none of the ITS2 deletions affect the production of mature 17 S rRNA . Since all of the deletions severely disturb the recently proposed secondary structure of ITS2, these findings suggest an important role for higher order structure of ITS2 in processing . Analysis of the effect of complete or partial replacement of S . cerevisiae ITS2 with its counterpart sequences from Saccharomyces rosei or Hansenula wingei, points to helix V of the secondary structure model as an important element for correct and efficient processing . Direct mutational analysis shows that disruption of base-pairing in the middle of helix V does not detectably affect 26 S rRNA formation . In contrast, introduction of clustered point mutations at the apical end of helix V that both disrupt base-pairing and change the sequence of the loop, severely reduces processing . Since a mutant containing only point mutations in the sequence of the loop produces normal amounts of mature 26 S rRNA, we conclude that the precise (secondary and/or primary) structure at the lower end of helix V, but excluding the loop, is of crucial importance for efficient removal of ITS2.

J Biol Chem, 1992 Feb 15, 267(5), 3282 - 8
The deduced sequence of the transcription factor TFIIIA from Saccharomyces cerevisiae reveals extensive divergence from Xenopus TFIIIA; Archambault J et al.; TFIIIA is an RNA polymerase III transcription factor that binds to the internal control region of the 5 S RNA gene as the first step in the assembly of a transcription complex . We have identified the gene encoding TFIIIA from Saccharomyces cerevisiae . Protein synthesized in vitro from the cloned gene has the same size, DNA-binding properties, and transcription factor activity as does purified yeast TFIIIA . Examination of the deduced sequence of the 50-kDa yeast transcription factor revealed the presence of nine zinc-finger motifs, a characteristic of Xenopus TFIIIA . Although the conservation of these nine putative DNA-binding domains is striking, the amino acid sequence throughout the corresponding fingers of the yeast and amphibian TFIIIAs has diverged extensively and in many instances the spacing between the residues that coordinate the zinc ions differs between the two proteins . A unique feature of the yeast protein is an 81-amino acid domain interrupting the repeated zinc-finger motifs between fingers 8 and 9 . Additionally, the yeast and amphibian proteins differ in both the size and sequence of the amino- and carboxyl-terminal domains flanking the zinc fingers . The gene encoding yeast TFIIIA is present in single copy in the S . cerevisiae genome and is essential for cell viability . A carboxyl-terminal truncated form of the protein containing 4.5 zinc-finger motifs retains the ability to bind to DNA but is no longer active in promoting transcription in vitro.

Gene, 1992 Feb 15, 111(2), 207 - 13
Efficient synthesis of secreted murine interleukin-2 by Saccharomyces cerevisiae: influence of 3'-untranslated regions and codon usage; Demolder J et al.; Several expression vectors were compared which directed the synthesis of secreted murine interleukin-2 (mIL2) in the culture medium of Saccharomyces cerevisiae . We used the prepro-sequence of the alpha 1 mating-factor precursor as a secretion signal in S . cerevisiae in combination with different promoters . The yield of mature mIL2 was significantly improved by deleting the major part of the 3'-untranslated region (UTR) . In Northern-blotting experiments we showed that a destabilizing sequence present in the 3' UTR might be responsible for rapid degradation of the mIL2 mRNA . The highest expression (about 10 micrograms/ml) was obtained under control of the GAL1 promoter in an S . cerevisiae strain where the regulatory GAL4 gene was overexpressed . No difference in expression level was observed in a construct wherein twelve consecutive codons were replaced by optimal codons for S . cerevisiae.

Mol Cell Biol, 1992 Feb, 12(2), 758 - 66
RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae; Ruggieri R et al.; The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A) . RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity . Deletion of RSR1 itself does not affect growth but causes a randomization of bud position . We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein) . rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed . rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number . These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p{GTP} and Cdc24p . Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells . As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S . cerevisiae Ras pathway . Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase . This interaction of Rsr1p with the effector of Ras in S . cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

J Virol, 1992 Feb, 66(2), 999 - 1006
Ribosomal frameshifting requires a pseudoknot in the Saccharomyces cerevisiae double-stranded RNA virus; Tzeng TH et al.; The large double-stranded RNA of the Saccharomyces cerevisiae (yeast) virus has two large overlapping open reading frames on the plus strand, one of which is translated via a -1 ribosomal frameshift . Sequences including the overlapping region, placed in novel contexts, can direct ribosomes to make a -1 frameshift in wheat germ extract, Escherichia coli and S . cerevisiae . This sequence includes a consensus slippery sequence, GGGUUUA, and has the potential to form a pseudoknot 3' to the putative frameshift site . Based on deletion analysis, a region of 71 nucleotides including the potential pseudoknot and the putative slippery sequence is sufficient for frameshifting . Site-directed mutagenesis demonstrates that the pseudoknot is essential for frameshifting.

Curr Genet, 1992 Feb, 21(2), 101 - 8
DNA integration into recipient yeast chromosomes by trans-kingdom conjugation between Escherichia coli and Saccharomyces cerevisiae; Nishikawa M et al.; IncQ-derived conjugative shuttle vectors, which carried the yeast gene URA3 and/or the yeast autonomously replicating sequence (ARS1), were constructed . Both the ars-plus plasmid pAY205 and the ars-less plasmid pAY201 were successfully transmitted from E . coli to S . cerevisiae by the action of mob and tra . In this trans-kingdom conjugation, plasmid pAY205 could replicate and be retained in transconjugants . Plasmid pAY201 caused the formation of "micro-colonies" of abortive transconjugants due to its transient expression and rapid disappearance . Nevertheless, one per about 10(3) colonies caused by transmitted pAY201 plasmids were uncurable by integration into the homologous region of a yeast chromosome . Analyses by restriction enzyme mapping and Southern hybridization indicate that this integration is primarily caused by a double crossover during conjugation and not by a single reciprocal recombination.

Glycobiology, 1992 Feb, 2(1), 77 - 84
Glycosylation in Saccharomyces cerevisiae: cloning and characterization of an alpha-1,2-mannosyltransferase structural gene; Hausler A et al.; A gene encoding an alpha-1,2-mannosyltransferase from Saccharomyces cerevisiae was cloned and sequenced . The alpha-1,2-mannosyltransferase which utilizes alpha-methylmannoside as acceptor of mannose from GDP-mannose was purified . The enzyme activity was shown to correspond to a 41 kDa protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis . This protein band was digested in situ with trypsin and amino acid sequence information was obtained from four peptides . Degenerate oligonucleotide primers corresponding to the amino acid sequences were designed and used for polymerase chain reactions on yeast genomic DNA . A specific reaction product was used to screen a genomic library of S.cerevisiae . A fragment of approximately 5.7 kb was isolated, of which a 2.9 kb fragment was sequenced . It contained a 1329 base pair open reading frame encoding the peptide sequences of the purified alpha-1,2-mannosyltransferase . The gene, designated MNT1, is located on the right arm of chromosome 4 . It encodes a 442 amino acid polypeptide with a calculated mol . wt of 51.4 kDa . The corresponding mRNA has a length of approximately 1.6 kb . Overexpression of the MNT1 gene increased this alpha-1,2-mannosyltransferase activity approximately 2.5-fold . The protein was shown to be modified with N-linked carbohydrate chains and its sequence contains one N-glycosylation site . The enzyme contains a putative membrane-spanning domain near its N-terminus and its topology is thus similar to that of mammalian Golgi glycosyltransferases . This is the first report of the cloning and sequencing of a yeast Golgi mannosyltransferase.

J Clin Microbiol, 1992 Feb, 30(2), 391 - 5
Pneumocystis carinii and specific fungi have a common epitope, identified by a monoclonal antibody; Lundgren B et al.; Because Pneumocystis carinii may be related to fungi, we evaluated the reactivities of monoclonal antibodies raised against P . carinii with a variety of fungi . Fifty-two fungi and six protozoa were evaluated by immunofluorescence . One of three monoclonal antibodies (MAbs) tested (MAb 7D7) reacted with 15 fungi but no protozoa . Saccharomyces cerevisiae showed the strongest reactivity by immunofluorescence . The reactive antigen was characterized for four fungi by the immunoblot technique . In all cases the antigen that was reactive with MAb 7D7 was larger than the P . carinii antigens that reacted with 7D7 . In further studies with P . carinii, Aspergillus species, and S . cerevisiae, we found that MAb 7D7 reacted with a carbohydrate component in all organisms . The presence of an epitope that is common to P . carinii and a number of fungi further supports the fungal nature of P . carinii.

Mol Microbiol, 1992 Feb, 6(3), 301 - 8
Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs; Gonzalez A et al.; A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library . Clones were obtained which still lacked NADP-GDH but showed GOGAT activity . Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA . Since the only GOGAT enzyme so far purified from S . cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit . Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs . Transformants were only recovered when the recipient strain was an E . coli GDH-less mutant lacking the small GOGAT subunit . These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2) . Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E . coli transformants is a hybrid comprising the large E . coli subunit and the small S . cerevisiae subunit.

EMBO J, 1992 Feb, 11(2), 497 - 505
Ubiquitin as a degradation signal; Johnson ES et al.; For many short-lived eukaryotic proteins, conjugation to ubiquitin, yielding a multiubiquitin chain, is an obligatory pre-degradation step . The conjugated ubiquitin moieties function as a 'secondary' signal for degradation, in that their posttranslational coupling to a substrate protein is mediated by amino acid sequences of the substrate that act as a primary degradation signal . We report that the fusion protein ubiquitin--proline--beta-galactosidase (Ub-P-beta gal) is short-lived in the yeast Saccharomyces cerevisiae because its N-terminal ubiquitin moiety functions as an autonomous, primary degradation signal . This signal mediates the formation of a multiubiquitin chain linked to Lys48 of the N-terminal ubiquitin in Ub-P-beta gal . The degradation of Ub-P-beta gal is shown to require Ubc4, one of at least seven ubiquitin-conjugating enzymes in S.cerevisiae . Our findings provide the first direct evidence that a monoubiquitin moiety can function as an autonomous degradation signal . This generally applicable, cis-acting signal can be used to manipulate the in vivo half-lives of specific intracellular proteins.

Biochem Biophys Res Commun, 1992 Jan 31, 182(2), 452 - 60
PRS3 encoding an essential subunit of yeast proteasomes homologous to mammalian proteasome subunit C5; Lee DH et al.; We found by computer analysis that a putative yeast proteasome subunit gene named PRS3 that encodes a protein very similar to subunit C5 of rat and human proteasomes is located immediately 3' to the ERD2 gene of Saccharomyces cerevisiae . The similarity of the primary structures of the two suggests that this subunit may have a common function in proteasomes of all eukaryotes . The protein, deduced from the open reading frame of PRS3, consists of 242 amino acid residues with a calculated molecular weight of 27,077 . Chromosomal disruption of the PRS3 gene created a recessive lethal mutation . Physical mapping by hybridization to intact S . cerevisiae chromosomal DNA showed that the PRS3 gene is located on chromosome II, unlike two other subunit genes, PRS1 and PRS2, which are located on chromosomes XV and VII, respectively . These findings indicate that the PRS3 protein is a subunit of yeast proteasomes that is essential for cell viability.

Cell, 1992 Jan 24, 68(2), 333 - 9
A position effect on the time of replication origin activation in yeast; Ferguson BM et al.; The chromosomes of eukaryotes are characterized by the mosaic nature of their replication--large regions of DNA that replicate early in S phase are interspersed with regions that replicate late . This pattern of early and late synthesis appears to be the consequence of a temporal program that activates replication origins at different times . The basis of this temporal regulation in the yeast S . cerevisiae has been investigated by changing the chromosomal locations of two origins, one activated early in the S phase (ARS1) and one activated late (ARS501) . We show that the cis-acting information controlling time of activation can be separated from the element that determines origin function . For the ARS501 origin, late activation appears to be a consequence of its proximity to the telomere.

Nature, 1992 Jan 23, 355(6358), 368 - 71
Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S . cerevisiae; Amon A et al.; Progression from G2 to M phase in eukaryotes requires activation of a protein kinase composed of p34cdc2/CDC28 associated with G1-specific cyclins . In some organisms the activation of the kinase at the G2/M boundary is due to dephosphorylation of a highly conserved tyrosine residue at position 15 (Y15) of the cdc2 protein . Here we report that in the budding yeast Saccharomyces cerevisiae, p34CDC28 also undergoes cell-cycle regulated dephosphorylation on an equivalent tyrosine residue (Y19) . However, in contrast to previous observations in S . pombe, Xenopus and mammalian cells, dephosphorylation of Y19 is not required for the activation of the CDC28/cyclin kinase . Furthermore, mutation of this tyrosine residue does not affect dependence of mitosis on DNA synthesis nor does it abolish G2 arrest induced by DNA damage . Our data imply that regulated phosphorylation of this tyrosine residue is not the 'universal' means by which the onset of mitosis is determined . We propose that there are other unidentified controls that regulate entry into mitosis.

J Biol Chem, 1992 Jan 15, 267(2), 871 - 5
Cloning human pyrroline-5-carboxylate reductase cDNA by complementation in Saccharomyces cerevisiae; Dougherty KM et al.; Pyrroline-5-carboxylate reductase (EC 1.5.1.2) catalyzes the NAD(P)H-dependent conversion of pyrroline-5-carboxylate to proline . We cloned a human pyrroline-5-carboxylate reductase cDNA by complementation of proline auxotrophy in a Saccharomyces cerevisiae mutant strain, DT1100 . Using a HepG2 cDNA library in a yeast expression vector, we screened 10(5) transformants, two of which gained proline prototrophy . The plasmids in both contained similar 1.8-kilobase inserts, which when reintroduced into strain DT1100, conferred proline prototrophy . The pyrroline-5-carboxylate reductase activity in these prototrophs was 1-3% that of wild type yeast, in contrast to the activity in strain DT1100 which was undetectable . The 1810-base pair pyrroline-5-carboxylate reductase cDNA hybridizes to a 1.85-kilobase mRNA in samples from human cell lines and predicts a 319-amino acid, 33.4-kDa protein . The derived amino acid sequence is 32% identical with that of S . cerevisiae . By genomic DNA hybridization analysis, the human reductase appears to be encoded by a single copy gene which maps to chromosome 17.

Nature, 1992 Jan 9, 355(6356), 179 - 82
New yeast actin-like gene required late in the cell cycle; Schwob E et al.; Actin, a major cytoskeletal component of all eukaryotic cells, is one of the most highly conserved proteins . It is involved in various cellular processes such as motility, cytoplasmic streaming, chromosome segregation and cytokinesis . The actin from the yeast Saccharomyces cerevisiae, encoded by the essential ACT1 gene, is 89% identical to mouse cytoplasmic actin and is involved in the organization and polarized growth of the cell surface . We report here the characterization of ACT2, a previously undescribed yeast split gene encoding a putative protein (391 amino acids, relative molecular mass (Mr) 44,073) that is 47% identical to yeast actin . The requirement of the ACT2 gene for vegetative growth of yeast cells and the existence of related genes in other eukaryotes indicate an important and conserved role for these actin-like proteins . Superimposition of the Act2 polypeptide onto the three-dimensional structure of known actins reveals that most of the divergence occurred in loops involved in actin polymerization, DNase I and myosin binding, leaving the core domain mainly unaffected . To our knowledge, the Act2 protein from S . cerevisiae is the first highly divergent actin molecule described . Structural and physiological data suggest that the Act2 protein might have an important role in cytoskeletal reorganization during the cell cycle.

Gene, 1992 Jan 2, 110(1), 33 - 9
Production of cytochrome P450 reductase yeast-rat hybrid proteins in Saccharomyces cerevisiae; Bligh HF et al.; We present a novel strategy for increasing the level of functional mammalian cytochrome P450 (Cyt.P450) and NADPH:cytochrome P450 reductase enzymes produced in yeast . A cDNA encoding the rat P450 reductase was modified by the addition of a sequence coding for the N-terminal region of P450 reductase from Saccharomyces cerevisiae . The addition of this hydrophobic tail greatly increased the apparent stability of the reductase protein produced in S . cerevisiae, as compared to the unmodified rat P450 reductase . When the rat hybrid reductase was produced simultaneously with one of two mammalian Cyt.P450s, the rat CYP2B1 or the human CYP2A6, there was a significant increase in the specific activity of each of the Cyt.P450s . The optimization of this approach and its extrapolation to other organisms should lead to a marked improvement in our ability to study and exploit the P450 system.

Curr Genet, 1992 Jan, 21(1), 13 - 6
Physical and biochemical characterization of the cloned LYS5 gene required for alpha-aminoadipate reductase activity in the lysine biosynthetic pathway of Saccharomyces cerevisiae; Rajnarayan S et al.; The LYS5 and LYS2 genes of Saccharomyces cerevisiae are required for the synthesis of alpha-aminoadipate reductase in the lysine pathway . The LYS5 gene, originally cloned as a DNA insert of the plasmid pSC5, has been subcloned on a 3.2 kb SphI-Sau3AI DNA fragment of the recombinant plasmid pSR7 . An internal 2.1 kb HpaI-HpaI DNA fragment of the subclone, upon Southern hybridization, exhibits homology with HpaI-restricted wild-type S . cerevisiae genomic DNA . The lys5+ transformants exhibited alpha-aminoadipate reductase activity similar to that of wild-type cells . S1 nuclease analysis localizes the transcription initiation site relative to the detailed restriction map, and reveals the direction of transcription, as well as the transcript size of the LYS5 gene which can be no greater than 1.65 kb . From this it is estimated that the encoded polypeptide is appreciably smaller than the 4 kb LYS2 gene product . These results provide a physical and biochemical characterization of the cloned LYS5 gene . Based on these observations, it is concluded that the LYS5 gene encodes a relatively small polypeptide of the large heteropolymeric alpha-aminoadipate reductase.

Crit Rev Biotechnol, 1992, 12(1-2), 157 - 88
Strategies for the genetic manipulation of Saccharomyces cerevisiae; Tuite MF; The budding yeast Saccharomyces cerevisiae is now widely used as a model organism in the study of gene structure, function, and regulation in addition to its more traditional use as a workhorse of the brewing and baking industries . In this article the plethora of methods available for manipulating the genome of S . cerevisiae are reviewed . This will include a discussion of methods for manipulating individual genes and whole chromosomes, and will address both classic genetic and recombinant DNA-based methods . Furthermore, a critical evaluation of the various genetic strategies for genetically manipulating this simple eukaryote will be included, highlighting the requirements of both the new and the more traditional biotechnology industries.

Genetics, 1992 Jan, 130(1), 51 - 8
Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway; Cherest H et al.; We have identified a mutation in a gene of Saccharomyces cerevisiae, STR1, that leads to a strict nutritional requirement for cysteine . The str1-1 mutation decreases to an undetectable level the cystathionine gamma-lyase activity . This enzyme catalyzes one of the two reactions involved in the transsulfuration pathway that yields cysteine from homocysteine with the intermediary formation of cystathionine . The phenotype induced by this mutation implies that, in S . cerevisiae, the sulfur atom of sulfide resulting from the reductive assimilation of sulfate is incorporated into a four carbon backbone yielding homocysteine, which, in turn, is the precursor of the biosynthesis of both cysteine and methionine . This also reveals that the direct synthesis of cysteine by incorporation of the sulfur atom into a three carbon backbone as found in Escherichia coli does not occur in S . cerevisiae . The study of the meiotic progeny of diploid strains heterozygous at the STR1 locus has shown that the str1-1 mutation undergoes a particularly high frequency of meiotic gene conversion.

