Protein Sci, 1993 Nov, 2(11), 1862 - 8 Kinetics of folding and association of differently glycosylated variants of invertase from Saccharomyces cerevisiae; Kern G et al.; A core-glycosylated form of the dimeric enzyme invertase has been isolated from secretion mutants of Saccharomyces cerevisiae blocked in transport to the Golgi apparatus . This glycosylation variant corresponds to the form that folds and associates during biosynthesis of the protein in vivo . In the present work, its largely homogeneous subunit size and well-defined quaternary structure were utilized to characterize the folding and association pathway of this highly glycosylated protein in comparison with the nonglycosylated cytoplasmic and the high-mannose-glycosylated periplasmic forms of the same enzyme encoded by the suc2 gene . Renaturation of core-glycosylated invertase upon dilution from guanidinium-chloride solutions follows a unibimolecular reaction scheme with consecutive first-order subunit folding and second-order association reactions . The rate constant of the rate-limiting step of subunit folding, as detected by fluorescence increase, is k1 = 1.6 +/- 0.4 x 10(-3) s-1 at 20 degrees C; it is characterized by an activation enthalpy of delta H++ = 65 kJ/mol . The reaction is not catalyzed by peptidyl-prolyl cis-trans isomerase of the cyclophilin type . Reactivation of the enzyme depends on protein concentration and coincides with subunit association, as monitored by size-exclusion high-pressure liquid chromatography . The association rate constant, estimated by numerical simulation of reactivation kinetics, increases from 5 x 10(3) M-1 s-1 to 7 x 10(4) M-1 s-1 between 5 and 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1993 Nov, 241(3-4), 327 - 34 An impaired RNA polymerase II activity in Saccharomyces cerevisiae causes cell-cycle inhibition at START; Drebot MA et al.; Saccharomyces cerevisiae cells harboring the temperature-sensitive mutation rpo21-4, in the gene encoding the largest subunit of RNA polymerase II, were shown to be partially impaired for cell-cycle progress at a permissive temperature, and to become permanently blocked at the cell-cycle regulatory step, START, at a restrictive temperature . The rpo21-4 mutation was lethal in combination with cdc28 mutations in the p34 protein kinase gene required for START . Transcripts of the CLN1 and CLN2 genes, encoding G1-cyclin proteins that, along with p34, are necessary for START, were decreased in abundance by the rpo21-4 mutation at a restrictive temperature . Increased G1-cyclin production, by expression of the CLN1 or CLN2 genes from a heterologous GAL promoter, overcame the rpo21-4-mediated START inhibition, but such mutant cells nevertheless remained unable to proliferate at a restrictive temperature . These findings reveal that START can be particularly sensitive to an impaired RNA polymerase II function, presumably through effects on G1-cyclin expression. Mol Gen Genet, 1993 Nov, 241(3-4), 305 - 11 The HYP2 gene of Saccharomyces cerevisiae is essential for aerobic growth: characterization of different isoforms of the hypusine-containing protein Hyp2p and analysis of gene disruption mutants; Wohl T et al.; In Saccharomyces cerevisiae, hypusine-containing proteins are encoded by two closely related genes, HYP1 and HYP2, which are regulated reciprocally by oxygen and heme . We have purified the aerobically expressed hypusine-containing proteins from yeast . The three proteins detected (two isoforms, which differ in their pI values, and a degradation product thereof, lacking the N-terminal 10 amino acid residues) are all encoded by HYP2 . The N-terminus of both isoforms is formed by acetylation of a serine residue after cleavage of the first methionine . Cells mutant for hyp2 are unable to grow aerobically . However, under anaerobic conditions these mutants display no obvious phenotype, presumably because the strictly anaerobically expressed HYP1 gene product (Hyp1p) is present . This implies that Hyp1p and Hyp2p fulfill very similar functions . In fact, Hyp1p can substitute for Hyp2p under aerobic conditions, when expressed under the control of the GAL1 promoter in hyp2 mutant cells. Mol Gen Genet, 1993 Nov, 241(3-4), 280 - 6 A dominant interfering mutation in RAS1 of Saccharomyces cerevisiae; Fujimura K et al.; A mutant allele of RAS1 that dominantly interferes with the wild-type Ras function in the yeast Saccharomyces cerevisiae was discovered during screening of mutants that suppress an ira2 disruption mutation . A single amino acid substitution, serine for glycine at position 22, was found to cause the mutant phenotype . The inhibitory effect of the RAS1Ser22 gene could be overcome either by overexpression of CDC25 or by the ira2 disruption