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 anoth