Mol Cell Biol, 1992 Jan, 12(1), 402 - 12
NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes; Boguta M et al.; We report the genetic characterization, molecular cloning, and sequencing of a novel nuclear suppressor, the NAM9 gene from Saccharomyces cerevisiae, which acts on mutations of mitochondrial DNA . The strain NAM9-1 was isolated as a respiration-competent revertant of a mitochondrial mit mutant which carries the V25 ochre mutation in the oxi1 gene . Genetic characterization of the NAM9-1 mutation has shown that it is a nuclear dominant omnipotent suppressor alleviating several mutations in all four mitochondrial genes tested and has suggested its informational, and probably ribosomal, character . The NAM9 gene was cloned by transformation of the recipient oxi1-V25 mutant to respiration competence by using a gene bank from the NAM9-1 rho o strain . Orthogonal-field alternation gel electrophoresis analysis and genetic mapping localized the NAM9 gene on the right arm of chromosome XIV . Sequence analysis of the NAM9 gene showed that it encodes a basic protein of 485 amino acids with a presequence that could target the protein to the mitochondrial matrix . The N-terminal sequence of 200 amino acids of the deduced NAM9 product strongly resembles the S4 ribosomal proteins from chloroplasts and bacteria . Significant although less extensive similarity was found with ribosomal cytoplasmic proteins from lower eucaryotes, including S . cerevisiae . Chromosomal inactivation of the NAM9+ gene is not lethal to the cell but leads to respiration deficiency and loss of mitochondrial DNA integrity . We conclude that the NAM9 gene product is a mitochondrial ribosomal counterpart of S4 ribosomal proteins found in other systems and that the suppressor acts through decreasing the fidelity of translation.

J Bacteriol, 1992 Jan, 174(1), 254 - 62
Drastic alteration of cycloheximide sensitivity by substitution of one amino acid in the L41 ribosomal protein of yeasts; Kawai S et al.; Cycloheximide is one of the antibiotics that inhibit protein synthesis in most eukaryotic cells . We have found that a yeast, Candida maltosa, is resistant to the drug because it possesses a cycloheximide-resistant ribosome, and we have isolated the gene responsible for this . In this study, we sequenced this gene and found that the gene encodes a protein homologous to the L41 ribosomal protein of Saccharomyces cerevisiae, whose amino acid sequence has already been reported . Two genes for L41 protein, named L41a and L41b, independently present in the genome of S . cerevisiae, were isolated . L41-related genes were also isolated from a few other yeast species . Each of these genes has an intron at the same site of the open reading frame . Comparison of their deduced amino acid sequences and their ability to confer cycloheximide resistance to S . cerevisiae, when introduced in a high-copy-number plasmid, suggested that the 56th amino acid residue of the L41 protein determines the sensitivity of the ribosome to cycloheximide; the amino acid is glutamine in the resistant ribosome, whereas that in the sensitive ribosome is proline . This was confirmed by constructing a cycloheximide-resistant strain of S . cerevisiae having a disrupted L41a gene and an L41b gene with a substitution of the glutamine codon for the proline codon.

Gene Expr, 1992, 2(2), 99 - 110
Identification of three mammalian proteins that bind to the yeast TATA box protein TFIID; Coulombe B et al.; The TATA box binding transcription factor TFIID of S . cerevisiae was used as a ligand for affinity chromatography . Polypeptides that bind specifically to yeast TFIID (TFIID-associated proteins, DAPs) were purified from human HeLa (heDAPs) and calf thymus (ctDAPs) whole cell extracts . Both heDAP and ctDAP fractions altered the binding of TFIID to the TATA element, and substituted for the TFIIA transcription activity in a reconstituted in vitro system . The heDAP fraction also behaved like TFIIA in its ability to form a promoter-TFIID-TFIIA complex and to recruit TFIIB to such a complex . The interaction of DAPs with TFIID can confer heat-resistance (47 degrees C) on recombinant yeast or human TFIID . SDS-PAGE analysis revealed that three polypeptides from HeLa extracts specifically bound to yTFIID columns (heDAP35, heDAP21, and heDAP12) . These data suggest that a multi-subunit transcription factor with the properties of TFIIA can bind to TFIID in the absence of DNA.

J Gen Microbiol, 1992 Jan, 138 ( Pt 1), 85 - 9
Excretion of anthranilate and 3-hydroxyanthranilate by Saccharomyces cerevisiae: relationship to iron metabolism; Lesuisse E et al.; Resting suspensions of cells of Saccharomyces cerevisiae grown in iron-rich or iron-deficient conditions were studied by following the fluorescence emission changes (lambda em . 400-460 nm, lambda exc . 300-340 nm) occurring in these suspensions upon addition of glucose and ferric iron . The results show that, in addition to NAD(P)H, metabolites of the aromatic amino acid pathway interfere with the fluorescence measurements, and that they could be involved in ferric iron reduction . Wild-type strains of S . cerevisiae are known to excreted anthranilic acid and 3-hydroxyanthranilic acid in response to glucose . The major fluorescing compound excreted by a chorismate-mutase-deficient mutant strain of S . cerevisiae was identified as anthranilic acid . The excretion of anthranilic and 3-hydroxyanthranilic acids was correlated with the ferric-reducing capacity of the extracellular medium . Excretion during growth was much greater by cells cultured in iron-rich medium than by cells grown in iron-deficient medium . The possibility was examined that a link could exist between the biosynthesis of aromatics and the ferri-reductase activity of the cells, via chorismate synthase and its putative diaphorase-associated activity . Two ferri-reductase-deficient mutants excreted much less 3-hydroxyanthranilate than did the parental wild-type strains . However, the ferri-reductase activity of a chorismate-synthase-deficient mutant was comparable to that of the parental strain.

Mol Cell Biol, 1992 Jan, 12(1), 347 - 59
Ornithine decarboxylase gene of Neurospora crassa: isolation, sequence, and polyamine-mediated regulation of its mRNA; Williams LJ et al.; Ornithine decarboxylase (ODC), which initiates the biosynthesis of the polyamines putrescine, spermidine, and spermine, is encoded by the spe-1 gene of the fungus Neurospora crassa . This gene and its cDNA have been cloned and sequenced . The gene has a single 70-nucleotide intron in the coding sequence . The cDNA, comprising the entire coding region, recognizes a single 2.4-kb mRNA in Northern (RNA) blots . The mRNA transcript, defined by S1 mapping, has an extremely long, 535-base leader without strong secondary-structure features or an upstream reading frame . The translational start of the protein is ambiguous: a Met-Val-Met sequence precedes the Pro known to be the N terminus of the ODC polypeptide . The polypeptide encoded by the N . crassa spe-1 gene (484 amino acids) has 46% amino acid identity with that of Saccharomyces cerevisiae (466 amino acids) and 42% with that of mouse (461 amino acids) . Alignment of the longer N . crassa sequence with S . cerevisiae and mouse sequences creates gaps in different sites in the S . cerevisiae and mouse sequences, suggesting that N . crassa ODC is closer to an ancestral form of the enzyme than that of either yeast or mouse ODC . N . crassa ODC, which turns over rapidly in vivo in the presence of polyamines, has two PEST sequences, found in most ODCs and other proteins with rapid turnover . In striking contrast to other eucaryotic organisms, the variation in the rate of ODC synthesis in response to polyamines in N . crassa is largely correlated with proportional changes in the abundance of ODC mRNA . Spermidine is the main effector of repression, while putrescine has a weaker effect . However, putrescine accumulation appears to increase the amount of active ODC that is made from a given amount of ODC mRNA, possibly by improving its translatability . Conversely, prolonged starvation for both putrescine and spermidine leads to the differentially impaired translation of ODC mRNA.

Folia Microbiol (Praha), 1992, 37(3), 176 - 80
Cloning of Candida boidinii DNA fragments promoting autonomous replication of plasmids in Saccharomyces cerevisiae; Janatova I et al.; Fragments of Candida boidinii chromosomal DNA were inserted into the integrative vector YIp-kanr and examined for the presence of sequences promoting autonomous replication of plasmids in Saccharomyces cerevisiae . Restriction maps of two plasmids, designated S6/4 and S6/5, originating from the same S . cerevisiae transformant, were constructed . Southern hybridization data confirmed that the plasmids carry sequences from the C . boidinii chromosome . Both plasmids transform S . cerevisiae strains at 4-5-fold higher frequency than cloning vectors based on the replication origin of the 2 microns plasmid . Mitotic stability of the constructed plasmids is similar to that of the 2 mu-based vector pNF2 in S . cerevisiae.

Biotechnology, 1992, 23, 69 - 87
Protein synthesis in Aspergillus nidulans; Martinelli SD et al.; In this review of protein synthesis, we have described a system for translation of mRNA using extracts of A . nidulans . This system is useful for characterizing mutants suspected to have defects in protein synthesis and for assessing the toxicity of various antibiotics and their effects on misreading the genetic code . The well developed genetical system of A . nidulans has enabled us to map at least 27 new genes whose mutation disturbs the level of translational accuracy . These mutants could be used to identify new components of translation or new roles for established components . The abundant fidelity mutations themselves could be used to elucidate the mechanism for maintaining the accuracy of protein synthesis . The large number of mutations in the control of fidelity indicate that mutations in other parts of the translation system could be easily obtained . This would be particularly important for initiation where many factors are thought to be needed and yet their exact roles are unknown . A . nidulans appears to have normal eukaryotic ribosomes and translation factors that can be used to study the mechanism of protein synthesis, its regulation, and the maintenance of its high fidelity . If highly purified factors were used, requirements for hitherto undiscovered factors could be seen . Since A . nidulans has typical eukaryotic responses to inhibitors of translation, it could be used to study new inhibitors, their mode of action, and their potency . Among the fungi, A . nidulans could be a worthy competitor to S . cerevisiae in the field of protein synthesis, particularly because so many translation genes have been identified . The system awaits further exploitation.

Dev Biol Stand, 1992, 77, 115 - 20
Assessment of the anti-viral effect of a short-term oral treatment of mice with live Saccharomyces cerevisiae cells; Fattal-German M et al.; For assessing the efficacy of antiviral treatments, influenza and herpes virus HSV-1 infections of varying degrees of severity have been produced . The infections proved to be reproducible with respect to both their course and death rate . These infections also exhibited a course slow enough to permit the assessment of treatments under conditions mimicking human infections and lent themselves to the choice of the best adapted strategy to treat an infection . A short-term oral treatment with live cells of S . cerevisiae was efficacious in protecting mice against mild influenza infection and partly but significantly against severe infection . On the other hand, it did not afford significant protection towards either mild or severe HSV-1 infections, but it significantly potentiated the effectiveness of the antiviral drug vidarabin . S . cerevisiae treatment induced the synthesis of IFN alpha but not that of TNF alpha.

Vopr Virusol, 1992 Jan-Feb, 37(1), 19 - 22
{The antigenic and immunogenic properties of a recombinant protein of the HIV-1 gag-pol gene secreted by Saccharomyces cerevisiae yeast cells}; Trushinskaia GN et al.; Analysis of the immunological properties of recombinant proteins of HIV-1 gene gag-pol secreted by yeast cells S . cerevisiae was carried out . The proteins under study interacted with antibodies from HIV-1-seropositive human subjects and with antibodies of rabbit immune serum to the native virus as effectively and specifically as natural HIV-1 proteins . The yeast gag-pol-protein complex was markedly immunogenic and induced in animals synthesis of antibodies of a certain specificity spectrum . A comparative immunochemical analysis of the properties of the recombinant proteins carried out by EIA and immune blot showed a certain degree of similarity between the yeast proteins and those of analogous construction produced in E . coli system.

Mol Cell Biol, 1992 Jan, 12(1), 155 - 63
Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex; Fien K et al.; A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication . One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork . To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae . RFC from S . cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA . Like its human-cell counterpart, RFC from S . cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity . By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex . Now that RFC from S . cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S . cerevisiae.

Ciba Found Symp, 1992, 170, 147 - 56; discussion 156-60
DNA replication and the cell cycle; Stillman B et al.; The replication of DNA in the eukaryotic cell cycle is one of the most highly regulated events in cell growth and division . Biochemical studies on the replication of the genome of the small DNA virus simian virus 40 (SV40) have resulted in the identification of a number of DNA replication proteins from human cells . One of these, Replication Protein A (RPA), was phosphorylated in a cell cycle-dependent manner, beginning at the onset of DNA replication . RPA was phosphorylated in vitro by the cell cycle-regulated cdc2 protein kinase . This kinase also stimulated the unwinding of the SV40 origin of DNA replication during initiation of DNA replication in vitro, suggesting a mechanism by which cdc2 kinase may regulate DNA replication . Functional homologues of the DNA replication factors have been identified in extracts from the yeast Saccharomyces cerevisiae, enabling a genetic characterization of the role of these proteins in the replication of cellular DNA . A cellular origin binding protein had not been characterized . To identify proteins that function like T antigen at cellular origins of DNA replication, we examined the structure of a yeast origin of DNA replication in detail . This origin consists of four separate functional elements, one of which is essential . A multiprotein complex that binds to the essential element has been identified and purified . This protein complex binds to all known cellular origins from S . cerevisiae and may function as an origin recognition complex.

Genetica, 1992, 86(1-3), 155 - 73
The population biology and evolutionary significance of Ty elements in Saccharomyces cerevisiae; Wilke CM et al.; The basic structure and properties of Ty elements are considered with special reference to their role as agents of evolutionary change . Ty elements may generate genetic variation for fitness by their action as mutagens, as well as by providing regions of portable homology for recombination . The mutational spectra generated by Ty1 transposition events may, due to their target specificity and gene regulatory capabilities, possess a higher frequency of adaptively favorable mutations than spectra resulting from other types of mutational processes . Laboratory strains contain between 25-35 elements, and in both these and industrial strains the insertions appear quite stable . In contrast, a wide variation in Ty number is seen in wild isolates, with a lower average number/genome . Factors which may determine Ty copy number in populations include transposition rates (dependent on Ty copy number and mating type), and stabilization of Ty elements in the genome as well as selection for and against Ty insertions in the genome . Although the average effect of Ty transpositions are deleterious, populations initiated with a single clone containing a single Ty element steadily accumulated Ty elements over 1,000 generations . Direct evidence that Ty transposition events can be selectively favored is provided by experiments in which populations containing large amounts of variability for Ty1 copy number were maintained for approximately 100 generations in a homogeneous environment . At their termination, the frequency of clones containing 0 Ty elements had decreased to approximately 0.0, and the populations had became dominated by a small number of clones containing > 0 Ty elements . No such reduction in variability was observed in populations maintained in a structured environment, though changes in Ty number were observed . The implications of genetic (mating type and ploidy) changes and environmental fluctuations for the long-term persistence of Ty elements within the S . cerevisiae species group are discussed.

Gene Expr, 1992, 2(3), 203 - 14
The MRS1 gene of S . douglasii: co-evolution of mitochondrial introns and specific splicing proteins encoded by nuclear genes; Herbert CJ et al.; We have developed a rapid and simple methodology to locate yeast genes within cloned inserts, obtain partial sequence information, and construct chromosomal disruptions of these genes . This methodology has been used to study a nuclear gene from the yeast S . douglasii (a close relative of S . cerevisiae), which is essential for the excision of the mitochondrial intron aI1 of S . douglasii (the first intron in the gene encoding subunit I of cytochrome oxidase), an intron which is not present in the mitochondrial genome of S . cerevisiae . We have shown that this gene is the homologue of the S . cerevisiae MRS1 gene, which is essential for the excision of the mitochondrial introns bI3 and aI5 beta of S . cerevisiae, but is unable to assure the excision of the intron aI1 from the coxI gene of S . douglasii . The two genes are very similar, with only 13% nucleotide substitutions in the coding region, transitions being 2.5 times more frequent than transvertions . At the protein level there are 86% identical residues and 7% conservative substitutions . The divergence of the MRS1 genes of S . cerevisiae and S . douglasii, and the concomitant changes in the structure of their mitochondrial genomes is an interesting example of the co-evolution of nuclear and mitochondrial genomes.

C R Acad Sci III, 1992, 315(2), 37 - 41
Two homologous mitochondrial introns from closely related Saccharomyces species differ by only a few amino acid replacements in their Open Reading Frames: one is mobile, the other is not; Lazowska J et al.; We have undertaken a comprehensive study of the gene conversion of all the mitochondrial introns of Saccharomyces capensis . The approach used involved the measurements of intron transmission amongst the progeny of crosses between a recipient strain (Saccharomyces cerevisiae intronless mitochondria) and various donor strains (Saccharomyces capensis, with various combinations of mitochondrial introns) . We have shown that the S . capensis second intron (bi2 of cytochrome b gene) is extremely active as a donor in gene conversion whereas its homologous S . cerevisiae intron is not . Determination of sequence of the S . capensis intron demonstrates that it differs from that of the homologous S . cerevisiae intron (bi2) by a very small number of nucleotide substitutions.

Appl Biochem Biotechnol, 1992 Jan-Mar, 32, 15 - 21
Ammonia assimilation in S . cerevisiae under chemostatic growth; Lacerda V et al.; Glutamate, glutamine, and ammonia pool size have been determined in two S . cerevisiae strains (GOGAT+ and GOGAT-) growing under ammonia excess and limitation at a dilution rate of 0.10/h . The biomass levels and glutamate dehydrogenase NADPH-dependent (NADPH-GDH) activities were also measured for both strains . The strain that lacks GOGAT activity showed lower levels of metabolites under both media and lower levels of biomass under carbon limitation (ammonia excess) compared to the GOGAT+ strain . Under nitrogen limitation, the biomass level was the same for both strains, but GOGAT- changed from rounded to ellipsoidal cells.

Arch Biochem Biophys, 1992 Jan, 292(1), 70 - 6
Phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, and the phosphoinositol sphingolipids are found in the plasma membrane and stimulate the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae; Patton JL et al.; Several plasma membrane phospholipids have been studied for their ability to modulate the activity of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae . We show here that phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2), and/or the phosphatidylinositol and PIP kinases are localized primarily in the plasma membrane . Previous in vivo studies with S . cerevisiae have shown that large, rapid, and reversible changes occur in the levels of PIP and PIP2 congruent with changes in cellular ATP levels . We demonstrate here that isolated plasma membranes exhibit the same changes in PIP and PIP2 content when they are supplied with or washed free of ATP . Using a mixed micellar assay we systematically studied the efficacy of the plasma membrane lipids in sustaining the activity of the plasma membrane H(+)-ATPase . We demonstrate for the first time that a number of plasma membrane glycerophospholipids effectively stimulate the ATPase, including PIP, PIP2, and cardiolipin . Phosphoinositol-containing sphingolipids, major components of the plasma membrane, are also shown to stimulate the ATPase at significantly lower levels than the glycerophospholipids and must also be considered as important effectors in vivo.

Nucleic Acids Res, 1991 Dec 25, 19(24), 6731 - 6
DNA polymerase delta: gene sequences from Plasmodium falciparum indicate that this enzyme is more highly conserved than DNA polymerase alpha; Ridley RG et al.; Genes encoding proteins homologous to the catalytic subunits of DNA polymerase alpha and delta have been cloned from the human malaria parasite Plasmodium falciparum . These are among the first cellular replicative DNA polymerase genes to be cloned and their sequences allow us to make new statements about the relative degrees of conservation of these two enzymes . The most important finding was that P . falciparum Pol delta showed considerable homology to the only other Pol delta enzyme for which published sequence is available, that of S . cerevisiae, displaying an overall amino acid identity of 45% and identity over a highly conserved central region of 59% . In contrast, the level of identity shown over the equivalent central region of Pol alpha between the P . falciparum and S . cerevisiae sequences is only 32% . The sequence data also allowed us to examine the degree of conservation in putative exonuclease domains of Pol delta . The Pol delta gene of P . falciparum maps to chromosome 10 and evidence is presented for the presence of different sized Pol delta mRNA's in the asexual and sexual erythrocytic stages of parasite development.