mutation . These results suggest that the RAS1Ser22 gene product interferes with the normal interaction of Ras with Cdc25 by forming a dead-end complex between Ras1Ser22 and Cdc25 proteins. Mol Gen Genet, 1993 Nov, 241(3-4), 241 - 54 Cloning of Saccharomyces cerevisiae STE5 as a suppressor of a Ste20 protein kinase mutant: structural and functional similarity of Ste5 to Far1; Leberer E et al.; The beta and gamma subunits of the mating response G-protein in the yeast Saccharomyces cerevisiae have been shown to transmit the mating pheromone signal to downstream components of the pheromone response pathway . A protein kinase homologue encoded by the STE20 gene has recently been identified as a potential G beta gamma target . We have searched multicopy plasmid genomic DNA libraries for high gene dosage suppressors of the signal transduction defect of ste20 mutant cells . This screen identified the STE5 gene encoding an essential component of the pheromone signal transduction pathway . We provide genetic evidence for a functional interrelationship between the STE5 gene product and the Ste20 protein kinase . We have sequenced the STE5 gene, which encodes a predicted protein of 917 amino acids and is specifically transcribed in haploid cells . Transcription is slightly induced by treatment of cells with pheromone . Ste5 has homology with Far1, a yeast protein required for efficient mating and the pheromone-inducible inhibition of a G1 cyclin, Cln2 . A STE5 multicopy plasmid is able to suppress the signal transduction defect of far1 null mutant cells suggesting that Ste5, at elevated levels, is able functionally to replace Far1 . The genetically predicted point of function of Ste5 within the pheromone signalling pathway suggests that Ste5 is involved in the regulation of a G beta gamma-activated protein kinase cascade which links a G-protein coupled receptor to yeast homologues of mitogen-activated protein kinases. FEBS Lett, 1993 Nov 1, 333(3), 238 - 42 Transcriptional regulation of the isocitrate lyase encoding gene in Saccharomyces cerevisiae; Fernandez E et al.; In this work, we studied the transcriptional regulation of isocitrate lyase synthesis . In Northern blot analyses we first showed that the steady-state ICL1 mRNA levels depend on the carbon source used for growth . In addition, we determined the kinetics of transcriptional repression upon a shift of ethanol-grown cells to glucose and of the induction when cells were transferred from glucose to ethanol . By deletion analyses as well as by studying the influence on expression of different fragments cloned into the heterologous CYC1 promoter lacking its own UAS sequences, we defined UAS and URS elements in the ICL1 promoter . A region mediating the control by CAT3, a gene also involved in the control of expression of other genes subject to carbon catabolite repression, was found to overlap with one of these UAS elements. EMBO J, 1993 Nov, 12(11), 4279 - 90 A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor; Muchardt C et al.; Several of the SNF and SWI genes of Saccharomyces cerevisiae code for proteins believed to assist transcriptional activators by relieving nucleosome repression . One of these proteins, SNF2/SWI2, has a homologue in Drosophila, a regulator of homeotic genes known as brahma or brm . In this report, we show that a counterpart of SNF2/SWI2 also exists in mice and humans . The human protein, designated hbrm, is a 180 kDa nuclear factor that can function as a transcriptional activator when fused to a heterologous DNA binding domain . The mouse homologue of hbrm is expressed in all mouse organs tested while hbrm was detected in some but not all investigated human cell lines . In cells failing to express the endogenous gene, transfected hbrm cooperates with the glucocorticoid receptor (GR) in transcriptional activation . However, hbrm had no effect on the activity of several other transcription factors, including the homeoprotein HNF-1 . The co-operation between hbrm and GR required the DNA binding domain of GR and two separated regions of the hbrm protein, including a domain with homology to known helicases. Ital J Biochem, 1993 Nov-Dec, 42(6), 373 - 87 Cell cycle and growth regulation in RAS2 mutant cells of Saccharomyces cerevisiae; Baroni MD et al.; Yeast cells carrying ras2 temperature-sensitive mutations undergo a specific arrest in the prereplicative unbudded phase of the cell cycle when they are shifted to non-permissive temperatures . At 36.5 degrees C, in spite of their abnormally large cell size, bulk protein synthesis and accumulation rates are depressed in ras2 temperature-sensitive cells in comparison with isogenic wild type . At the same temperature, total RNA synthesis and accumulation rates are much more