Gene, 1991 Dec 20, 109(1), 99 - 105
Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements; Wright MC et al.; We have developed a transformation system for the filamentous ascomycete fungus Ashbya gossypii . Mycelial protoplasts were transformed to geneticin-resistance with plasmids containing the Escherichia coli kanamycin-resistance gene as a selectable marker and autonomously replicating sequences (ARS) from Saccharomyces cerevisiae (ARS1, 2 mu ARS) . Transformation frequencies of up to 63 transformants per microgram of plasmid DNA were obtained . The transformants were unstable under nonselective conditions . Southern analysis of DNA separated by conventional and pulsed-field-gel electrophoresis showed that the transforming DNA was present as autonomously replicating plasmid . Plasmid integration into chromosomal DNA was not detected . We concluded that the S . cerevisiae ARS elements are functional in A . gossypii, since vectors lacking such elements did not yield transformants.

FEMS Microbiol Lett, 1991 Dec 15, 69(1), 73 - 8
Physiological studies on the utilization of oleic acid by Saccharomyces cerevisiae in relation to microbody development; Evers ME et al.; We studied the influence of specific growth conditions on the induction of beta-oxidation enzymes and rate of microbody proliferation in S . cerevisiae by oleic acid . Of all conditions tested, highest enzyme levels and microbody numbers were achieved in glucose-limited continuous cultures, supplemented with oleic acids second carbon source . Comparable enzyme levels were observed in identical cultures of peroxisome-deficient (pas) mutants of S . cerevisiae . These experiments showed chemostat cultures on glucose/oleic acid mixtures to be a method of choice for future studies on microbody biogenesis/assembly in constructed, conditional pas mutants.

J Biol Chem, 1991 Dec 15, 266(35), 23796 - 801
The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase; Feng ZH et al.; The glc7 mutant of the yeast Saccharomyces cerevisiae does not accumulate glycogen due to a defect in glycogen synthase activation (Peng, Z., Trumbly, R . J., and Reimann, E.M . (1990) J . Biol . Chem . 265, 13871-13877) whereas wild-type strains accumulate glycogen as the cell cultures approach stationary phase . We isolated the GLC7 gene by complementation of the defect in glycogen accumulation and found that the GLC7 gene is the same as the DIS2S1 gene (Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T., and Yanagida, M . (1989) Cell 57, 997-1007) . The protein product predicted by the GLC7 DNA sequence has a sequence that is 81% identical with rabbit protein phosphatase 1 catalytic subunit . Protein phosphatase 1 activity was greatly diminished in extracts from glc7 mutant cells . Two forms of protein phosphatase 1 were identified after chromatography of extracts on DEAE-cellulose . Both forms were diminished in the glc7 mutant and were partly restored by transformation with a plasmid carrying the GLC7 gene . Southern blots indicate the presence of a single copy of GLC7 in S . cerevisiae, and gene disruption experiments showed that the GLC7 gene is essential for cell viability . The GLC7 mRNA was identified as a 1.4-kilobase RNA that increases 4-fold at the end of exponential growth in wild-type cells, suggesting that activation of glycogen synthase is mediated by increased expression of protein phosphatase 1 as cells reach stationary phase.

Nature, 1991 Dec 12, 354(6353), 496 - 8
Cloning and expression of the essential gene for poly(A) polymerase from S . cerevisiae; Lingner J et al.; Poly(A) polymerase is essential for the maturation of messenger RNA, adding tracts of adenosine residues to the 3' end of precursor RNA generated by endonucleolytic cleavage . This mechanism of mRNA 3' processing seems to be similar in yeast and in higher eucaryotes, although there are differences in the recognition signals in the pre-mRNA . Here we describe the cloning of the gene for yeast poly(A) polymerase . The enzyme is encoded by a single and essential gene located near the centromere on the left arm of chromosome 11 . Poly(A) polymerase purified from recombinant Escherichia coli has the same physical and biochemical properties as the yeast enzyme . The yeast poly(A) polymerase shares features of sequence with its mammalian homologue.

Nucleic Acids Res, 1991 Dec 11, 19(23), 6427 - 32
Induction of S.cerevisiae MAG 3-methyladenine DNA glycosylase transcript levels in response to DNA damage; Chen J et al.; We previously showed that the expression of the Saccharomyces cerevisiae MAG 3-methyladenine (3MeA) DNA glycosylase gene, like that of the E . coli alkA 3MeA DNA glycosylase gene, is induced by alkylating agents . Here we show that the MAG induction mechanism differs from that of alkA, at least in part, because MAG mRNA levels are not only induced by alkylating agents but also by UV light and the UV-mimetic agent 4-nitroquinoline-1-oxide . Unlike some other yeast DNA-damage-inducible genes, MAG expression is not induced by heat shock . The S . cerevisiae MGT1 O6-methylguanine DNA methyltransferase is not involved in regulating MAG gene expression since MAG is efficiently induced in a methyltransferase deficient strain; similarly, MAG glycosylase deficient strains and four other methylmethane sulfonate sensitive strains were normal for alkylation-induced MAG gene expression . However, de novo protein synthesis is required to elevate MAG mRNA levels because MAG induction was abolished in the presence of cycloheximide . MAG mRNA levels were equally well induced in cycling and G1-arrested cells, suggesting that MAG induction is not simply due to a redistribution of cells into a part of the cell cycle which happens to express MAG at high levels, and that the inhibition of DNA synthesis does not act as the inducing signal.

Eur J Biochem, 1991 Dec 5, 202(2), 657 - 64
Primary structure and processing of the Candida tsukubaensis alpha-glucosidase . Homology with the rabbit intestinal sucrase-isomaltase complex and human lysosomal alpha-glucosidase; Kinsella BT et al.; The nucleotide sequence of a 4.39-kb DNA fragment encoding the alpha-glucosidase gene of Candida tsukubaensis is reported . The cloned gene contains a major open reading frame (ORF 1) which encodes the alpha-glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa . N-terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the alpha-glucosidase precursor is proteolytically processed by removal of an N-terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63-65 kDa and 50-52 kDa, respectively . Both subunits are secreted by the heterologous host S . cerevisiae in a glycosylated form . Coincident with its efficient expression in the heterologous host, the C . tsukubaensis alpha-glucosidase gene contains many of the canonical features of highly expressed S . cerevisiae genes . There is considerable sequence similarity between C . tsukubaensis alpha-glucosidase, the rabbit sucrase-isomaltase complex (proSI) and human lysosomal acid alpha-glucosidase . The cloned DNA fragment from C . tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids . The function and identity of the polypeptide encoded by ORF 2 is not known.

J Bacteriol, 1991 Dec, 173(23), 7429 - 35
The yeast heat shock response is induced by conversion of cells to spheroplasts and by potent transcriptional inhibitors; Adams CC et al.; We report here that procedures commonly used to measure transcription and mRNA decay rates in Saccharomyces cerevisiae induce the heat shock response . First, conversion of cells to spheroplasts with lyticase, a prerequisite for nuclear runoff transcription, induces the expression of HSP70 and HSP90 heat shock genes . The transcript levels of the non-heat-shock gene ACT1 are slightly depressed, consistent with the general yeast stress response . Second, the DNA intercalator, 1,10-phenanthroline, widely employed as a general transcriptional inhibitor in S . cerevisiae, enhances the mRNA abundance of certain heat shock genes (HSP82, SSA1-SSA2) although not of others (HSC82, SSA4, HSP26) . Third, the antibiotic thiolutin, previously demonstrated to inhibit all three yeast RNA polymerases both in vivo and in vitro, increases the RNA levels of HSP82 5- to 10-fold, those of SSA4 greater than 25-fold, and those of HSP26 greater than 50-fold under conditions in which transcription of non-heat-shock genes is blocked . By using an episomal HSP82-lacZ fusion gene, we present evidence that lyticase and thiolutin induce heat shock gene expression at the level of transcription, whereas phenanthroline acts at a subsequent step, likely through message stabilization . We conclude that, because of the exquisite sensitivity of the yeast heat shock response, procedures designed to measure the rate of gene transcription or mRNA turnover can themselves impact upon each process.

Genes Dev, 1991 Dec, 5(12A), 2166 - 75
A novel cyclin gene from Drosophila complements CLN function in yeast; Lahue EE et al.; In the yeast Saccharomyces cerevisiae three G1-S cyclins, or CLNs, have been identified that trigger the G1-S transition of the cell cycle . The regulation of the G1-S transition is particularly intriguing in Drosophila both because G1 is added to the cell cycle developmentally and G1-S regulators may drive the polytene cell cycle . To identify potential G1-S regulators from Drosophila, a cDNA expression library was constructed in which Kc cell cDNAs were placed in a high-copy S . cerevisiae vector under the control of the constitutive ADH1 promoter . Following transformation into an S . cerevisiae strain lacking all three CLN gene products, we identified one Drosophila cDNA that complemented the yeast G1 cyclins and restored growth to near wild-type levels . The CLNDm gene is present as a single copy in the Drosophila genome and encodes a 1.2-kb mRNA . DNA sequence analysis reveals that although this gene has cyclin homology, it is a new member of the cyclin gene family . CLNDm mRNA expression correlates with periods of maximal cell division throughout Drosophila development . The transcript is most abundant in early embryos, and it is present in low levels in larvae, pupae, and adults . Drosophila embryos hybridized in situ to this cyclin gene show uniform expression of the message throughout the embryo, with diminishing expression as embryogenesis proceeds.

Yeast, 1991 Dec, 7(9), 971 - 9
The nucleotide sequence of a third cyclophilin-homologous gene from Saccharomyces cerevisiae; Franco L et al.; The nucleotide sequence of a 1558 bp DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae contains an open reading frame of 954 nucleotides with coding potential for a protein with high similarity to the ubiquitous cyclophilins which are both peptidyl-prolyl cis-trans isomerases and cyclosporin A-binding proteins . It should, therefore, represent the third gene (SCC3) of this kind from S . cerevisiae . SCC3 is present in a single copy in the genome of S . cerevisiae and results in a constitutively expressed 1.2 kb transcript during cell growth . Its putative protein product (Scc3) contains two hydrophobic cores, one at the amino terminal, 20 amino acids long, which could serve as a signal peptide, and the other one at the carboxyl end with a structure similar to a transmembrane helix . These findings suggest that Scc3 could be a secretory or, more likely, a transmembrane protein . The only cyclophilin with similar structure to that of Scc3 is ninaA from Drosophila melanogaster, a transmembrane protein which seems to be implicated in the correct folding and/or intercalation of rhodopsin in the endoplasmic reticulum of the fly photoreceptors (Stamnes, M.A . et al., Cell 65, 219-227, 1991) . In addition, the amino and the carboxy regions of Scc3 and ninaA share a significant level of homology, which suggests that they have a similar function, albeit for different target proteins.

Yeast, 1991 Dec, 7(9), 943 - 52
On the level of plasmid-bearing cells in transformed cultures of Saccharomyces cerevisiae; Guerrini AM et al.; LC1, a YIP5-derived plasmid containing a human DNA fragment with ARS activity in yeast, has been used to study the replication of ARS plasmids in Saccharomyces cerevisiae . ARS plasmids carried in yeast hosts are normally mitotically unstable . In transformed cultures the fraction of cells that contain plasmid, measured by plating on selective media, is lower than would be expected from measured rates of plasmid loss . In the case of S . cerevisiae carrying either the plasmid LC1 or YRP17, the assay yields values of the order of 10-20% or 30-50% respectively . We have found that by doing a double nutritional upshift that involves conditioned medium and casamino acids, a population of cells can be defined that carry plasmid but are unable to grow on media that select for the plasmid marker . Thus the total fraction of cells that can be shown to contain plasmid increases to greater than 70% . To distinguish between the inability of plasmid to replicate in these cells and lack of expression of the selectable gene, cultures grown from single cells were analysed for the presence of plasmid DNA . In a substantial fraction of the population, plasmid DNA could be detected only by polymerase chain reaction and not by standard blotting and hybridization . These results suggest that plasmid is unable to replicate in these cells . Growth kinetics experiments with transformed cultures are consistent with the notion that only a small fraction of the cells contains plasmid capable of replication upon dilution into selective medium . Possible explanations for the phenomena observed are discussed.

Yeast, 1991 Dec, 7(9), 891 - 911
Structure of the yeast endoplasmic reticulum: localization of ER proteins using immunofluorescence and immunoelectron microscopy; Preuss D et al.; The endoplasmic reticulum (ER) and other secretory compartments of Saccharomyces cerevisiae have biochemical functions that closely parallel those described in higher eukaryotic cells, yet the morphology of the yeast organelles is quite distinct . In order to associate ER functions with the corresponding cellular structures, we localized several proteins, each of which is expected to be associated with the ER on the basis of enzymatic activity, biological function, or oligosaccharide content . These marker proteins were visualized by immunofluorescence or immunoelectron microscopy, allowing definition of the S . cerevisiae ER structure, both in intact cells and at the ultrastructural level . Each marker protein was most abundant within the membranes that envelop the nucleus and several were also found in extensions of the ER that frequently juxtapose the plasma membrane . Double-labeling experiments were entirely consistent with the idea that the marker proteins reside within the same compartment . This analysis has permitted, for the first time, a detailed characterization of the ER morphology as yeast cells proceed through their growth and division cycles.

Curr Genet, 1991 Dec, 20(6), 457 - 63
Isolation and characterization of S . cerevisiae mutants deficient in amino acid-inducible peptide transport; Island MD et al.; The transport of small peptides into the yeast Saccharomyces cerevisiae is subject to complex regulatory control . In an effort to determine the number, and to address the function, of the components involved in peptide transport and its regulation, spontaneous mutants resistant to toxic di- and tripeptides were isolated under inducing conditions . Twenty-four mutant strains were characterized in detail and fell into two phenotypic groups; one group deficient in amino acid-inducible peptide uptake, the other with a pleiotropic phenotype including a loss of peptide transport . Complementation analysis of recessive mutations in 12 of these strains defined three groups; ptr1 (nine strains), ptr2 (two strains), and ptr3 (one strain) . Isolation and screening of 31 additional N-methyl-N-nitro-N-Nitrosoguanidine (MNNG)-induced, peptide transport-deficient mutants produced one ptr3 and 30 ptr2 strains: no additional complementation groups were detected . Uptake of radiolabeled dileucine was negligible in ptr1 and ptr2 strains and was reduced by 65% and 90% in the two ptr3 mutants, indicating that all strains were defective at the transport step . We conclude that the S . cerevisiae amino acid-inducible peptide transport system recognizes a broad spectrum of peptide substrates and involves at least three components . One gene, PTR3, may play an indirect or regulatory role since mutations in this gene cause a pleiotropic phenotype.

Gene, 1991 Dec 1, 108(1), 81 - 9
Protein disulfide isomerase is essential for viability in Saccharomyces cerevisiae; Farquhar R et al.; Protein disulfide isomerase (PDI) is an enzyme involved in the catalysis of disulfide bond formation in secretory and cell-surface proteins . Using an oligodeoxyribonucleotide designed to detect the conserved 'thioredoxin-like' active site of vertebrate PDIs, we have isolated a gene encoding PDI from the lower eukaryote, Saccharomyces cerevisiae . The nucleotide sequence and deduced open reading frame of the cloned gene predict a 530-amino-acid (aa) protein of Mr 59,082 and a pI of 4.1, physical properties characteristic of mammalian PDIs . Furthermore, the aa sequence shows 30-32% identity with mammalian and avian PDI sequences and has a very similar overall organisation, namely the presence of two approx . 100-aa segments, each of which is repeated, with the most significant homologies to mammalian and avian PDIs being in the regions (a, a') that contain the conserved 'thioredoxin-like' active site . The N-terminal region has the characteristics of a cleavable secretory signal sequence and the C-terminal four aa (-His-Asp-Glu-Leu) are consistent with the protein being a component of the S . cerevisiae endoplasmic reticulum . Transformants carrying multiple copies of this gene (designated PDI1) have tenfold higher levels of PDI activity and overproduce a protein of the predicted Mr . The PDI1 gene is unique in the yeast genome and encodes a single 1.8-kb transcript that is not found in stationary phase cells . Disruption of the PDI1 gene is haplo-lethal indicating that the product of this gene is essential for viability.

Gene, 1991 Dec 1, 108(1), 157 - 62
Isolation and nucleotide sequence of the ribosomal protein S16-encoding gene from Aspergillus nidulans; Bradshaw RE et al.; A genomic clone has been isolated from Aspergillus nidulans which is homologous to the ribosomal (r) protein S16-encoding gene of Saccharomyces cerevisiae (S16A) and the r-protein S19-encoding gene of rat (S19) . The amino acid (aa) sequences, deduced from nucleotide (nt) sequence analysis, show that in both cases more than 63% of the aa are conserved . The proposed A . nidulans r-protein S16 gene (rps16) differs from that of S . cerevisiae in that it occurs as a single copy in the haploid genome (rather than two copies as in yeast) and contains two putative introns (rather than one) . The mRNA leader is long compared to many Aspergillus genes, commencing 293 nt upstream from the coding region, and contains an open reading frame of 13 codons.

Jpn J Genet, 1991 Dec, 66(6), 709 - 18
Physical mapping and RFLP analysis of mtDNAs from the ascosporogenous yeasts: Saccharomyces exiguus, S . kluyveri and Hansenula wingei; Okamoto K et al.; Mitochondrial DNAs from the ascosporogenous yeasts S . exiguus, S . kluyveri and H . wingei were prepared by a new rapid method without CsCl isopycnic centrifugation . The mtDNA RFLPs were identified and clearly distinguished each species from the other . The physical maps were constructed by single and double digestion with nine restriction endonucleases . The location of rRNA genes was assigned to the maps by Southern hybridization with synthetic consensus probes . The genome sizes of these mtDNAs were estimated to be 45 kb for S . exiguus, 54 kb for S . kluyveri and 27 kb for H . wingei . The mtDNA RFLP analysis indicates a phylogenetic relationship among these yeasts . This indicates that S . cerevisiae is closer to H . wingei than S . kluyveri . However, the derived phylogenetic tree is completely consistent with that which was previously constructed on amino acid replacement in mating pheromones and electrophoretic karyotypes (Yoshida et al., 1989).

Nature, 1991 Nov 28, 354(6351), 311 - 4
Catalysis of guanine nucleotide exchange on the CDC42Hs protein by the dbl oncogene product; Hart MJ et al.; THE superfamily of low molecular mass GTP-binding proteins, for which the ras proteins are prototypes, has been implicated in the regulation of diverse biological activities including protein trafficking, secretion, and cell growth and differentiation . One member of this family, CDC42Hs (originally referred to as Gp or G25K), seems to be the human homologue of the Saccharomyces cerevisiae cell-division-cycle protein, CDC42Sc . A second S . cerevisiae protein, CDC24, which is known from complementation studies to act with CDC42Sc to regulate the development of normal cell shape and the selection of nonrandom budding sites in yeast, contains a region with sequence similarity to the dbl oncogene product . Here we show that dbl specifically catalyses the dissociation of GDP from CDC42Hs and thereby qualifies as a highly selective guanine nucleotide exchange factor for the GTP-binding protein . Although guanine nucleotide exchange activities have been previously described for other members of the Ras-related GTP-binding protein family, this is the first demonstration, to our knowledge, of the involvement of a human oncogenic protein in catalysing exchange activity.

Biochemistry, 1991 Nov 26, 30(47), 11249 - 54
Functional analysis of the domains of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae; Lawson JE et al.; The LAT1 gene encoding the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase (PDH) complex from Saccharomyces cerevisiae was disrupted, and the lat1 null mutant was used to analyze the structure and function of the domains of E2 . Disruption of LAT1 did not affect the viability of the cells . Apparently, flux through the PDH complex is not required for growth of S . cerevisiae under the conditions tested . The wild-type and mutant PDH complexes were purified to near-homogeneity and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme assays . Mutant cells transformed with LAT1 on a unit-copy plasmid produced a PDH complex very similar to that of the wild-type PDH complex . Deletion of most of the putative lipoyl domain (residues 8-84) resulted in loss of about 85% of the overall activity, but did not affect the acetyltransferase activity of E2 or the binding of pyruvate dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), and protein X to the truncated E2 . Similar results were obtained by deleting the lipoyl domain plus the first hinge region (residues 8-145) and by replacing lysine-47, the putative site of covalent attachment of the lipoyl moiety, by arginine . Although the lipoyl domain of E2 and/or its covalently bound lipoyl moiety were removed, the mutant complexes retained 12-15% of the overall activity of the wild-type PDH complex . Replacement of both lysine-47 in E2 and the equivalent lysine-43 in protein X by arginine resulted in complete loss of overall activity of the mutant PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1991 Nov 25, 19(22), 6139 - 44
Interactions of transfer RNA pseudouridine synthases with RNAs substituted with fluorouracil; Samuelsson T; We have previously purified and characterized two different S . cerevisiae enzymes that produce pseudouridine specifically in nucleotide positions 13 and 55, respectively, in their tRNA substrates . The interactions of these enzymes with fluorinated tRNAs have now been studied . Such RNAs were produced by in vitro transcription using as templates synthetic genes that encode variants of a yeast glycine tRNA . RNAs substituted with fluorouracil were found to markedly inhibit pseudouridine synthase activity and the inhibitory effect of a tRNA was to a large extent dependent on the presence of fluorouracil in the nucleotide position where normally pseudouridylation occurs . Pseudouridine synthases were shown to form highly stable, non-covalent complexes with fluorinated tRNAs and we demonstrate that this interaction may be used to further characterize and purify these enzymes . The use of 5-fluorouracil as a cancer therapeutic agent is discussed in relation to our results.

J Biol Chem, 1991 Nov 25, 266(33), 22199 - 205
NAD(+)-dependent isocitrate dehydrogenase . Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae; Cupp JR et al.; NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 (Mr approximately 40,000) and IDH2 (Mr approximately 39,000) . We have isolated and characterized a yeast genomic clone containing the IDH2 gene . The amino acid sequence deduced from the gene indicates that IDH2 is synthesized as a precursor of 369 amino acids (Mr 39,694) and is processed upon mitochondrial import to yield a mature protein of 354 amino acids (Mr 37,755) . Amino acid sequence comparison between S . cerevisiae IDH2 and S . cerevisiae NADP(+)-dependent isocitrate dehydrogenase shows no significant sequence identity, whereas comparison of IDH2 and Escherichia coli NADP(+)-dependent isocitrate dehydrogenase reveals a 33% sequence identity . To confirm the identity of the IDH2 gene and examine the relationship between IDH1 and IDH2, the IDH2 gene was disrupted by genomic replacement in a haploid yeast strain . The disruption strain expressed no detectable IDH2, as determined by Western blot analysis, and was found to lack NAD(+)-dependent isocitrate dehydrogenase activity, indicating that IDH2 is essential for a functional enzyme . Overexpression of IDH2, however, did not result in increased NAD(+)-dependent isocitrate dehydrogenase activity, suggesting that both IDH1 and IDH2 subunits are required for catalytic activity . The disruption strain was unable to utilize acetate as a carbon source and exhibited a 2-fold slower growth rate than wild type strains on glycerol or lactate . This growth phenotype is consistent with NAD(+)-dependent isocitrate dehydrogenase performing an essential role in the oxidative function of the citric acid cycle.

Cell, 1991 Nov 15, 67(4), 701 - 16
Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase; Simon MA et al.; We have conducted a genetic screen for mutations that decrease the effectiveness of signaling by a protein tyrosine kinase, the product of the Drosophila melanogaster sevenless gene . These mutations define seven genes whose wild-type products may be required for signaling by sevenless . Four of the seven genes also appear to be essential for signaling by a second protein tyrosine kinase, the product of the Ellipse gene . The putative products of two of these seven genes have been identified . One encodes a ras protein . The other locus encodes a protein that is homologous to the S . cerevisiae CDC25 protein, an activator of guanine nucleotide exchange by ras proteins . These results suggest that the stimulation of ras protein activity is a key element in the signaling by sevenless and Ellipse and that this stimulation may be achieved by activating the exchange of GTP for bound GDP by the ras protein.

J Bacteriol, 1991 Nov, 173(21), 6807 - 10
The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair; Impellizzeri KJ et al.; Uracil-DNA-glycosylase has been proposed to function as the first enzyme in strand-directed mismatch repair in eukaryotic organisms, through removal of uracil from dUMP residues periodically inserted into the DNA during DNA replication (Aprelikova, O . N., V . M . Golubovskaya, T . A . Kusmin, and N . V . Tomilin, Mutat . Res . 213:135-140, 1989) . This hypothesis was investigated with Saccharomyces cerevisiae . Mutation frequencies and spectra were determined for an ung1 deletion strain in the target SUP4-o tRNA gene by using a forward selection scheme . Mutation frequencies in the SUP4-o gene increased about 20-fold relative to an isogenic wild-type S . cerevisiae strain, and the mutator effect was completely suppressed in the ung1 deletion strain carrying the wild-type UNG1 gene on a multicopy plasmid . Sixty-nine independently derived mutations in the SUP4-o gene were sequenced . All but five of these were due to GC----AT transitions . From this analysis, we conclude that the mutator phenotype of the ung1 deletion strain is the result of a failure to repair spontaneous cytosine deamination events occurring frequently in S . cerevisiae and that the UNG1 gene is not required for strand-specific mismatch repair in S . cerevisiae.

Mol Cell Biol, 1991 Nov, 11(11), 5710 - 7
Mutations in SPT16/CDC68 suppress cis- and trans-acting mutations that affect promoter function in Saccharomyces cerevisiae; Malone EA et al.; SPT16 was previously identified as a high-copy-number suppressor of delta insertion mutations in the 5' regions of the HIS4 and LYS2 genes of Saccharomyces cerevisiae . We have constructed null mutations in the SPT16 gene and have demonstrated that it is essential for growth . Temperature-sensitive-lethality spt16 alleles have been isolated and shown to be pleiotropic; at a temperature permissive for growth, spt16 mutations suppress delta insertion mutations, a deletion of the SUC2 upstream activating sequence, and mutations in trans-acting genes required for both SUC2 and Ty expression . In addition, SPT16 is identical to CDC68, a gene previously shown to be required for passage through the cell cycle control point START . However, at least some transcriptional effects caused by spt16 mutations are independent of arrest at START . These results and those in the accompanying paper (A . Rowley, R . A . Singer, and G . C . Johnston, Mol . Cell . Biol . 11:5718-5726, 1991) indicate that SPT16/CDC68 is required for normal transcription of many loci in S . cerevisiae.

Pharmacogenetics, 1991 Nov, 1(2), 86 - 93
Progesterone metabolism in recombinant yeast simultaneously expressing bovine cytochromes P450c17 (CYP17A1) and P450c21 (CYP21B1) and yeast NADPH-P450 oxidoreductase; Sakaki T et al.; Simultaneous expression plasmids were constructed for bovine adrenal cytochromes P450c17 and P450c21 (pA gamma alpha) and for both P450s together with NADPH-cytochrome P450 reductase (pAR gamma alpha) . On introduction of each of the plasmids into Saccharomyces cerevisiae AH22 cells, the transformed yeast strains AH22/pA gamma alpha and AH22/pAR gamma alpha produced about 10(5) molecules per cell of P450c17 and 2 x 10(3) molecules per cell of P450c21 . The expression levels of NADPH-cytochrome P450 reductase was about 3 x 10(4) and 6 x 10(5) molecules per cell in the strains AH22/pA gamma alpha and AH22/pAR gamma alpha, respectively . When progesterone was added to growing cell cultures of the transformed yeast strains, the substrate was metabolized more rapidly in the AH22/pAR gamma alpha cells than AH22/pA gamma alpha cells, probably due to overproduction of the reductase . In the AH22/pAR gamma alpha cells, progesterone was first converted into 17 alpha-hydroxyprogesterone to the extent of 82% by the catalysis of P450c17 . 17 alpha-hydroxyprogesterone was further converted into 11-deoxycortisol by P450c21 to the extent of 60% of the added substrate . The conversion of progesterone into androstenedione through 17 alpha-hydroxyprogesterone was estimated to be less than 3%, suggesting very low C17,20-lyase activity of P450c17, although other hydroxylation products were detected . Androstenedione was further converted into testosterone by an unknown pathway present in S . cerevisiae cells.

Mol Microbiol, 1991 Nov, 5(11), 2663 - 71
Targeting and assembly of an oligomeric bacterial enterotoxoid in the endoplasmic reticulum of Saccharomyces cerevisiae; Schonberger O et al.; A hybrid protein consisting of the Escherichia coli lipoprotein signal sequence attached to the mature sequence of the B subunit of heat-labile enterotoxin (Lipo-EtxB) was expressed in yeast and E . coli . Analyses of cell lysates from Saccharomyces cerevisiae and E . coli expressing the protein revealed that both organisms were able to assemble Lipo-EtxB into oligomers that were (i) stable in the presence of sodium dodecyl sulphate, (ii) resistant to proteinase K degradation, and (iii) able to bind to GM1-ganglioside receptors . Each of these properties are characteristic of the wild-type B subunit pentamer produced in E . coli . Assembly of Lipo-EtxB was found to be unaffected in a sec18 mutant of S . cerevisiae, which possesses a temperature-sensitive defect in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, but was found not to assemble in a sec53 mutant, which causes the misfolding of proteins targeted to the ER . A kar2-1 mutation with a defect in the yeast homologue of BiP caused an 18-fold reduction in Lipo-EtxB assembly at the non-permissive temperature in S . cerevisiae . However, introduction of the wild-type KAR2 gene on a plasmid into the kar2-1 mutant completely suppressed the inhibition of Lipo-EtxB assembly . This provides the first evidence that KAR2 facilitates the assembly of an oligomeric protein in yeast and thus implicates KAR2 as a 'molecular chaperone' . The possible mechanisms of enterotoxoid assembly in E . coli and S . cerevisiae are discussed.

Gene, 1991 Oct 30, 107(1), 161 - 4
Molecular cloning of the Candida maltosa ADE1 gene; Sasnauskas K et al.; The structural gene (ADE1) encoding phosphoribosyl-aminoimidazole-succinocarboxamide synthetase (SAICAR synthetase; EC 6.3.2.6) in Candida maltosa has been isolated by functional complementation of an ade1 strain of Saccharomyces cerevisiae . The gene was localized on a 2.5-kb BamHI DNA fragment . Nucleotide sequence analysis of the cloned gene has revealed an open reading frame encoding a protein (SAICAR synthetase) with an Mr of 32,751 . The codon bias index, 0.68, indicates that the ADE1 gene is a moderately highly expressed gene . The cloned gene shows 63.5% nt identity and 65.2% deduced amino acid identity with the S . cerevisiae ADE1 gene which encodes the same enzymatic activity . The gene may be used as a convenient genetic marker for construction of a new host-vector system for C . maltosa.

Gene, 1991 Oct 30, 107(1), 155 - 60
The gene encoding squalene epoxidase from Saccharomyces cerevisiae: cloning and characterization; Jandrositz A et al.; The gene (ERG1) encoding squalene epoxidase (ERG) from Saccharomyces cerevisiae was cloned . It was isolated from a gene library, prepared from an allylamine-resistant (AlR) S . cerevisiae mutant, by screening transformants in a sensitive strain for AlR colonies . The ERG tested in a cell-free extract from one of these transformants proved to be resistant to the Al derivative, terbinafine . From this result, we concluded that the recombinant plasmid in the transformant carried an allelic form of the ERG1 gene . The nucleotide sequence showed the presence of one open reading frame coding for a 55,190-Da peptide of 496 amino acids . Southern hybridization experiments allowed us to localize the ERG1 gene on yeast chromosome 15.

Gene, 1991 Oct 30, 107(1), 111 - 8
Selection of secretory protein-encoding genes by fusion with PHO5 in Saccharomyces cerevisiae; Sidhu RS et al.; Secretory protein-encoding genes of Saccharomyces cerevisiae have been cloned by a novel procedure that is based on the functional selection of their fusions with acid phosphatase (APase) at the DNA level . DNA fragments that functionally replace the promoter and signal sequence-encoding regions of the PHO5 gene (encoding APase) have been obtained by positive selection from a pool of cloned random DNA fragments . Five unique DNA sequences containing the promoter, and encoding signal sequences have been isolated . We have also isolated the complete gene, SSP120, encoding one of these S . cerevisiae secretory proteins, SSP120 . Gene disruption studies have shown that the SSP120 gene is not essential for viability and growth . The SSP120 amino acid (aa) sequence has 13.5% identity with the middle 88-250 aa residues of the chicken glycosylation site-binding protein . However, SSP120 disruption did not affect protein glycosylation in yeast . The present study provides an alternative approach for the isolation of genes encoding secretory proteins, in contrast to classical genetic approaches that require isolation of functionally defective mutations followed by gene isolation by functional complementation . The present procedure should contribute to our understanding of protein sorting by permitting the cloning of genes encoding proteins targeted to different organelles in the secretory pathway.

Biochim Biophys Acta, 1991 Oct 25, 1080(2), 135 - 7
Purification and determination of the NH2-terminal amino acid sequence of mouse alpha-amylase secreted from Saccharomyces cerevisiae: correct processing of the secretion signal from pGKL killer 28 kDa precursor protein; Tokunaga M et al.; We have previously reported the construction of recombinant mouse salivary alpha-amylase secretion vector in Saccharomyces cerevisiae utilizing novel yeast secretion signal derived from killer 28 kDa precursor protein . Here, we have first purified recombinant mouse alpha-amylase to homogeneity from the culture medium of S . cerevisiae, and determined its NH2-terminal amino acid sequence . The sequencing data indicated that the 28 kDa killer secretion signal-alpha-amylase fusion protein was cleaved accurately at its native processing site, and that both the core-glycosylated and non-glycosylated alpha-amylases possessed the same NH2-terminal amino acid sequences.

FEBS Lett, 1991 Oct 21, 291(2), 355 - 8
A method to study the rapid phosphorylation-related modulation of neutral trehalase activity by temperature shifts in yeast; De Virgilio C et al.; Heat shock enhanced the synthesis of neutral trehalase in growing cells of Saccharomyces cerevisiae, as detected by immunological methods . The activity of the enzyme was measured in extracts obtained by two methods: cells were either harvested by filtration and subsequent disruption with glass beads at 0-4 degrees C or immediately frozen with liquid nitrogen in the presence of Triton X-100, followed by thawing at 30 degrees C . The first procedure yielded artificially high activities of neutral trehalase in heat-shocked cells due to rapid (less than 1 min) activation during handling at 4 degrees C before homogenization . Activity of the enzyme in these homogenates decreased 75-90% upon a treatment with alkaline phosphatase, indicating that activation was due to phosphorylation . The second procedure yielded low trehalase activities for heat-shock treated cells, much higher activities for cells shifted back for some seconds to 27 degrees C, and very low activities again for cells shifted from 27 to 40 degrees C for a second time . Thus, permeabilization of cells following rapid freezing in Triton X-100 is a method of choice to study post-translational modulation of the neutral trehalase of S . cerevisiae by phosphorylation and dephosphorylation.

FEBS Lett, 1991 Oct 21, 291(2), 269 - 72
Metabolic regulation of the trehalose content of vegetative yeast; Winkler K et al.; We have investigated the mechanism by which heat shock conditions lead to a reversible accumulation of trehalose in growing yeast . When cells of S . cerevisiae M1 growing exponentially at 30 degrees C were shifted to 45 degrees C for 20 min, or to 39 degrees C for 40 min, the concentration of trehalose increased by about 25-fold; an effect reversed upon lowering the temperature to 30 degrees C . This was compared to the more than 50-fold rise in trehalose levels obtained upon transition from the exponential to the stationary growth phase . Whereas the latter was paralleled by a 12-fold increase in the activity of trehalose-6-phosphate synthase, no significant change in the activities of trehalose-synthesizing and -degrading enzymes was measured under heat shock conditions . Accordingly, cycloheximide did not prevent the heat-induced accumulation of trehalose . However, the concentrations of the substrates for trehalose-6-phosphate synthase, i.e . glucose-6-phosphate and UDP-glucose, were found to rise during heat shock by about 5-10-fold . Since the elevated levels of both sugars are still well below the Km-values determined for trehalose-6-phosphate synthase in vitro, they are likely to contribute to the increase in trehalose under heat shock conditions . A similar increase in the steady-state levels was obtained for other intermediates of the glycolytic pathway between glucose and triosephosphate, including ATP . This suggests that temperature-dependent changes in the kinetic parameters of glycolytic enzymes vary in steady-state levels of intermediates of sugar metabolism, including an increase of those that are required for trehalose synthesis . Trehalose, glucose-6-phosphate, UDP-glucose, and ATP, were all found to increase during the 40 min heat treatment at 39 degrees C . Since this also occurs in a mutant lacking the heat shock-induced protein HSP104 (delta hsp104), this protein cannot be involved in the accumulation of trehalose under these heat shock conditions . However, mutant delta hsp104, in contrast to the parental wild-type, was sensitive towards a 20 min incubation at 50 degrees C . Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not towards a 20 min incubation at 50 degrees C . Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not the accumulation of trehalose, protects S . cerevisiae from the damage caused by a 50 degrees C treatment.

Cell, 1991 Oct 18, 67(2), 389 - 402
S . cerevisiae alpha pheromone receptors activate a novel signal transduction pathway for mating partner discrimination; Jackson CL et al.; Wild-type S . cerevisiae cells of both mating types prefer partners producing high levels of pheromone and mate very infrequently to cells producing no pheromone . However, some mutants that are supersensitive to pheromone lack this ability to discriminate . In this study, we provide evidence for a novel role of alpha pheromone receptors in mating partner discrimination that is independent of the known G protein-mediated signal transduction pathway . Furthermore, in response to pheromone, receptors become localized to the emerging region of morphogenesis that is positioned adjacent to the nucleus, suggesting that receptor localization may be involved in mating partner discrimination . Actin, myosin 2, and clathrin heavy chain are involved in mating partner discrimination, since strains carrying mutations in the genes encoding these proteins result in a small but significant defect in mating partner discrimination.

Cell, 1991 Oct 18, 67(2), 355 - 64
RNA-mediated recombination in S . cerevisiae; Derr LK et al.; The existence of pseudogenes and the observation of intron loss suggest that RNA can serve as an intermediate in recombination . We used a HIS3 reporter gene to show that RNA-mediated recombination occurs in yeast . His3+ prototroph formation required transcription and expression of the retrotransposon Ty . Two RNA-mediated recombination events were detected: homologous recombination between the cDNA and plasmid his3 sequences, resulting in intron loss, and insertion of the cDNA into the chromosome in the absence of HIS3 homology . The chromosomal His3+ prototrophs showed many hallmarks of naturally occurring pseudogenes . They integrated at novel sites in the chromosome, lacked introns, and possessed poly(A) tracts . Additionally, their 5' ends corresponded with the site of initiation of the GAL1 transcript.

Cell, 1991 Oct 4, 67(1), 131 - 44
The signal recognition particle in S . cerevisiae; Hann BC et al.; We have identified the Saccharomyces cerevisiae homolog of the signal recognition particle (SRP) and characterized its function in vivo . S . cerevisiae SRP is a 16S particle that includes a homolog of the signal sequence-binding protein subunit of SRP (SRP54p) and a small cytoplasmic RNA (scR1) . Surprisingly, the genes encoding scR1 and SRP54p are not essential for growth, though SRP-deficient cells grow poorly, suggesting that SRP function can be partially by-passed in vivo . Protein translocation across the ER membrane is impaired in SRP-deficient cells, indicating that yeast SRP, like its mammalian counterpart, functions in this process . Unexpectedly, the degree of the translocation defect varies for different proteins . The ability of some proteins to be efficiently targeted in SRP-deficient cells may explain why previous genetic and biochemical analyses in yeast and bacteria did not reveal components of the SRP-dependent protein targeting pathway.