inhibited, suggesting that a defective Ras2/cAMP pathway alters the coordination between RNA and protein synthesis rates . The preferential RNA synthesis inhibition is correlated to a specific inhibition of the synthesis of the 35S rRNA precursor . These findings, taken together with the results of previous analyses, are in favour of a control by the cAMP pathway on rRNA biosynthesis. Yeast, 1993 Nov, 9(11), 1273 - 7 Sequence and function analysis of a 2.73 kb fragment of Saccharomyces cerevisiae chromosome II; Miosga T et al.; The nucleotide sequence of a fragment of 2728 base pairs of Saccharomyces cerevisiae chromosome II has been determined . The sequence contains two open reading frames, one of them being incomplete . Deletion mutants of YBR11.21 are viable . YBR11.20 is identical to the recessive omnipotent suppressor SUP45 (SUP1). Yeast, 1993 Nov, 9(11), 1259 - 65 The DNA sequence analysis of the HAP4-LAP4 region on chromosome XI of Saccharomyces cerevisiae suggests the presence of a second aspartate aminotransferase gene in yeast; Cheret G et al.; The nucleotide sequence of a 19,000 base pair region from the left arm of chromosome XI of Saccharomyces cerevisiae has been determined and analysed . It covers the HAP4-GFA1-LAP4 loci already described . As expected HAP4, GFA1 and LAP4 genes have been found and six new open reading frames (ORFs) with a coding capacity of more than 100 amino acid residues have been identified . One of them (YKL461) shows a high degree of identity with an aspartate aminotransferase gene . This raises the question of a second aspartate aminotransferase gene in yeast . A second ORF (YKL462) shows features compatible with a membranous localization . The other ORFs do not show a similarity with any known gene . A member of the highly repetitive 'CAT' DNA sequence is present. Yeast, 1993 Nov, 9(11), 1241 - 9 Lysine144 is essential for the catalytic activity of Saccharomyces cerevisiae transaldolase; Miosga T et al.; Replacement of lysine144 by glutamine in the pentose phosphate pathway enzyme transaldolase of Saccharomyces cerevisiae is associated with the complete loss of activity indicating the essential role in catalysis . Neither histidine nor cysteine is important for catalytic activity as proposed for the Candida utilis enzyme . Also we could not find any evidence for a half-site character of the enzyme as described for transaldolase of C . utilis . Therefore, the reaction mechanisms for the two enzymes are different. Yeast, 1993 Nov, 9(11), 1177 - 87 The ERG3 gene in Saccharomyces cerevisiae is required for the utilization of respiratory substrates and in heme-deficient cells; Smith SJ et al.; ERG3 is the structural gene in Saccharomyces cerevisiae for the sterol delta 5 desaturase that introduces the C5 = 6 unsaturation in ergosterol biosynthesis . The ERG3 gene has been mapped on chromosome XII, 13.7 centimorgans from GAL2 toward SPT8 . The essentiality of the gene is dependent on the conditions used for the cultivation of the mutants . Insertionally inactivated mutants of ERG3 fail to grow without 'sparking' levels of delta 5 sterols in heme-deficient cells, and are unable to grow on the respiratory substrates glycerol and ethanol. Mol Microbiol, 1993 Nov, 10(3), 585 - 96 A proline-rich protein, verprolin, involved in cytoskeletal organization and cellular growth in the yeast Saccharomyces cerevisiae; Donnelly SF et al.; A gene (VRP1) encoding a novel proline-rich protein (verprolin) has been isolated from the yeast Saccharomyces cerevisiae as a result of its hybridization to a chick vinculin cDNA probe . The deduced protein sequence contains 24% proline residues present as proline-rich motifs throughout the verprolin sequence . Several of these motifs resemble recently identified sequences shown to bind Src homology 3 (SH3) domains in vitro . Replacement of the wild-type VRP1 allele with a mutant allele results in strains that grow slower than wild-type strains and are temperature sensitive . The vrp1 mutants are impaired in both cell shape and size and display aberrant chitin and actin localization . We propose that verporlin is involved in the maintenance of the yeast actin cytoskeleton, through interactions with other proteins, possibly containing SH3 domains. Appl Microbiol Biotechnol, 1993 Nov, 40(2-3), 333 - 40 Saccharomyces cerevisiae can release hepatitis B virus surface antigen (HBsAg) particles into the medium by its secretory apparatus; Kuroda S et al.; We constructed a plasmid that directs the synthesis and secretion of hepatitis B virus (HBV) surface antigen (HBsAg) particles by Saccharomyces cerevisiae . This plasmid contains a proteinase-resistant HBsAg M (M-P31c) gene fused at its 5'-terminus with a chicken-lysozyme signal peptide (C-SIG) gene, which