DNA Cell Biol, 1991 Oct, 10(8), 613 - 21
Expression of bovine adrenodoxin and NADPH-adrenodoxin reductase cDNAs in Saccharomyces cerevisiae; Akiyoshi-Shibata M et al.; Expression of both bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR) were examined in Saccharomyces cerevisiae . Three ADX and two ADR expression plasmids were constructed by inserting each of the corresponding cDNA fragments between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5N . Plasmids pAX and pMX contained the coding region for the precursor and mature ADX, respectively, while pCMX carried the mature ADX preceded by the mitochondrial signal of yeast cytochrome c oxidase subunit IV (COX IV) . Similarly, pMR and pCMR coded for mature ADR without and with the mitochondrial signal of yeast COX IV, respectively . Transformed S . cerevisiae AH22{rho 0}/pAX cells produced the ADX precursor, while AH22{rho 0}/pMX and AH22{rho 0}/pCMX cells produced mature ADX (mat-ADX) and modified ADX (mat-COX/ADX), respectively . Mat-ADX and mat-COX/ADX were found mainly in the cytosolic and mitochondrial fractions, respectively, and showed cytochrome c reductase activity . AH22{rho+}/pMR and AH22{rho+}/pCMR cells produced mature ADR (mat-ADR) and modified ADR (mat-COX/ADR), respectively . Mat-ADR lacking the mitochondrial signal was found in the cytosolic fraction and exhibited cytochrome c reductase activity, while mat-COX/ADR was localized in the mitochondrial fraction, but showed no reductase activity . In an in vitro reconstituted system consisting of both mat-COX/ADX- and mat-ADR-containing fractions, bovine P450scc converted cholesterol into pregnenolone . Thus mat-COX/ADX and mat-ADR produced in the yeast can transfer electrons from NADPH to P450scc.

Mol Cell Biol, 1991 Oct, 11(10), 5330 - 7
Yeast glycolytic mRNAs are differentially regulated; Moore PA et al.; The regulation of glycolytic genes in response to carbon source in the yeast Saccharomyces cerevisiae has been studied . When the relative levels of each glycolytic mRNA were compared during exponential growth on glucose or lactate, the various glycolytic mRNAs were found to be induced to differing extents by glucose . No significant differences in the stabilities of the PFK2, PGK1, PYK1, or PDC1 mRNAs during growth on glucose or lactate were observed . PYK::lacZ and PGK::lacZ fusions were integrated independently into the yeast genome at the ura3 locus . The manner in which these fusions were differentially regulated in response to carbon source was similar to that of their respective wild-type loci . Therefore, the regulation of glycolytic mRNA levels is mediated at the transcriptional level . When the mRNAs are ordered with respect to the glycolytic pathway, two peaks of maximal induction are observed at phosphofructokinase and pyruvate kinase . These enzymes (i) catalyze the two essentially irreversible steps on the pathway, (ii) are the two glycolytic enzymes that are circumvented during gluconeogenesis and hence are specific to glycolysis, and (iii) are encoded by mRNAs that we have shown previously to be coregulated at the translational level in S . cerevisiae (P . A . Moore, A . J . Bettany, and A . J . P . Brown, NATO ASI Ser . Ser . H Cell Biol . 49:421-432, 1990) . This differential regulation of glycolytic mRNA levels might therefore have a significant influence upon glycolytic flux in S . cerevisiae.

J Steroid Biochem Mol Biol, 1991 Oct, 39(4A), 487 - 92
Expression of rat 5 alpha-reductase in Saccharomyces cerevisiae; Ordman AB et al.; Dihydrotestosterone (DHT) is the principle androgen in certain tissues such as the prostate . DHT is formed from testosterone by the NADPH-dependent enzyme 5 alpha-reductase (5AR) . In this paper we report the expression of catalytically active steroid 5AR from the rat in Saccharomyces cerevisiae . A full length cDNA coding for 5AR was isolated from a rat liver cDNA library and fixed in frame to the signal sequence of yeast acid phosphatase . A constitutive short promoter fragment of the acid phosphatase gene (PHO5) and the PHO5 transcriptional terminator were added and the expression cassette ligated into the yeast 2 mu vector pDP34 . S . cerevisiae transformed with the 5AR expression plasmid pDP34/PHO5AR exhibited about 100-fold more activity per gram wet weight than rat prostate.

Arch Biochem Biophys, 1991 Oct, 290(1), 197 - 201
Electron microscopic localization of pyruvate carboxylase in rat liver and Saccharomyces cerevisiae by immunogold procedures; Rohde M et al.; The intracellular location of pyruvate carboxylase (EC 6.4.1.1) in rat liver and Saccharomyces cerevisiae was investigated using the antibody-gold and protein A-gold techniques carried out as a postembedding immunoelectron microscopic procedure . The vast majority of gold particles (greater than 98%), indicative of the presence of antigenic sites of pyruvate carboxylase, were found in the mitochondria of rat liver . No other cellular compartment was labeled except the cytosol which did not account for more than 2% of the total labeling of a rat hepatocyte . Furthermore, 60% of labeled pyruvate carboxylase molecules within a mitochondrion were found adjacent to the matrix side of the inner mitochondrial membrane . In contrast, in S . cerevisiae, pyruvate carboxylase was found exclusively in the cytosol.

Mol Microbiol, 1991 Oct, 5(10), 2331 - 8
Yeast dsRNA viruses: replication and killer phenotypes; Tipper DJ et al.; The cytoplasmic L-A dsRNA virus of Saccharomyces cerevisiae consists of a 4.5 kb dsRNA and the two gene products it encodes; the capsid (cap) and at least one copy of the capsid-polymerase (cap-pol) fusion protein . Virion cap-pol catalyses transcription of the plus (sense)-strand; this is extruded from the virus and serves as messenger for synthesis of cap and cap-pol . Nascent cap-pol binds to a specific domain in the plus strand to initiate encapsidation and then catalyses minus-strand synthesis to complete the replication cycle . Products of at least three host genes are required for replication, and virus copy number is kept at tolerable levels by the SKI antivirus system . S . cerevisiae killer viruses are satellite dsRNAs that use a similar encapsidation domain to parasitize the L-A replication machinery . They encode precursors of secreted polypeptide toxins and immunity (specific resistance) determinants and are self-selecting . Three unique killer types, K1, K2 and K28, are currently recognized . They are distinguished by an absence of cross-immunity and by toxin properties and lethal mechanisms; while K1 and K2 toxins bind to cell-wall glucan and disrupt membrane functions, K28 toxin binds to mannoprotein and causes inhibition of DNA synthesis.

Cell, 1991 Sep 20, 66(6), 1279 - 87
Modifiers of position effect are shared between telomeric and silent mating-type loci in S . cerevisiae; Aparicio OM et al.; Genes placed near telomeres in S . cerevisiae succumb to position-effect variegation . SIR2, SIR3, SIR4, NAT1, ARD1, and HHF2 (histone H4) were identified as modifiers of the position effect at telomeres, since transcriptional repression near telomeres was no longer observed when any of the modifier genes were mutated . These genes, in addition to SIR1, have previously been shown to repress transcription at the silent mating loci, HML and HMR . However, there were differences between transcriptional silencing at telomeres and the HM loci, as demonstrated by suppressor analysis and the lack of involvement of SIR1 in telomeric silencing . These findings provide insights into telomeric structure and function that are likely to apply to many eukaryotes . In addition, the distinctions between telomeres and the HM loci suggest a hierarchy of chromosomal silencing in S . cerevisiae.

Cell, 1991 Sep 20, 66(6), 1239 - 56
Temporal comparison of recombination and synaptonemal complex formation during meiosis in S . cerevisiae; Padmore R et al.; In synchronous cultures of S . cerevisiae undergoing meiosis, an early event in the meiotic recombination pathway, site-specific double strand breaks (DSBs), occurs early in prophase, in some instances well before tripartite synaptonemal complex (SC) begins to form . This observation, together with previous results, supports the view that events involving DSBs are required for SC formation . We discuss the possibility that the mitotic pathway for recombinational repair of DSBs served as the primordial mechanism for connecting homologous chromosomes during the evolution of meiosis . DSBs disappear during the period when tripartite SC structure is forming and elongating (zygotene); presumably, they are converted to another type of recombination intermediate . Neither DSBs nor mature recombinant molecules are present when SCs are full length (pachytene) . Mature reciprocally recombinant molecules arise at the end of or just after pachytene . We suggest that the SC might coordinate recombinant maturation with other events of meiosis.

Cell, 1991 Sep 20, 66(6), 1217 - 28
Human cyclin E, a new cyclin that interacts with two members of the CDC2 gene family; Koff A et al.; A new human cyclin, named cyclin E, was isolated by complementation of a triple cln deletion in S . cerevisiae . Cyclin E showed genetic interactions with the CDC28 gene, suggesting that it functioned at START by interacting with the CDC28 protein . Two human genes were identified that could interact with cyclin E to perform START in yeast containing a cdc28 mutation . One was CDC2-HS, and the second was the human homolog of Xenopus CDK2 . Cyclin E produced in E . coli bound and activated the CDC2 protein in extracts from human G1 cells, and antibodies against cyclin E immunoprecipitated a histone H1 kinase from HeLa cells . The interactions between cyclin E and CDC2, or CDK2, may be important at the G1 to S transition in human cells.

Cell, 1991 Sep 20, 66(6), 1197 - 206
Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast; Lew DJ et al.; We have isolated a number of cDNAs derived from human mRNAs that are able to substitute for G1 cyclin genes in S . cerevisiae . Several of these encode human cyclins A, B1, and B2 . Three novel genes have been identified, which we call cyclins C, D, and E . The novel proteins are sufficiently distantly related to the other members of the cyclin family and to each other as to constitute three new classes of cyclins . Cyclin C and E mRNAs accumulate periodically through the cell cycle, peaking at different times in G1.

Biochem Pharmacol, 1991 Sep 12, 42(7), 1367 - 72
Heterologous expression of human microsomal epoxide hydrolase in Saccharomyces cerevisiae . Study of the valpromide-carbamazepine epoxide interaction; Eugster HP et al.; A cDNA of human microsomal epoxide hydrolase (hmEH) was constitutively and inducibly expressed in Saccharomyces cerevisiae . The heterologous enzyme was located mainly in the microsomal fraction of yeast cells . Yeast microsomes containing hmEH exerted styrene oxide hydrolase activity (Km = 300 microM; Vmax = 22 nmol/mg min) as well as carbamazepine epoxide hydrolase activity . The hmEH catalysed exclusively the formation of carbamazepine-10,11-transdihydrodiol, since no carbamazepine-10,11-cisdihydrodiol was detected . Inhibition studies using these microsomes revealed unequivocally hmEH as the target for inhibition by the antiepileptic drug valpromide . A Ki value of 27 microM was determined for the inhibitor valpromide with styrene oxide as substrate . For carbamazepine epoxide, a Ki value of 8.6 microM was obtained, which is well in line with data published for hmEH determined with human liver microsomes . Our results demonstrate the potential of heterologous gene expression in S . cerevisiae and its application to the in vitro study of pharmacological and toxicological problems.

Biochim Biophys Acta, 1991 Sep 11, 1085(2), 217 - 22
Isolation and characterization of carnitine acetyltransferase from S . cerevisiae; Kispal G et al.; Carnitine acetyltransferase was isolated from yeast Saccharomyces cerevisiae with an apparent molecular weight of 400,000 . The enzyme contains identical subunits of 65,000 Da . The Km values of the isolated enzyme for acetyl-CoA and for carnitine were 17.7 microM and 180 microM, respectively . Carnitine acetyltransferase is an inducible enzyme, a 15-fold increase in the enzyme activity was found when the cells were grown on glycerol instead of glucose . Carnitine acetyltransferase, similarly to citrate synthase, has a double localization (approx . 80% of the enzyme is mitochondrial), while acetyl-CoA synthetase was found only in the cytosol . In the mitochondria carnitine acetyltransferase is located in the matrix space . The incorporation of 14C into CO2 and in lipids showed a similar ratio, 2.9 and 2.6, when the substrate was {1-14C}acetate and {1-14C}acetylcarnitine, respectively . Based on these results carnitine acetyltransferase can be considered as an enzyme necessary for acetate metabolism by transporting the activated acetyl group from the cytosol into the mitochondrial matrix.

Mutat Res, 1991 Sep-Oct, 250(1-2), 229 - 37
Genotoxic effects of ethylene oxide and propylene oxide: a comparative study; Agurell E et al.; The two alkylating agents ethylene oxide (EO) and propylene oxide (PO) were compared for genotoxic effectiveness in various test systems . The study was undertaken partly to shed light on the difference between the compounds found after chronic exposure of monkeys (Lynch et al., 1984) where EO but not PO was able to induce SCE and chromosomal aberrations . In the present study EO was found to be 5-10 times more effective than PO with respect to gene conversion and reverse mutation in Saccharomyces cerevisiae D7 and sister-chromatid conversion in S . cerevisiae RS112 . In contrast, the abilities of the two compounds to induce point mutation in S . typhimurium strains and SCE in human lymphocytes were approximately equal . One possible cause of EO being more effective than PO in certain respects, discussed on the basis of inference from earlier studies, is an expected difference in ability to cause strand breaks via alkylation of DNA-phosphate groups.

Eur J Biochem, 1991 Sep 1, 200(2), 487 - 93
Identification and characterization of a Saccharomyces cerevisiae gene (PAR1) conferring resistance to iron chelators; Schnell N et al.; o-Phenanthroline (1,10-phenanthroline) is a chemical known to chelate iron and other transition metal ions . This compound was added to solid yeast media to reduce the concentration of biologically available iron . Other essential divalent cations, like Zn2+ or Cu2+, which could also be bound, were supplemented . Growth of wild-type yeast strains was totally inhibited at specific concentrations of the chelator . However, several cells containing plasmids of a multicopy vector genomic library of S . cerevisiae could be selected by growth on these media . All of the resistant clones carried a single additional gene, PAR1 on their multicopy plasmids . Plasmid-directed overexpression of PAR1 increased the resistance of transformants to o-phenanthroline and additionally conferred resistance to 1-nitroso-2-naphthol, an iron(III)-binding molecule with different coordinating ligands . By supplementing the o-phenanthroline-containing media with several different metal ions, it could be proved that the selection plates really caused a specific iron limitation . These observations clearly demonstrated that the overexpressed PAR1 gene enables the cell to compete with iron-chelating organic molecules . PAR1 null mutants, constructed by insertion of the LEU2 gene into the open reading frame, showed a remarkable phenotype: they did not grow on slightly alkaline buffered media (pH greater than 7) and became hypersensitive to oxidative stress by hydrogen peroxide . Of several heavy metal ions, such as Fe3+, Fe2+, Co2+, Ni2+, Cu2+ and Zn2+, tested for supplementation of the alkaline growth deficiency, only iron, either added in the ferrous or ferric form, was able to restore cellular growth . It can be concluded from the DNA sequence that PAR1 encodes a highly acidic protein of 650 residues with mostly hydrophilic character . Some interesting repetitive amino acid motifs, such as (Asp-Asn)4 or Cys-Ser-Glu, may act as metal-binding sites . The possible role of PAR1 is discussed.

Genes Dev, 1991 Sep, 5(9), 1589 - 600
Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase; Brill SJ et al.; Replication factor-A (RF-A) is a three-subunit protein complex originally purified from human cells as an essential component for SV40 DNA replication in vitro . We have previously identified a functionally homologous three-subunit protein complex from the yeast Saccharomyces cerevisiae . Here we report the cloning and characterization of the genes encoding RF-A from S . cerevisiae . Each of the three subunits is encoded by a single essential gene . Cells carrying null mutations in any of the three genes arrest as budded and multiply budded cells . All three genes are expressed in a cell-cycle-dependent manner; the mRNA for each subunit peaks at the G1/S-phase boundary . A comparison of protein sequences indicates that the human p34 subunit is 29% identical to the corresponding RFA2 gene product . However, expression of the human protein fails to rescue the rfa2::TRP1 disruption.

Mol Microbiol, 1991 Sep, 5(9), 2143 - 52
Molecular cloning, characterization and analysis of the regulation of the ARO2 gene, encoding chorismate synthase, of Saccharomyces cerevisiae; Jones DG et al.; We describe here the cloning, characterization and analysis of the regulation of the ARO2 gene of Saccharomyces cerevisiae, the first reported study of a eukaryotic gene encoding chorismate synthase (E.C . 4.6.1.4) . The gene contains an ORF of 1128 bp, encoding a protein with a calculated molecular mass of 40.8 kDa . ARO2 is regulated under the 'general control system' for amino acid biosynthesis by the transcriptional activator GCN4 which binds in vitro at three sites within the ARO2 promoter . The ARO2 gene product is highly similar to its Escherichia coli counterpart, with a 47% identity distributed over the entire length of the peptide . We therefore suggest that the S . cerevisiae chorismate synthase, in contrast to the Neurospora crassa enzyme, but like other chorismate synthases, is a monofunctional peptide, solely possessing chorismate synthase activity.

Chromosoma, 1991 Sep, 100(8), 519 - 23
Ultrastructural localization of nucleic acid sequences in Saccharomyces cerevisiae nucleoli; Dvorkin N et al.; The putative nucleolus in Saccharomyces cerevisiae is visible in electron micrographs as a darkly stained, crescent-shaped structure associated with the nuclear envelope . The haploid yeast genome contains 100-200 tandem copies of a 9.1 kb ribosomal DNA (rDNA) repeat predicted to reside in this structure . We combined in situ hybridization of non-isotopically labeled probes to isolated S . cerevisiae nuclei with immunogold detection to localize rDNA and rDNA precursor sequences in nuclei at the electron microscope (EM) level . Gold particles are restricted to defined regions of nuclei which appear more electron dense than the bulk of the nucleus and which generally exhibit the crescent shape typical of the structure thought to be the nucleolus . In addition, snR17, the yeast homolog of mammalian U3, a nucleolar-restricted small nuclear RNA (snRNA), was localized to the same electron dense region of the nucleus . These data, in conjunction with published immunofluorescent localizations of nucleolar-associated antigens, provide definitive proof that the dense crescent is the nucleolus . Finally, the technique described is applicable to probing nuclear organization in a genetically manipulable system.

Mol Cell Biol, 1991 Sep, 11(9), 4537 - 44
Cellular role of yeast Apn1 apurinic endonuclease/3'-diesterase: repair of oxidative and alkylation DNA damage and control of spontaneous mutation; Ramotar D et al.; The APN1 gene of Saccharomyces cerevisiae encodes the major apurinic/apyrimidinic endonuclease and 3'-repair DNA diesterase in yeast cell extracts . The Apn1 protein is a homolog of Escherichia coli endonuclease IV, which functions in the repair of some oxidative and alkylation damages in that organism . We show here that yeast strains lacking Apn1 (generated by targeted gene disruption or deletion-replacement) are hypersensitive to both oxidative (hydrogen peroxide and t-butylhydroperoxide) and alkylating (methyl- and ethylmethane sulfonate) agents that damage DNA . These cellular hypersensitivities are correlated with the accumulation of unrepaired damages in the chromosomal DNA of apn1 mutant yeast cells . Hydrogen peroxide-treated APN1+ but not apn1 mutant cells regenerate high-molecular-weight DNA efficiently after the treatment . The DNA strand breaks that accumulate in the Apn1-deficient mutant contain lesions that block the action of DNA polymerase but can be removed in vitro by purified Apn1 . An analogous result with DNA from methylmethane sulfonate-treated cells corresponded to the accumulation of unrepaired DNA apurinic sites in the apn1 mutant cells . The rate of spontaneous mutation in apn1 mutant S . cerevisiae was 6- to 12-fold higher than that measured for wild-type yeast cells . This increase indicates that under normal growth conditions, the production of DNA damages that are targets for Apn1 is substantial and that such lesions can be mutagenic when left unrepaired.

Curr Genet, 1991 Sep, 20(4), 265 - 7
The CDC8 gene product is required for transformation with episomal and integrative plasmids in Saccharomyces cerevisiae; Lecka-Czernik B et al.; The product of the yeast CDC8 gene (thymidylate kinase), which is required for chromosomal, mitochondrial and 2 mu plasmid replication, also participates in plasmid transformation processes in S . cerevisiae . The thermosensitive cdc8-1 mutant strain was transformed with episomal pDQ9 and integrative pDQ9-1 plasmids both of which carry the CDC8 gene . The results suggest that thymidylate kinase is essential for the expression of genes carried on transforming episomal plasmid DNA (probably through its replication) and is also essential for homologous recombination between chromosomal and linearized integrative plasmid DNA.

Arch Biochem Biophys, 1991 Sep, 289(2), 281 - 8
Characterization of a soluble inorganic pyrophosphatase from Microcystis aeruginosa and preparation of its antibody; Kang CB et al.; A soluble inorganic pyrophosphatase was isolated from a crude extract of Microcystis aeruginosa by adsorption chromatography . The enzyme was purified to homogeneity as judged by sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide gel electrophoresis and N-terminal amino acid analysis . The molecular mass was estimated to be 80 kDa by gel filtration chromatography, 87 kDa by nondenaturing polyacrylamide gel electrophoresis, and 28 kDa by SDS-polyacrylamide gel electrophoresis . The enzyme has an isoelectric point of 4.5, which is similar to the pI values reported for other soluble inorganic pyrophosphatases . The sequence of 29 N-terminal amino acids was determined; only 4 of these amino acids are identical to those in the sequence of Saccharomyces cerevisiae inorganic pyrophosphatase . M . aeruginosa inorganic pyrophosphatase is a Mg(2+)-dependent enzyme exhibiting a pH optimum of around 7.5 . Its KM value for inorganic pyrophosphate was estimated to be 1.30 mM . A specific antibody was raised in chicken to M . aeruginosa inorganic pyrophosphatase . No immunological cross-reactivity was seen when Western blots of partially purified S . cerevisiae or Escherichia coli inorganic pyrophosphatase were probed with the antibody.

J Biol Chem, 1991 Aug 25, 266(24), 15602 - 7
Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled; Farkas I et al.; In previous work, we identified a Saccharomyces cerevisiae glycogen synthase gene, GSY1, which codes for an 85-kDa polypeptide present in purified yeast glycogen synthase (Farkas, I., Hardy, T.A., DePaoli-Roach, A.A., and Roach, P.J . (1990) J . Biol . Chem . 265, 20879-20886) . We have now cloned another gene, GSY2, which encodes a second S . cerevisiae glycogen synthase . The GSY2 sequence predicts a protein of 704 residues, molecular weight 79,963, with 80% identity to the protein encoded by GSY1 . Amino acid sequences obtained from a second polypeptide of 77 kDa present in yeast glycogen synthase preparations matched those predicted by GSY2 . GSY1 resides on chromosome VI, and GSY2 is located on chromosome XII . Disruption of the GSY1 gene produced a strain retaining about 85% of wild type glycogen synthase activity at stationary phase, while disruption of the GSY2 gene yielded a strain with only about 10% of wild type enzyme activity . The level of glycogen synthase activity in yeast cells disrupted for GSY1 increased in stationary phase, whereas the activity remained at a constant low level in cells disrupted for GSY2 . Disruption of both genes resulted in a viable haploid that totally lacked glycogen synthase activity and was defective in glycogen deposition . In conclusion, yeast expresses two forms of glycogen synthase with activity levels that behave differently in the growth cycle . The GSY2 gene product appears to be the predominant glycogen synthase with activity linked to nutrient depletion.

J Biol Chem, 1991 Aug 25, 266(24), 15890 - 4
RAD6 gene product of Saccharomyces cerevisiae requires a putative ubiquitin protein ligase (E3) for the ubiquitination of certain proteins; Sharon G et al.; The RAD6 (UBC2) gene of Saccharomyces cerevisiae which is involved in DNA repair, induced mutagenesis, and sporulation, encodes a ubiquitin-conjugating enzyme (E2) . Since the RAD6 gene product can transfer ubiquitin directly to histones in vitro without the participation of a ubiquitin protein ligase (E3), it has been suggested that in vivo it also acts by the unassisted conjugation of ubiquitin to histones or to other target proteins . Here we show that the RAD6 protein can ligate ubiquitin in vitro to a hitherto unknown set of exogenous target proteins (alpha-, beta-, and kappa-casein and beta-lactoglobulin) when supplemented by a putative ubiquitin protein ligase (E3-R) from S . cerevisiae . RAD6 supplemented with E3-R ligates 1 or, sometimes, 2 ubiquitin molecules to the target protein molecule . UBC3 (CDC34) protein in the presence of E3-R has barely detectable activity on the non-histone substrates . Other ubiquitin-conjugating enzymes tested (products of the UBC1 and UBC4 genes) do not cooperate with E3-R in conjugating ubiquitin to the same substrates . Thus, E3-R apparently interacts selectively with RAD6 protein . These findings suggest that some of the in vivo activities of the RAD6 gene may involve E3-R.

Cell, 1991 Aug 23, 66(4), 743 - 58
The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S . cerevisiae transcription factor SWI5; Moll T et al.; The intracellular localization of the S . cerevisiae transcription factor SWI5 is cell cycle dependent . The protein is nuclear in G1 cells but cytoplasmic in S, G2, and M phase cells . We have identified SWI5's nuclear localization signal (NLS) and show that it can confer cell cycle-dependent nuclear entry to a heterologous protein . Located within or close to the NLS are three serine residues, mutation of which results in constitutive nuclear entry . These residues are phosphorylated in a cell cycle-dependent manner in vivo, being phosphorylated when SWI5 is in the cytoplasm and dephosphorylated when it is in the nucleus . As all three serines are phosphorylated by purified CDC28-dependent H1 kinase activity in vitro, we propose a model in which the CDC28 kinase acts directly to control nuclear entry of SWI5.

Biochem Biophys Res Commun, 1991 Aug 15, 178(3), 1064 - 71
Different substrate specificities of lanosterol 14a-demethylase (P-45014DM) of Saccharomyces cerevisiae and rat liver for 24-methylene-24,25-dihydrolanosterol and 24,25-dihydrolanosterol; Aoyama Y et al.; The purified lanosterol 14a-demethylase (P-45014DM) of S . cerevisiae catalyzed the 14a-demethylation of 24-methylene-24,25-dihydrolanosterol (24-methylenelanost-8-en-3 beta-ol, 24-methylene-DHL), the natural substrate of the demethylase of filamentous fungi, as well as its natural substrate, lanosterol . Lanosterol 14a-demethylase of rat liver microsomes also catalyzed the 14a-demethylation of 24-methylene-DHL, but the activity was considerably lower than that for lanosterol . The activity of the rat liver enzyme for 24-methylene-DHL was also lower than that for 24,25-dihydrolanosterol (DHL), while the activity of yeast P-45014DM for 24-methylene-DHL was considerably higher than that for DHL . Since 24-substituted sterols are not found in mammals and DHL is not an intermediate of ergosterol biosynthesis by yeast, above-mentioned different substrate specificities between the yeast and the mammalian 14a-demethylases may reflect certain evolutional alteration in their active sites in relation to the difference in their sterol biosynthetic pathways.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7434 - 7
Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast; Hamilton AJ et al.; The ethylene-forming enzyme (EFE), which catalyzes the last step in the biosynthesis of the plant hormone ethylene, has never been purified and no molecular probes are available . Recently, a putative cDNA clone for tomato EFE (pTOM13) has been identified by inhibiting ethylene synthesis with an antisense gene expressed in transgenic plants . A direct test of its function has been made by expression of a pTOM13 gene in Saccharomyces cerevisiae . After cloning artefacts were discovered in the 5' region of the cDNA, a corrected cDNA (pRC13) was created by the fusion of the 5' end of a genomic clone to the 3' end of the cDNA and expressed in S . cerevisiae . Cultures of transformed yeast converted 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, whereas control cells did not . This EFE activity displays similar characteristics to EFE found in plant tissue: it converts the trans isomer of the ACC analogue 1-amino-2-ethylcyclopropane-1-carboxylic acid to 1-butene in preference to the cis isomer, and it is strongly inhibited by cobaltous ions and 1,10-phenanthroline . Furthermore, information gained from the activity of effectors on yeast EFE activity supports the hypothesis that EFE is one of a group of hydroxylase enzymes.

J Biol Chem, 1991 Aug 15, 266(23), 15120 - 7
Glycoprotein biosynthesis in Saccharomyces cerevisiae . Isolation and characterization of the gene encoding a specific processing alpha-mannosidase; Camirand A et al.; We have isolated the gene from Saccharomyces cerevisiae encoding an alpha-mannosidase of unique specificity which catalyzes the removal of one mannose residue from Man9GlcNAc to produce a single isomer of Man8GlcNAc (Jelinek-Kelly, S., and Herscovics, A . (1988) J . Biol . Chem . 263, 14757-14763) . Amino acid sequence information was obtained and corresponding degenerate oligonucleotide primers were synthesized for polymerase chain reactions on yeast genomic DNA . The labeled polymerase chain reaction products were used to screen a S . cerevisiae genomic library in YEp24, and positive clones of different lengths with similar restriction maps were isolated . A 4.6-kilobase fragment which hybridized with the probes was sequenced . It contained a 1650-base pair open reading frame encoding peptide sequences corresponding to the amino acid sequences of the purified alpha-mannosidase . The gene, designated MNS1, encodes a 549-amino acid polypeptide of calculated molecular size 63,017 Da produced by an mRNA species of approximately 1.7 kilobases . The protein possesses a putative noncleavable signal sequence near its N-terminal region which probably acts as a transmembrane domain . It has three potential N-glycosylation sites and a calcium-binding consensus sequence . Its amino acid sequence is homologous to the recently isolated cDNA from rabbit liver alpha-1,2 mannosidase which can transform Man9GlcNAc to Man5GlcNAc (Moremen, K . W., Schutzbach, J . S., Forsee, W . T., Neame, P., Bishoff, J., Lodish, H . F., and Robbins, P . W . (1990) Glycoconjugate J . 7, 401) . Overexpression of the MNS1 gene caused an 8-10-fold increase in specific alpha-mannosidase activity . Disruption of the MNS1 gene resulted in undetectable specific alpha-mannosidase activity but no apparent effect on growth . These results demonstrate that MNS1 is the structural gene for the specific alpha-mannosidase and that its activity is not essential for viability.

Cell, 1991 Aug 9, 66(3), 497 - 505
Evidence for a functional link between profilin and CAP in the yeast S . cerevisiae; Vojtek A et al.; CAP is a component of the S . cerevisiae adenylyl cyclase complex . The N-terminal domain is required for cellular RAS responsiveness . Loss of the C-terminal domain is associated with morphological and nutritional defects . Here we report that cap- cells bud randomly and are defective in actin distribution . The morphological and nutritional defects associated with loss of the CAP C-terminal domain are suppressed by over-expression of PFY, the gene encoding profilin, an actin- and polyphosphoinositide-binding protein . The phenotype of cells lacking PFY resembles that of cells lacking the CAP C-terminal domain . Study of mutated yeast profilins and profilins from Acanthamoeba suggests that the ability of profilin to suppress cap- cells is dependent upon a property other than, or in addition to, its ability to bind actin . This property may be its ability to bind polyphosphoinositides . We propose that CAP and profilin provide a link between growth signals and remodeling of the cellular cytoskeleton.

Cell, 1991 Aug 9, 66(3), 507 - 17
S . cerevisiae genes required for cell cycle arrest in response to loss of microtubule function; Hoyt MA et al.; We have identified mutant strains of S . cerevisiae that fail to properly arrest their cell cycles at mitosis in response to the loss of microtubule function . New bud emergence and DNA replication (but not cytokinesis) occur with high efficiency in the mutants under conditions that inhibit these events in wild-type cells . The inability to halt cell cycle progression is specific for impaired microtubule function; the mutants respond normally to other cell cycle-blocking treatments . Under microtubule-disrupting conditions, the mutants neither achieve nor maintain the high level of histone H1 kinase activity characteristic of wild-type cells . Our studies have defined three genes required for normal cell cycle arrest . These findings are consistent with the existence of a surveillance system that halts the cell cycle in response to microtubule perturbation.

J Biol Chem, 1991 Aug 5, 266(22), 14603 - 10
Sequence dependence of protein isoprenylation; Moores SL et al.; Several proteins have been shown to be post-translationally modified on a specific C-terminal cysteine residue by either of two isoprenoid biosynthetic pathway metabolites, farnesyl diphosphate or geranylgeranyl diphosphate . Three enzymes responsible for protein isoprenylation were resolved chromatographically from the cytosolic fraction of bovine brain: a farnesyl-protein transferase (FTase), which modified the cell-transforming Ras protein, and two geranyl-geranyl-protein transferases, one (GGTase-I) which modified a chimeric Ras having the C-terminal amino acid sequence of the gamma-6 subunit of heterotrimeric GTP-binding proteins, and the other (GGTase-II) which modified the Saccharomyces cerevisiae secretory GTPase protein YPT1 . In a S . cerevisiae strain lacking FTase activity (ram1), both GGTases were detected at wild-type levels . In a ram2 S . cerevisiae strain devoid of FTase activity, GGTase-I activity was reduced by 67%, suggesting that GGTase-I and FTase activities derive from different enzymes but may share a common genetic feature . For the FTase and the GGTase-I activities, the C-terminal amino acid sequence of the protein substrate, the CAAX box, appeared to contain all the critical determinants for interaction with the transferase . In fact, tetrapeptides with amino acid sequences identical to the C-terminal sequences of the protein substrates for FTase or GGTase-I competed for protein isoprenylation by acting as alternative substrates . Changes in the CAAX amino acid sequence of protein substrates markedly altered their ability to serve as substrates for both FTase and GGTase-I . In addition, it appeared that FTase and GGTase-I had complementary affinities for CAAX protein substrates; that is, CAAX proteins that were good substrates for FTase were, in general, poor substrates for GGTase-I, and vice versa . In particular, a leucine residue at the C terminus influenced whether a CAAX protein was either farnesylated or geranylgeranylated preferentially . The YPT1 C terminus peptide, TGGGCC, did not compete or serve as a substrate for GGTase-II, indicating that the interaction between GGTase-II and YPT1 appeared to depend on more than the 6 C-terminal residues of the protein substrate sequence . These results identify three different isoprenyl-protein transferases that are each selective for their isoprenoid and protein substrates.

Mol Cell Biol, 1991 Aug, 11(8), 4196 - 206
The AGA1 product is involved in cell surface attachment of the Saccharomyces cerevisiae cell adhesion glycoprotein a-agglutinin; Roy A et al.; Saccharomyces cerevisiae a and alpha cells express the complementary cell surface glycoproteins a-agglutinin and alpha-agglutinin, respectively, which interact with one another to promote cellular aggregation during mating . Treatment of S . cerevisiae a cells with reducing agents releases the binding subunit of a-agglutinin, which has been purified and characterized; little biochemical information on the overall structure of a-agglutinin is available . To characterise a-agglutinin structure and function, we have used a genetic approach to clone an a-agglutinin structural gene (AGAI) . Mutants with a-specific agglutination defects were isolated, the majority of which fell into a single complementation group, called aga1 . The aga1 mutants showed wild-type pheromone production and response, efficient mating on solid medium, and a mating defect in liquid medium; these phenotypes are characteristic of agglutinin mutants . The AGA1 gene was cloned by complementation; the gene sequence indicated that it could encode a protein of 725 amino acids with high serine and threonine content, a putative N-terminal signal sequence, and a C-terminal hydrophobic sequence similar to signals for the attachment to glycosyl phosphatidylinositol anchors . Active a-agglutinin binding subunit is secreted by aga1 mutants, indicating that AGA1 is involved in cells surface attachment of a-agglutinin . This result suggests that AGA1 encodes a protein with functional similarity to the core subunits of a-agglutinin analogs from other budding yeasts . Unexpectedly, the AGA1 transcript was expressed and induced by pheromone in both a and alpha cells, suggesting that the a-specific expression of active a-agglutinin results only from a-specific regulation of the a-agglutinin binding subunit.

EMBO J, 1991 Aug, 10(8), 2215 - 21
The ribosomal protein L2 in S . cerevisiae controls the level of accumulation of its own mRNA; Presutti C et al.; The expression of the yeast L2 r-protein gene is controlled at the level of mRNA accumulation . The product of the gene appears to participate in this regulation by an autogenous feedback mechanism . This control does not operate at the level of transcription but instead affects L2 mRNA accumulation . This autogenous regulation of mRNA accumulation provides an interesting analogy to the autogenous translational regulation of r-proteins in Escherichia coli.

EMBO J, 1991 Aug, 10(8), 2187 - 93
Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N-end-recognizing E3 enzyme; Sung P et al.; The RAD6 gene of Saccharomyces cerevisiae encodes a 20 kd ubiquitin conjugating (E2) enzyme that is required for DNA repair, DNA damage-induced mutagenesis, and sporulation . Here, we demonstrate a novel activity of RAD6 protein--its ability to mediate protein degradation dependent on the N-end-recognizing ubiquitin protein ligase (E3) . In reaction mixtures containing E1, E3 and the ubiquitin specific protease from rabbit reticulocytes, RAD6 is as effective as mammalian E214k in E3 dependent ubiquitin--protein conjugate formation and subsequent protein degradation . The ubiquitin conjugating activity of RAD6 is required for these reactions as indicated by the ineffectiveness of the rad6 Ala88 and rad6 Val88 mutant proteins, which lack the ability to form a thioester adduct with ubiquitin and therefore do not conjugate ubiquitin to substrates . We also show that the highly acidic carboxyl-terminus of RAD6 is dispensable for the interaction with E3, and that purified S . cerevisiae E2(30k), product of the UBC1 gene, does not function with E3 . These findings demonstrate a specific interaction between RAD6 and E3, and highlight the strong conservation of the ubiquitin conjugating system in eukaryotes . We suggest a function for RAD6 mediated E3 dependent protein degradation in sporulation, and discuss the possible role of this activity during vegetative growth.

EMBO J, 1991 Aug, 10(8), 2179 - 86
Primary sequence and biological functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA repair methyltransferase gene; Xiao W et al.; We previously identified and characterized biochemically an O6-methylguanine (O6MeG) DNA repair methyltransferase (MTase) in the yeast Saccharomyces cerevisiae and showed that it recognizes both O6MeG and O4-methylthymine (O4MeT) in vitro . Here we characterize the cloned S . cerevisiae O6MeG DNA MTase gene (MGT1) and determine its in vivo role in protecting yeast from DNA alkylation damage . We isolated a yeast DNA fragment that suppressed alkylation-induced killing and mutation in Escherichia coli ada ogt MTase deficient mutants and produced in these cells a protein similar to the yeast MTase . The cloned yeast fragment was mapped to chromosome IV and DNA sequencing identified an open reading frame, designated MGT1, which encodes a 188 amino acid protein with a molecular weight of 21,500 daltons . An 88 amino acid stretch of the MGT1 protein displays remarkable homology with four bacterial MTases and the human DNA MTase . S.cerevisiae mutants bearing an insertion in the MGT1 gene lacked DNA MTase activity and were very sensitive to alkylation induced killing and mutation . MGT1 transcript levels are not increased in response to DNA alkylation damage, nor is the MGT1 MTase involved in the regulation of the yeast 3-methyladenine DNA glycosylase gene (MAG) . Expression of the MGT1 gene in E.coli prevented the induction by alkylating agents of both G:C to A:T and A:T to G:C transition mutations indicating that this eukaryotic MTase repairs both O6MeG and O4MeT in vivo.