is placed under the yeast GLD (glyceraldehyde-3-phosphate dehydrogenase gene) promoter . The products encoded by the "C-SIG+M-P31c" (LM-P31c) gene were synthesized and assembled themselves into HBsAg particles in yeast cells, and the particles were released into the medium along with poly-HSA (polymerized human serum albumin) binding activity . The HBsAg particles purified from the medium were very similar in density (1.19 g cm-3), size (19.2 +/- 0.8 nm in diameter) and shape (sphere) to human-plasma-derived HBsAg particles . When several sec (temperature-sensitive secretion-defective) mutants were used as host cells, the release of HBsAg particles into the medium was blocked at 37 degrees C but not at 25 degrees C, indicating that the HBsAg particles are exported through the normal yeast secretion pathway . To our knowledge, this is the first report that yeast cells are capable of secreting particles into the medium. Biotechnol Prog, 1993 Nov-Dec, 9(6), 594 - 9 Copy number modulation in an autoselection system for stable plasmid maintenance in Saccharomyces cerevisiae; Compagno C et al.; Efficient expression of a foreign gene requires a stable vector present at a high number of copies per cell . We have constructed an autoselection system for the stable maintenance of expression vector in the yeast Saccharomyces cerevisiae that uses the fructose 1,6-bisphosphate aldolase gene (FBA1) to stabilize plasmids in cells bearing a disruption of the chromosomal FBA1 gene . This system allowed us to obtain stable production of a reporter heterologous enzyme (Escherichia coli beta-galactosidase) in rich media . By using an inducible promoter to regulate the expression of FBA1 gene, we have also obtained the modulation of plasmid copy number by carbon source. Genes Dev, 1993 Nov, 7(11), 2161 - 71 The Saccharomyces cerevisiae DNA repair gene RAD25 is required for transcription by RNA polymerase II; Qiu H et al.; The RAD25 gene of Saccharomyces cerevisiae is required for excision repair of ultraviolet-damaged DNA and, in addition, is essential for viability . RAD25 shares a high degree of homology with the human ERCC3/XPBC-encoded protein, and the yeast and human proteins resemble one another in containing the conserved ATPase/DNA helicase sequence motifs . To determine the nature of the essential role of RAD25, we have isolated a recessive temperature-sensitive conditional lethal mutation of the gene and have examined its effect on transcription . Upon shift to the nonpermissive temperature, the rad25 temperature-sensitive (ts) mutant stops growth rapidly and shows a large decrease in the synthesis of poly(A)+ RNA . Transcription of a large number of yeast genes, including HIS3, TRP3, STE2, MET19, RAD23, CDC9, and ACT1 is inhibited at the restrictive temperature in the rad25 ts mutant, and the galactose-inducible synthesis of GAL7 and GAL10 mRNAs is also severely affected by the loss of RAD25 activity . These findings implicate a general requirement of RAD25 in RNA polymerase II transcription. Genetics, 1993 Nov, 135(3), 731 - 40 An efficient positive selection procedure for the isolation of peroxisomal import and peroxisome assembly mutants of Saccharomyces cerevisiae; Elgersma Y et al.; To study peroxisome biogenesis, we developed a procedure to select for Saccharomyces cerevisiae mutants defective in peroxisomal protein import or peroxisome assembly . For this purpose, a chimeric gene was constructed encoding the bleomycin resistance protein linked to the peroxisomal protein luciferase . In wild-type cells this chimeric protein is imported into the peroxisome, which prevents the neutralizing interaction of the chimeric protein with its toxic phleomycin ligand . Peroxisomal import and peroxisome assembly mutants are unable to import this chimeric protein into their peroxisomes . This enables the bleomycin moiety of the chimeric protein to bind phleomycin, thereby preventing its toxicity . The selection is very efficient: upon mutagenesis, 84 (10%) of 800 phleomycin resistant colonies tested were unable to grow on oleic acid . This rate could be increased to 25% using more stringent selection conditions . The selection procedure is very specific; all oleic acid non utilizing (onu) mutants tested were disturbed in peroxisomal import and/or peroxisome assembly . The pas (peroxisome assembly) mutants that have been used for complementation analysis represent 12 complementation groups including three novel ones, designated pas20, pas21 and pas22. FEBS Lett, 1993 Oct 25, 333(1-2), 169 - 74 Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae; Tsukada M et al.; Autophagy in the yeast is similar to that in mammalian cells . A mutant designated as apg1 (autophagy) defective in accumulation of autophagic