FEMS Microbiol Lett, 1991 Aug 1, 66(2), 149 - 52
Transformation of the yeast Saccharomyces kluyveri by Saccharomyces cerevisiae-based plasmids; Fujimura H; For the transformation of the yeast Saccharomyces kluyveri, ura3 mutants were obtained by 5-fluoro-orotic acid selection . By utilizing the method based on treatment of intact cells with alkali cations, the ura3 strains of S . kluyveri were transformed by Saccharomyces cerevisiae-based plasmids . In the transformed cells, a S . cerevisiae centromere-based plasmid was stably replicated autonomously . Thus, this system will permit the study of gene expression and its regulation in S . kluyveri in relationship to that in S . cerevisiae.

Eur J Cell Biol, 1991 Aug, 55(2), 336 - 45
Comparison of two cytochromes P-450 from Candida maltosa: primary structures, substrate specificities and effects of their expression in Saccharomyces cerevisiae on the proliferation of the endoplasmic reticulum; Schunck WH et al.; cDNAs were cloned, sequenced and expressed which encode two different cytochrome P-450 forms of the alkane-assimilating yeast Candida maltosa, designated as P-450Cm1 and P-450Cm2 . The amino acid sequences deduced were about 55% identical . Expression in Saccharomyces cerevisiae resulted in the formation of intact microsomal P-450 systems catalyzing the hydroxylation of n-hexadecane and lauric acid with significantly different substrate preferences . A massive proliferation of the endoplasmic reticulum was observed in the S . cerevisiae cells which produced P-450 . Depending on the P-450 form expressed, distinctly organized stacks of paired membranes appeared and occupied considerable areas of the cytoplasm . As shown by immunoelectron microscopy for P-450Cm1, the protein expressed was highly concentrated within these newly formed membrane structures.

Curr Genet, 1991 Aug, 20(3), 189 - 94
Polymorphism within the nuclear and 2 micron genomes of Saccharomyces cerevisiae; Rank GH et al.; Seven strains of bakers' yeast were obtained as a representative sample of the Spanish baking industry . The nuclear genome was monitored for polymorphism by transverse alternating field electrophoresis (TAFE) and restriction maps of 2 micron DNA were produced . All seven strains were uniquely different when evaluated by their total chromosomal lengths whereas only two 2 micron variants were defined . There was no apparent correlation between chromosomal and plasmid polymorphism . The extensive chromosomal polymorphism within one 2 micron DNA type indicates the rapid and relatively recent evolution of the nuclear genome . The hybrid origin (S . cerevisiae-S . monacensis) of lager yeast was critically evaluated by TAFE analysis of S . cerevisiae and S . carlsbergensis chromosomes . The absence of corresponding S . cerevisiae chromosomes III and XIII in S . carlsbergensis argued against the hybrid origin of lager strains . We discuss limitations of the hybrid origin hypothesis of industrial yeasts and propose that the molecular coevolution observed in 2 micron DNA serves as a useful additional mechanism for rationalization of some of the structural polymorphism of the nuclear genome.

J Cell Biol, 1991 Aug, 114(3), 515 - 32
Studies concerning the temporal and genetic control of cell polarity in Saccharomyces cerevisiae; Snyder M et al.; The establishment of cell polarity was examined in the budding yeast, S . cerevisiae . The distribution of a polarized protein, the SPA2 protein, was followed throughout the yeast cell cycle using synchronized cells and cdc mutants . The SPA2 protein localizes to a patch at the presumptive bud site of G1 cells . Later it concentrates at the bud tip in budded cells . At cytokinesis, the SPA2 protein is at the neck between the mother and daughter cells . Analysis of unbudded haploid cells has suggested a series of events that occurs during G1 . The SPA2 patch is established very early in G1, while the spindle pole body residues on the distal side of the nucleus . Later, microtubules emanating from the spindle pole body intersect the SPA2 crescent, and the nucleus probably rotates towards the SPA2 patch . By middle G1, most cells contain the SPB on the side of the nucleus proximal to the SPA2 patch, and a long extranuclear microtubule bundle intersects this patch . We suggest that a microtubule capture site exists in the SPA2 staining region that stabilizes the long microtubule bundle; this capture site may be responsible for rotation of the nucleus . Cells containing a polarized distribution of the SPA2 protein also possess a polarized distribution of actin spots in the same region, although the actin staining is much more diffuse . Moreover, cdc4 mutants, which form multiple buds at the restrictive temperature, exhibit simultaneous staining of the SPA2 protein and actin spots in a subset of the bud tips . spa2 mutants contain a polarized distribution of actin spots, and act1-1 and act1-2 mutants often contain a polarized distribution of the SPA2 protein suggesting that the SPA2 protein is not required for localization of the actin spots and the actin spots are not required for localization of the SPA2 protein . cdc24 mutants, which fail to form buds at the restrictive temperature, fail to exhibit polarized localization of the SPA2 protein and actin spots, indicating that the CDC24 protein is directly or indirectly responsible for controlling the polarity of these proteins . Based on the cell cycle distribution of the SPA2 protein, a "cytokinesis tag" model is proposed to explain the mechanism of the non-random positioning of bud sites in haploid yeast cells.

Semin Cell Biol, 1991 Aug, 2(4), 205 - 12
Saccharomyces cerevisiae cell cycle: cdc28 and the G1 cyclins; Futcher AB; Two families of cyclin-like proteins have been found in S . cerevisiae . The clb proteins are the mitotic cyclins . The cln proteins provide an essential function, are required for the G1/S transition, and appear to be rate-limiting for START, but have no obvious role elsewhere in the cycle . The cln proteins are unstable; they form complexes with cdc28; the complexes have protein kinase activity; and at least one of the clns oscillates in abundance through the cell cycle . The action of the cln cyclins at START suggests that they may be 'G1 cyclins'.

Yeast, 1991 Aug-Sep, 7(6), 609 - 15
A family of low and high copy replicative, integrative and single-stranded S . cerevisiae/E . coli shuttle vectors; Bonneaud N et al.; We describe a set of replicative, integrative and single-stranded shuttle vectors constructed from the pUC19 plasmid that we use routinely in our experiments . They bear a yeast selectable marker: URA3, TRP1 or LEU2 . Replicative vectors carrying different yeast replication origins have been constructed in order to have plasmids based on the same construction with a high or low copy number per cell and with different mitotic stabilities . All the vectors are small in size, provide a high yield in Escherichia coli and efficiently transform Saccharomyces cerevisiae . These plasmids have many of the unique sites of the pUC19 multicloning region and many of them allow for the screening of plasmids with an insert by alpha-complementation . The nucleotide sequence of each of them is completely known.

Mol Cell Biol, 1991 Aug, 11(8), 4121 - 7
SHI, a new yeast gene affecting the spacing between TATA and transcription initiation sites; Furter-Graves EM et al.; In a genetic selection for Saccharomyces cerevisiae genes involved in transcription start site specification, two mutant genes which restore alcohol dehydrogenase activity to a functionally defective S . pombe ADH gene were recovered . Examination of S . pombe ADH initiation sites showed that mutations in the SHI gene shift the location of the transcription initiation window closer to TATA . The shi mutant also affected initiation site selection for two S . cerevisiae genes that were tested . For H2B mRNA, initiation occurred in the shi mutant at a series of initiation sites located 43 to 80 bp 3' of the histone H2B TATA sequence and at the usual initiation sites 102 and 103 bp downstream of the TATA sequence . Weakly used initiation sites ranging from 51 to 80 bp downstream of the TATA sequence were observed for the S . cerevisiae ADH1 gene in shi strains, in addition to the normal ADH1 initiation sites 89 and 99 bp from the TATA sequence . Restoration of function to the defective S . pombe ADH gene occurs only when this gene contains a TATA sequence; a single-base-pair TATA-to-TAGA change is sufficient to prevent this restoration of function . Genetic mapping placed the SHI locus on the left arm of chromosome VII, 22.3 centimorgans from cyh2; it does not correspond to any previously mapped gene.

Nucleic Acids Res, 1991 Jul 25, 19(14), 3857 - 60
Genetic depletion indicates a late role for U5 snRNP during in vitro spliceosome assembly; Seraphin B et al.; The pre-mRNA splicing pathway is highly conserved from yeast (S . cerevisiae) to mammals . Of the four snRNPs involved in splicing three (U1, U2 and U4/U6) have been shown to be essential for in vitro splicing . To examine the remaining snRNP, we utilized our previously described genetic procedures (Seraphin and Rosbash, 1989) to prepare yeast extracts depleted of U5 snRNP . The results show that U5 snRNP is necessary for both steps of pre- mRNA splicing and for proper spliceosome assembly, i.e., addition of the U4/U5/U6 triple snRNP . The prior steps of U1 and U2 snRNP addition occur normally in the absence of U5 snRNP.

Cell, 1991 Jul 12, 66(1), 149 - 59
A fission yeast B-type cyclin functioning early in the cell cycle; Bueno A et al.; We have cloned a fission yeast gene, cig1+, encoding a 48 kd product that is most similar to cyclin B proteins . The cig1+ protein has a "cyclin box" approximately 40% identical to B-type cyclins of other species, but lacks the "destruction box" required for proteolysis of mitotic cyclins . Deletion of cig1+ had no observable effect on cell viability or progression through G2 or M phase, but instead caused a marked lag in the progression from G1 to S phase . G1 constituted approximately 70% of the cell cycle in cig1 deletion strains, as compared with less than 10% in cig1+ strains . Constitutive cig1+ overexpression was lethal, causing cessation of growth and arrest in G1 . Expression of cig1+ failed to rescue an S . cerevisiae strain lacking CLN Start cyclins . Thus, cig1+ identifies a new class of B-type cyclin acting in G1 or S phase that appears to be functionally distinct from all previously described cyclin proteins.

Nucleic Acids Res, 1991 Jul 11, 19(13), 3701 - 8
Photoaffinity polyamines: sequence-specific interactions with DNA; Xiao L et al.; ANB-spermine is a photoaffinity analog of the naturally-occurring polyamine, acetylspermine . ANB-spermine was used to determine its binding sites on naked double stranded DNA, at the nucleotide level, using a modification of the primer extension technique . A total of 1,275 nucleotides was examined in 5 sequences of DNA from Saccharomyces cerevisiae . Binding sites were non-random . The primary determinant of binding was the presence of a thymidine residue . Secondary determinants appeared to depend on the secondary structure of the DNA, with runs of thymidines providing unusually poor binding sites while TA and, especially, TATA providing the strongest binding sites . The 'TATA element' upstream of the URA3 gene from S . cerevisiae was the strongest binding site . The data indicate that ANB-spermine binding to DNA is a probe for DNA secondary structure and suggest a role for polyamines in regulating the structure of chromatin in vivo.

J Biol Chem, 1991 Jul 5, 266(19), 12772 - 8
Molecular cloning and characterization of W double-stranded RNA, a linear molecule present in Saccharomyces cerevisiae . Identification of its single-stranded RNA form as 20 S RNA; Rodriguez-Cousino N et al.; Most strains of the yeast Saccharomyces cerevisiae harbor a double-stranded RNA (dsRNA) molecule, called W . We obtained W cDNA clones by random priming of denatured W dsRNA followed by reverse transcription . Sequence data of W shows that only one strand ((+)-strand) has coding capacity for a protein with 829 amino acids which spans almost the entire length of the molecule (2.5 kilobases) . Within this protein we found a sequence pattern characteristic of RNA dependent RNA polymerases of (+)-strand and double-stranded RNA viruses . W has no homology with other dsRNAs found in S . cerevisiae, such as L-A, L-BC or M1 . However, a (+)-strand-specific probe for W hybridized with 20-S RNA . Furthermore, W (+)-strands comigrated with 20 S RNA in strand separation gels . These results suggest that 20 S RNA is a (+)-single-stranded RNA form of W dsRNA itself or a closely related molecule.

J Biol Chem, 1991 Jul 5, 266(19), 12406 - 11
Heat shock factor-independent heat control of transcription of the CTT1 gene encoding the cytosolic catalase T of Saccharomyces cerevisiae; Wieser R et al.; Transcription of the Saccharomyces cerevisiae CTT1 gene encoding the cytosolic catalase T has been previously shown to be derepressed by nutrient stress . To investigate whether expression of this gene is also affected by other types of stress, the influence of heat shock on CTT1 expression was studied . The results obtained show that expression of the gene is low at 23 degrees C and is induced rapidly at 37 degrees C . By deletion analysis, a promoter element necessary for high level induction by heat shock was located between base pairs -340 and -364 upstream of the translation start codon . This region was demonstrated to be sufficient for heat shock control by placing it upstream of a S . cerevisiae LEU2-lacZ fusion gene . Mutagenesis of the region showed that the response to heat shock is not mediated by a sequence similar to canonical heat shock elements, but by DNA elements also involved in nutrient control of transcription . Catalase T appears to have a function in protecting yeast cells against oxidative damage under stress conditions . Catalase T-containing strains are less sensitive to exposure to 50 degrees C ("lethal heat shock") than isogenic catalase T-deficient mutants, and catalase T-containing strains pretreated by incubation at 37 degrees C are less sensitive to H2O2 than pretreated catalase-deficient mutants.

Curr Genet, 1991 Jul, 20(1-2), 45 - 52
Molecular cloning and characterization of a Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae; Kinsella BT et al.; The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported . The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S . cerevisiae . The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides . On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C . 3.2.1.20) . It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C) . Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts . A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S . cerevisiae transformants, was developed.

Mol Cell Biol, 1991 Jul, 11(7), 3676 - 81
Role of heat shock transcription factor in yeast metallothionein gene expression; Yang WM et al.; The induction of Saccharomyces cerevisiae metallothionein gene transcription by Cu and Ag is mediated by the ACE1 transcription factor . In an effort to detect additional stimuli and factors that regulate metallothionein gene transcription, we isolated a Cu-resistant suppressor mutant of an ACE1 deletion strain . Even in the absence of metals, the suppressor mutant exhibited high basal levels of metallothionein gene transcription that required upstream promoter sequences . The suppressor gene was cloned, and its predicted product was shown to correspond to yeast heat shock transcription factor with a single-amino-acid substitution in the DNA-binding domain . The mutant heat shock factor bound strongly to metallothionein gene upstream promoter sequences, whereas wild-type heat shock factor interacted weakly with the same region . Heat treatment led to a slight but reproducible induction of metallothionein gene expression in both wild-type and suppressor strains, and Cd induced transcription in the mutant strain . These studies provide evidence for multiple pathways of metallothionein gene transcriptional regulation in S . cerevisiae.

Yeast, 1991 Jul, 7(5), 445 - 54
Analysis of the expression and secretion of the Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae; Kinsella BT et al.; The alpha-glucosidase gene of Candida tsukubaensis is contained within a 3.47 kb BamH1-Mlul fragment which, when introduced into Saccharomyces cerevisiae AH22 on a yeast-Escherichia coli shuttle vector, allows the transformants to utilize maltose as sole carbon source . Thus, the cloned gene confers a dominant selectable phenotype on transformed strains of S . cerevisiae which are otherwise unable to grow in nutrient media containing maltose, dextrin or other alpha-1.4-linked alpha-D-glucopyranosides, specifically hydrolysed by the alpha-glucosidase . The cloned enzyme expressed in yeast is secreted into the extracellular medium in a glycosylated form which accounts for up to 60% of the secreted protein and has a molecular size of 70-80 kilodalton (kDa) . Deglycosylation of the alpha-glucosidase showed that the enzyme is composed of two distinct polypeptides with subunit molecular weights of 63-65 kDa (peptide 1) and 50-52 kDa (peptide 2) . An increase in the level of expression of the alpha-glucosidase by yeast transformants in selective minimal medium was obtained by using a vector with increased copy number containing the leu2-d gene as selectable marker . The alpha-glucosidase gene promoter functions more effectively than the Gall-10 promoter in directing alpha-glucosidase expression in S . cerevisiae . It also directs the expression of high levels of beta-galactosidase activity in yeast when fused to a promoterless E . coli lacZ gene . Expression of the alpha-glucosidase gene under the control of its own promoter is constitutive, orientation dependent and not subject to catabolite repression.

Oncogene, 1991 Jul, 6(7), 1099 - 104
C-myc and the yeast transcription factor PHO4 share a common CACGTG-binding motif; Fisher F et al.; The basic-helix-loop-helix (b-HLH) motif is common to a number of proteins involved in transcriptional regulation and cell-type determination . The b-HLH motif is also present in the S . cerevisiae transcription factor PHO4 which positively regulates the acid phosphatase gene PHO5 . In this report we show that the b-HLH region of PHO4 is sufficient to confer specific DNA-binding to the sequence CACGTG and, by comparison of the basic regions of PHO4 with those of other recently isolated CACGTG-binding proteins, we identify a specific subset of conserved amino acids in the basic region likely to confer DNA-binding specificity . On the basis of these observations we predict successfully the effect of substituting the PHO4 basic region with that from c-myc and show that the chimaeric protein activates transcription from the CACGTG elements present in the PHO5 UAS . From these data it is clear that the myc basic region confers specific binding to the sequence CACGTG.

J Gen Microbiol, 1991 Jul, 137 ( Pt 7), 1701 - 8
Induction of increased thermotolerance in Saccharomyces cerevisiae may be triggered by a mechanism involving intracellular pH; Coote PJ et al.; Incubation of Saccharomyces cerevisiae at sub-lethal temperatures results in an increase in thermotolerance . This process is dependent not only on the sub-lethal temperature but also on the duration of sub-lethal heating . This indicates that the mechanism inducing thermotolerance is a time/temperature dose response . Other factors that induce thermotolerance include exposure to ethanol, sorbic acid and low external pH values . These factors induce thermotolerance after incubation in the presence of protein synthesis inhibitors, and they are all known to affect the intracellular pH (pHi) . The acquisition of increased thermotolerance is minimal with sub-lethal heating under neutral external pH conditions . However, when the external pH is reduced to 4.0 the level of induced thermotolerance increases to a maximum value . Using a specific ATPase inhibitor, diethylstilboestrol (DES), ATPase activity was shown to be essential for the cell to survive heat stress . In addition, measurement of acid efflux, or ATPase activity, revealed that proton pumping from the cell increased by approximately 50% at sublethal temperatures that induce thermotolerance . This work has clearly implicated pHi perturbation as the triggering mechanism conferring thermotolerance on S . cerevisiae.

Mol Cell Biol, 1991 Jul, 11(7), 3419 - 24
The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities; Burd CG et al.; The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae . The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus . We produced different parts of the S . cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro . We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently . Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity . Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV) . We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).