bodies in the vacuoles was isolated by selection using a light microscope from a mutagenized proteinase-deficient strain . In the apg1 strain, which has normal vacuolar proteinases, nitrogen starvation did not induce protein degradation . The apg1 mutant lost its viability faster than wild-type cells during nitrogen starvation . By using the loss of viability as a first screening test, 75 other apg mutants were selected . These apg mutants including apg1 fell into 15 complementation groups . Genetic analyses of representative apg mutants revealed that they all had single recessive chromosomal mutations . Strains with each apg mutation were defective in protein degradation in the vacuoles induced by nitrogen starvation and homozygous diploids for each apg mutation did not sporulate . These results on the apg mutants suggest that autophagy via autophagic bodies is indispensable for protein degradation in the vacuoles under starvation conditions, and that at least 15 APG genes are involved in autophagy in yeast. Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9688 - 92 Pheromone action regulates G-protein alpha-subunit myristoylation in the yeast Saccharomyces cerevisiae; Dohlman HG et al.; Myristic acid (C14:0) is added to the N-terminal glycine residue of the alpha subunits of certain receptor-coupled guanine nucleotide-binding regulatory proteins (G proteins) . The G alpha subunit (GPA1 gene product) coupled to yeast pheromone receptors exists as a pool of both myristoylated and unmyristolyated species . After treatment of MATa cells with alpha factor, the myristoylated form of Gpa1p increases dramatically, and the unmyristoylated form decreases concomitantly . This pheromone-stimulated shift depends on the function of STE2 (alpha-factor receptor), STE11 (a protein kinase in the response pathway), and NMT1 (myristoyl-CoA:protein N-myristoyltransferase) genes and uses the existing pool of fatty acids (is not blocked by cerulenin) . Myristoylated Gpa1p persists long after pheromone is removed . Because myristoylation is essential for proper G alpha-G beta gamma association and receptor coupling, pheromone-dependent stimulation of Gpa1p myristoylation may be an important contributing factor in adaptation after signal transmission. Proc Natl Acad Sci U S A, 1993 Oct 15, 90(20), 9315 - 9 Multiple nucleosome positioning with unique rotational setting for the Saccharomyces cerevisiae 5S rRNA gene in vitro and in vivo; Buttinelli M et al.; A simple no-background assay was developed for high-resolution in vivo analysis of yeast chromatin . When applied to Saccharomyces cerevisiae 5S rRNA genes (5S rDNA), this analysis shows that nucleosomes completely cover this chromosomal region, occupying alternative positions characterized by a unique helical phase . This supports the notion that sequence-intrinsic rotational signals are the major determinant of nucleosome localization . Nucleosomal core particles reconstituted in vitro occupy the same positions and have the same helically phased distribution observed in vivo, as determined by mapping of exonuclease III-resistant borders, mapping by restriction cleavages, and by DNase I and hydroxyl-radical digestion patterns. J Biol Chem, 1993 Oct 15, 268(29), 22156 - 63 A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that resembles an Escherichia coli fatty acyltransferase; Nagiec MM et al.; Saccharomyces cerevisiae normally requires sphingolipid biosynthesis for growth; however, mutant strains lacking sphingolipids have been isolated by suppression of a genetic defect in sphingolipid long chain base biosynthesis . To begin to understand the nature of the suppressor(s) we isolated and characterized a suppressor gene, SLC1 (sphingolipid compensation) . DNA sequence analysis showed that the wild type SLC1 allele differs from the suppressor allele by a single nucleotide which changes Gln-44 in the predicted wild type protein to Leu4-4 in the predicted SLC1-1 suppressor protein . The predicted SLC1 protein sequence is homologous to the 1-acyl-sn-glycerol-3-phosphate acyltransferase of Escherichia coli encoded by the plsC gene . The homology extends to function as well since the SLC1 gene complements the growth defect in an E . coli strain mutated in plsC . These results suggest that the SLC1 protein has a fatty acyltransferase activity . SLC1 thus may be the first eucaryotic sn2-acylglyceride fatty acyltransferase gene to be cloned . SLC strains grown in the absence of long chain base make novel phosphatidylinositol derivatives (Lester, R . L., Wells, G . B., Oxford, G., and Dickson, R . C . (1993) J . Biol . Chem . 