Exp Lung Res, 1991 Jul-Aug, 17(4), 687 - 705
Liposomes and pulmonary alveolar macrophages: functional and morphologic interactions; Gonzalez-Rothi RJ et al.; In vitro toxicity of liposomes and their functional and morphologic interactions with rat pulmonary alveolar macrophage (AMs) were investigated using viability (trypan blue exclusion), phagocytic and killing activity (uptake and digestion of live S . cerevisiae), surface adherence, respiratory burst (nitro-blue tetrazolium reduction), and morphometry (computerized image analysis) as indicators . Liposome stability in physiologic solutions and uptake of liposome-encapsulated carboxyfluorescein (CF) by AMs was assessed by fluorescence spectroscopy and microscopy . Liposomes made from saturated phospholipids and cholesterol were stable, whereas liposomes consisting of unsaturated phospholipids without cholesterol lost 30% to 40% of their content over 24 h . However, CF uptake was highest with unsaturated phospholipid preparations, whereas uptake of the three other formulations was comparable . Although liposome exposure did not affect macrophage viability, a reduction in the number of phagocytizing macrophages to 73% of control was noted after 24-h incubation with the highest lipid concentration tested (10 mumol/ml) . Phagocytic killing was similar under all circumstances observed . The fraction of intracellularly killed yeast ranged from 32% to 42% for both control and experimental samples . An increase in cell surface area from 166.1 +/- 39.9 microns 2 on day O (n = 709) to 196.3 +/- 57.6 microns 2 on day 1 (n = 516) and 211.2 +/- 48.0 microns 2 on day 4 (n = 834) was observed after liposome treatment . The corresponding average cell areas of control samples did not change during the observation period . There was no net cell loss of adherence from monolayers as determined by protein assay . The respiratory burst, indicating generation of intracellular superoxide, was also similar--84% to 92% of experimental and control cells under all conditions showed a strong nitro-blue tetrazolium reduction . In summary, in vitro exposure of AMs to large concentrations of liposomes, although producing an increase in macrophage size, was not associated with aberrant macrophage morphologic features, function, or toxicity for the parameters examined.

Yeast, 1991 Jul, 7(5), 463 - 73
Expression of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Hansenula polymorpha; Fellinger AJ et al.; The methylotrophic yeast Hansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the alpha-galactosidase from the plant Cyamopsis tetragonoloba (guar) . The yeast/Escherichia coli shuttle expression vector used is based on the origin of replication of the endogenous 2 microns plasmid of Saccharomyces cerevisiae and the LEU2 gene of S . cerevisiae for selection in H . polymorpha . In the expression vector, the alpha-galactosidase is controlled by the methanol-regulated promoter from the methanol oxidase gene, MOX, of H . polymorpha . The signal sequence of SUC2 (invertase) from the yeast S . cerevisiae, was used to ensure secretion of the alpha-galactosidase enzyme . After transformation and stabilization, the expression vector was stably integrated in the genome . The active alpha-galactosidase enzyme was efficiently secreted (greater than 85%) and after methanol induction, the expression level was 42 mg/l . Amino-terminal sequencing of the purified alpha-galactosidase enzyme synthesized by H . polymorpha showed that the S . cerevisiae invertase signal sequence was correctly processed by H . polymorpha . The secreted alpha-galactosidase was glycosylated and had a sugar content of 9.5% . The specific activity of the alpha-galactosidase produced by H . polymorpha was 38 U mg-1 compared to 100 U mg-1 for the guar alpha-galactosidase . Deglycosylation of the H . polymorpha alpha-galactosidase restored the specific activity completely.

Cell, 1991 Jun 28, 65(7), 1225 - 31
Functional cloning of BUD5, a CDC25-related gene from S . cerevisiae that can suppress a dominant-negative RAS2 mutant; Powers S et al.; By searching for genes that behave like CDC25 of S . cerevisiae in their ability to counteract a dominant-negative RAS2 mutant in a wild-type RAS-dependent manner, we have isolated a CDC25-like homolog, BUD5 . BUD5 is tightly linked to the MAT locus . Although overexpressed BUD5 cannot substitute for CDC25 function, we present evidence that its gene product can bind to the guanine nucleotide binding-deficient RAS2val19ala22 gene product and thereby counteract its dominant-negative effect . We propose that BUD5 is a member of a family of CDC25-related genes that encode activators of RAS and RAS-like proteins.

Cell, 1991 Jun 28, 65(7), 1213 - 24
Yeast BUD5, encoding a putative GDP-GTP exchange factor, is necessary for bud site selection and interacts with bud formation gene BEM1; Chant J et al.; Cells of the yeast S . cerevisiae choose bud sites in an axial or bipolar spatial pattern depending on their cell type . We have identified a gene, BUD5, that resembles BUD1 and BUD2 in being required for both patterns; bud5- mutants also exhibit random budding in all cell types . The BUD5 nucleotide sequence predicts a protein of 538 amino acids that has similarity to the S . cerevisiae CDC25 product, an activator of RAS proteins that catalyzes GDP-GTP exchange . Two potential targets of BUD5 are known: BUD1 (RSR1) and CDC42, proteins involved in bud site selection and bud formation, respectively, that have extensive similarity to RAS . We also show that BUD5 interacts functionally with a gene, BEM1, that is required for bud formation . This interaction provides further support for the view that products involved in bud site selection guide the positioning of a complex necessary for bud formation.

Cell, 1991 Jun 28, 65(7), 1093 - 6
Development of cell polarity in budding yeast; Drubin DG; The development of cell polarity involves virtually every aspect of cell biology . Yeast are less complex than cells traditionally used for studies on cell polarity and are amendable to sophisticated genetic analysis . This has resulted in a growing number of molecular markers for yeast cell polarity and an increasingly well-defined progression of molecular events required for bud formation . Together, these factors provide a favorable context in which to understand how the interplay between a large number of processes can polarize a cell . Many genes required for morphogenesis have been identified, and genetic interactions provide evidence that the products of these genes function together . Studies on cell polarity development in S . cerevisiae have demonstrated a requirement for small GTP-binding proteins and have established functional relationships between temporally coincident events . With the continued identification and analysis of genes required for morphogenesis, and the pursuit of these studies on a cytological and biochemical level, studies on yeast will continue to contribute to our understanding of cell polarity development.

Eur J Biochem, 1991 Jun 15, 198(3), 651 - 7
Alpha-factor-leader-directed secretion of recombinant human-insulin-like growth factor I from Saccharomyces cerevisiae . Precursor formation and processing in the yeast secretory pathway; Steube K et al.; A synthetic gene coding for human-insulin-like growth factor I (IGFI) was fused to the leader sequence of yeast prepro-alpha-factor and expressed in Saccharomyces cerevisiae under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter fragment . Recombinant IGFI was found inside yeast cells and secreted into the medium . The secreted IGFI migrated on SDS gels with the same electrophoretic mobility as authentic IGFI, i.e . at about 7.5 kDa . HPLC analysis of secreted IGFI revealed the presence of the correctly folded, genuine molecule as well as an isomeric byproduct of equal molecular mass but with two of the three disulfide bonds interchanged . Inside exponentially growing cells the 7.5-kDa IGFI was also found, along with up to four additional IGFI-related polypeptides of higher molecular mass . By endoglycosidase F treatment the three polypeptides between 19-26 kDa were converted to a single peptide of 17 kDa . Since this peptide also reacted with an anti-alpha-factor antibody, it represents most likely the unglycosylated alpha-factor--IGFI fusion precursor . Pulse-chase experiments established the precursor nature of the intracellular higher-molecular-mass IGFI species . Conversion of the primary translation product to the differently glycosylated IGFI precursor proteins and into the mature form occurred very rapidly, within 2 min . Rapid maturation was, however, not followed by an equally rapid secretion of the mature form into the medium: only after 30-40 min did IGFI appear outside the cells . We therefore postulate the presence of an as yet undefined Golgi or post-Golgi bottleneck representing a major obstacle in secretion of recombinant IGFI from S . cerevisiae cells.

Biochem J, 1991 Jun 15, 276 ( Pt 3), 759 - 64
Further studies on the localization of the reactive lysyl residue of pyruvate carboxylase; Chapman-Smith A et al.; We have shown the increase in the acetyl-CoA-independent activity of sheep liver pyruvate carboxylase following trinitrophenylation of a specific lysine residue (designated Lys-A) to be the result of a large stimulation of the first partial reaction and a slight stimulation of the second partial reaction catalysed by this enzyme . Like acetyl-CoA, the activators adenosine 3',5'-bisphosphate and CoA did not stimulate the catalytic activity of the trinitrophenylated enzyme in either the overall reaction or the first partial reaction . Conversely, trinitrophenylation had no effect on activation of the overall reaction and the second partial reaction by acetyl-phosphopantetheine . Protection experiments demonstrated that the presence of both acetyl-CoA and adenosine 3',5'-bisphosphate decreased the rate of loss of activity during exposure of sheep liver pyruvate carboxylase to trinitrobenzenesulphonic acid (TNBS), whereas acetyl-phosphopantetheine did not . 5'-AMP and acetyl-dephospho-CoA did not protect the enzyme against loss of activity, whereas the presence of adenosine 2',5'-bisphosphate only slightly decreased the rate of modification . This suggests that Lys-A interacts with the adenosine nucleotide portion of the acetyl-CoA molecule, specifically the 3'-phosphate moiety . Acetyl-CoA and adenosine 3',5'-bisphosphate were shown to protect pyruvate carboxylase from Saccharomyces cerevisiae against inhibition by TNBS . A {14C}acetyl-CoA-binding assay demonstrated that modification of Lys-A inhibits the binding of acetyl-CoA to S . cerevisiae pyruvate carboxylase, indicating that Lys-A is at or near the acetyl-CoA-binding site.

Cell, 1991 Jun 14, 65(6), 1023 - 31
Yeast histone H4 N-terminal sequence is required for promoter activation in vivo; Durrin LK et al.; To search for histone domains that may regulate transcription in vivo, we made deletions and amino acid substitutions in the histone N-termini of S . cerevisiae . Histone H4 N-terminal residues 4-23, which include the extremely conserved, reversibly acetylated lysines (at positions 5, 8, 12, and 16), were found to encompass a region required for the activation of the GAL1 promoter . Deletions in the H4 N-terminus reduce GAL1 activation 20-fold . This effect is specific to histone H4 in that large deletions in the N-termini of H2A, H2B, and H3 do not similarly decrease induction . Activation of the PHO5 promoter is reduced approximately 4- to 5-fold by these H4 deletions . Mutations in histone H4 acetylation sites and surrounding residues can cause comparable and, in some cases, even greater effects on induction of these two promoters . We postulate that the H4 N-terminus may interact with a component of the transcription initiation complex, allowing nucleosome unfolding and subsequent initiation.

Biochim Biophys Acta, 1991 Jun 13, 1089(2), 269 - 72
Protein phosphatase 2Bw and protein phosphatase Z are Saccharomyces cerevisiae enzymes; Da Cruz e Silva EF et al.; cDNAs encoding three protein phosphatases, termed PP2Bw (Da Cruz e Silva, E.F . and Cohen, P.T.W . (1989) Biochim . Biophys . Acta 1009, 293-296), PPZ1 and PPZ2 that have been isolated from a Clontech 'rabbit brain' library are shown to be Saccharomyces cerevisiae clones . PPZ1 and PPZ2 are two novel yeast phosphatases showing 93% amino acid sequence identity to one another . PPZ1 shows approx . 60% sequence identity to S . cerevisiae or mammalian PP1 and approx . 40% identity to S . cerevisiae or mammalian PP2A . These and other observations suggest that the two isoforms of PPZ have functions distinct from those of PP1.

Biochim Biophys Acta, 1991 Jun 13, 1089(2), 206 - 12
The overexpression of the 3' terminal region of the CDC25 gene of Saccharomyces cerevisiae causes growth inhibition and alteration of purine nucleotides pools; Frascotti G et al.; The CDC25 gene is transcribed at a very low level in S . cerevisiae cells . We have studied the effects of an overexpression of this regulatory gene by cloning either the whole CDC25 open reading frame (pIND25-2 plasmid) or its 3' terminal portion (pIND25-1 plasmid) under the control of the inducible strong GAL promoter . The strain transformed with pIND25-2 produced high levels of CDC25 specific mRNA, induced by galactose . This strain does not show any apparent alteration of growth, both in glucose and in galactose . Instead the yeast cells transformed with pIND25-1, that overexpress the 3' terminal part of CDC25 gene, grow very slowly in galactose medium, while they grow normally in glucose medium . The nucleotides were extracted from transformed cells, separated by HPLC and quantitated . The ATP/ADP and GTP/GDP ratios were almost identical in control and in pIND25-2 transformed strains growing in glucose and in galactose, while the strain that overexpresses the 3' terminal portion of CDC25 gene showed a decrease of ATP/ADP ratio and a partial depletion of the GTP pool . The disruption of RAS genes was only partially able to 'cure' this phenotype . A ras2-ts1, ras1::URA3 strain, transformed with pIND25-1 plasmid, was able to grow in galactose at 36 degrees C . These results suggest that the carboxy-terminal domain of the CDC25 protein could stimulate an highly unregulated GTPase activity in yeast cells by interacting not only with RAS gene products but also with some other yeast G-proteins.

J Biol Chem, 1991 Jun 5, 266(16), 10498 - 504
Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase by co-expressing it with mammalian G protein alpha subunits in Escherichia coli; Duronio RJ et al.; A dual plasmid system was used to examine the protein and acyl-CoA specificities of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by co-expressing it in Escherichia coli with each of four homologous alpha subunits of the signal-transducing, heterotrimeric G proteins . Exogenous {3H}myristate was incorporated into rat Gi alpha 1 and rat Go alpha but not into bovine Gs alpha or human Gz alpha . Oxygen for methylene group substitutions in myristate result in analogs with comparable chain length and stereochemistry but marked reductions in hydrophobicity . Metabolic labeling studies with 6-, 11-, or 13-{3H}oxatetradecanoic acid indicated that they were incorporated into rat Gi alpha 1 and Go alpha with an efficiency that could be correlated with their accumulation into E . coli and their interactions with purified NMT in vitro . Octapeptides derived from the NH2-terminal sequences of these four G alpha polypeptides were tested as substrates for purified S . cerevisiae NMT . None were bound by the enzyme . Acidic residues at positions 7 and 8 appear to contribute to this effect; deletion of these two amino acids or addition of the next 9 residues of rat Go alpha produced active substrates . These results imply that productive interactions between NMT and G alpha protein substrates in vivo require structural features that are not fully represented within their NH2-terminal 8 residues.

Mutat Res, 1991 Jun, 260(2), 165 - 80
Reevaluation of the 9 compounds reported conclusive positive in yeast Saccharomyces cerevisiae aneuploidy test systems by the Gene-Tox Program using strain D61.M of Saccharomyces cerevisiae; Albertini S; The state of aneuploidy test methodology was appraised by the U.S . Environmental Protection Agency in 1986 in analyzing published data . In Saccharomyces cerevisiae 9 chemicals were reported to be conclusive positive for aneuploidy induction in either mitotic or meiotic cells . We reevaluated these 9 chemicals using Saccharomyces cerevisiae D61.M, a strain that detects mitotic chromosome malsegregation . Acetone (lowest effective dose (LED): 40 microliters/ml), bavistan (LED: 5 micrograms/ml), benomyl (LED: 30 micrograms/ml) and oncodazole (LED: 4 micrograms/ml) induced a dose-dependent increase in the frequencies of chromosomal malsegregation . Ethyl methanesulfonate (EMS; highest tested dose (HTD): 1000 micrograms/ml) and methyl methanesulfonate (MMS; HTD: 100 micrograms/ml) did not induce malsegregation but were both potent inducers of other genetic events, detected by an increase in the frequencies of cyhR cells . No increases in both endpoints (malsegregation and other genetic events) were observed after treatment of S . cerevisiae D61.M with cyclophosphamide (CP; HTD: 16 mg/ml) in the absence of S9, p-D,L-fluorophenylalanine (p-FPA; HTD: 250 micrograms/ml) and phorbol-12-myristate-13-acetate (TPA; HTD: 50 micrograms/ml) . A marginal increase in the frequency of mitotic chromosome malsegregation was obtained with cyclophosphamide in the presence of S9 . Thus our test results largely disagree with those previously published by various authors and taken as conclusive by EPA . We interpret the discrepancies to be due to lack of properly controlled testing (e.g., no check for multiple mutational events) . Only with a careful test design it is possible to discriminate between chemicals inducing only chromosome loss and no other genetic effects (e.g., acetone, oncodazole), chemicals inducing a variety of genetic damage but no chromosome loss (e.g., EMS, MMS) and chemicals inducing neither chromosome loss nor other genetic events in yeast (e.g., TPA, p-FPA).

J Cell Biol, 1991 Jun, 113(6), 1313 - 30
Myristic acid auxotrophy caused by mutation of S . cerevisiae myristoyl-CoA:protein N-myristoyltransferase; Duronio RJ et al.; The S . cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT1) is essential for vegetative growth . NMT1 was found to be allelic with a previously described, but unmapped and unidentified mutation that causes myristic acid (C14:0) auxotrophy . The mutant (nmt1-181) is temperature sensitive, but growth at the restrictive temperature (36 degrees C) is rescued with exogenous C14:0 . Several analogues of myristate with single oxygen or sulfur for methylene group substitutions partially complement the phenotype, while others inhibit growth even at the permissive temperature (24 degrees C) . Cerulenin, a fatty acid synthetase inhibitor, also prevents growth of the mutant at 24 degrees C . Complementation of growth at 36 degrees C by exogenous fatty acids is blocked by a mutation affecting the acyl:CoA synthetase gene . The nmt1-181 allele contains a single missense mutation of the 455 residue acyltransferase that results in a Gly451----Asp substitution . Analyses of several intragenic suppressors suggest that Gly451 is critically involved in NMT catalysis . In vitro kinetic studies with purified mutant enzyme revealed a 10-fold increase in the apparent Km for myristoyl-CoA at 36 degrees C, relative to wild-type, that contributes to an observed 200-fold reduction in catalytic efficiency . Together, the data indicate that nmt-181 represents a sensitive reporter of the myristoyl-CoA pools utilized by NMT.

J Lipid Res, 1991 Jun, 32(6), 1025 - 38
Use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae to modify the lipid composition and function of mitochondrial membranes; Tung BS et al.; KD115 (ol1), an unsaturated fatty acid auxotroph of S . cerevisiae, was grown in a semi-synthetic medium supplemented with 3.3 x 10(-4) M palmitoleic (cis 16:1) or palmitelaidic (trans 16:1) acids . The parent strain S288C was studied as a control . The lipid composition (fatty acids, neutral lipids, and phospholipids), respiratory activity (O2 consumption), and ultrastructure were compared in mutant yeast grown with each unsaturated fatty acid supplement . The fatty acid supplement represented 70-80% of the yeast fatty acids . Yeast grown in trans 16:1 contained more squalene, a higher ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC), and had 10-20% of the respiratory activity compared to the same yeast grown in cis 16:1 . The mitochondrial morphology of yeast in each growth supplement was notably different . The use of mixtures of cis and trans 16:1 in different proportions revealed that the PE/PC ratio, the squalene content, the respiratory defect, and the mitochondrial morphology were all similarly dependent on the fraction of trans 16:1 in the mixtures . As little as 10-20% of cis 16:1 in the mixture was sufficient to abrogate the physiological effects of trans 16:1 on each of the parameters noted above . The combined effects of high content of trans unsaturated fatty acid and the altered phospholipid composition seem to account for the decrease in lipid fluidity, the defective structure and function of the mitochondrial membrane.

Immunol Lett, 1991 Jun, 28(3), 181 - 5
Identification of a 200-kDa glycoprotein antigen of Saccharomyces cerevisiae; Heelan BT et al.; SDS-PAGE analysis of the soluble proteins of the yeast Saccharomyces cerevisiae revealed a single predominant moiety of apparent size 200 kDa which was strongly periodic acid Schiff-positive . In immunoblotting experiments, human sera containing anti-S . cerevisiae antibodies were reactive only with this glycoprotein (gp200); this was demonstrated using sera containing IgG and IgA isotype-specific anti-S . cerevisiae antibodies . The antigenicity of gp200 was substantially reduced by periodate treatment . These results show that gp200 is a major glycoprotein antigen of S . cerevisiae which is immunogenic in man.

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