268, 845-856) having a C26 fatty acid at the sn-2 position and the same polar head groups as normal sphingolipids . We postulate that the SLC1 suppressor allele encodes a variant enzyme with an altered substrate specificity that enables it to use a C26 in place of a C16/18 fatty acid precursor to acylate the sn-2 position of inositol-containing glycerolipids. J Biol Chem, 1993 Oct 15, 268(29), 21844 - 53 Purification and characterization of VDE, a site-specific endonuclease from the yeast Saccharomyces cerevisiae; Gimble FS et al.; The 119-kDa primary translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes a self-catalyzed rearrangement ("protein splicing") that excises an internal 50-kDa segment of the polypeptide and joins the amino-terminal and carboxyl-terminal segments to generate the 69-kDa subunit of the vacuolar membrane-associated H(+)-ATPase . We have shown previously that the internal segment is a site-specific endonuclease (Gimble, F . S., and Thorner, J . (1992) Nature 357, 301-306) . Here we describe methods for the high level expression and purification to near homogeneity of both the authentic VMA1-derived endonuclease (or VDE) from yeast (yield 18%) and a recombinant form of VDE made in bacteria (yield 29%) . Detailed characterization of these preparations demonstrated that the yeast-derived and bacterially produced enzymes were indistinguishable, as judged by: (a) behavior during purification; (b) apparent native molecular mass (50 kDa); (c) immunological reactivity; and (d) catalytic properties (specific activity; cleavage site recognition; and optima for pH, temperature, divalent cation and ionic strength) . The minimal site required for VDE cleavage was delimited to a 30-base pair sequence within its specific substrate (the VMA1 delta vde allele). J Biol Chem, 1993 Oct 15, 268(29), 21783 - 90 Purification and characterization of a DNA helicase from Saccharomyces cerevisiae; Bean DW et al.; A novel DNA helicase, scHelI, has been purified from whole cell extracts of Saccharomyces cerevisiae using biochemical assays to monitor the fractionation . The enzyme unwinds partial duplex DNA substrates, as long as 343 base pairs in length, in a reaction that is dependent on either ATP or dATP hydrolysis . scHelI also catalyzes a single-stranded DNA-dependent ATP hydrolysis reaction; the apparent Km for ATP is 325 microM . The unwinding reaction on circular partial duplex substrates is biphasic, with a fast component occurring within 5 min of the initiation of the reaction and a slow component continuing to 60 min . This is in contrast to the ATP hydrolysis reaction, which exhibits linear kinetics for 60 min . The direction of the unwinding reaction is 5' to 3' with respect to the strand of DNA on which the enzyme is bound . The unwinding reaction is strongly stimulated by the addition of Escherichia coli single-stranded DNA-binding protein when long partial duplex substrates are used . The enzymatic activity of scHelI copurifies with a polypeptide of 135 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . The polypeptide sediments as a monomer in a glycerol gradient in the presence of 0.2 M NaCl. Gene, 1993 Oct 15, 132(2), 175 - 83 The Saccharomyces cerevisiae MTS1 gene encodes a putative RNA-binding protein involved in mitochondrial protein targeting; Ellis EM et al.; Most proteins present in the mitochondrion are nuclear encoded, and are directed to the organelle by virtue of a targeting sequence at the N terminus of the precursor protein . Mitochondrial (mt) protein targeting appears to require several accessory proteins that recognise mt precursors both in the cytoplasm and at the mt surface . We describe here the use of yeast genetics to identify a protein that is required for mt protein targeting . Two yeast mutants (mts1 and mts2) were isolated as extragenic suppressors of a known targeting defect in the presequence of the beta-subunit of ATP synthase . We have cloned and sequenced the wild-type allele of one of these genes (MTS1) and shown that it encodes a member of a family of RNA-binding proteins that is essential for growth. Eur J Biochem, 1993 Oct 15, 217(2), 657 - 60 Interactions of calcium and magnesium with the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae; Uribe S et al.; The activity of the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae was measured in the presence of increasing concentrations of magnesium and calcium . Calcium pyrophosphate (dissociation constant Kd = 1.9 microM) inhibited pyrophosphatase by competition with magnesium pyrophosphate (Kd = 50 microM) . The small movements of calcium detected in mitochondria from yeast may be physiologically significant for the control of inorganic pyrophosphatase activity and the concentration of pyrophosphate in the matrix of yeast mitochondria. J Mol Biol, 1993 Oct 5, 233(3), 372 - 88 The gene clusters ARC and COR on chromosomes 5 and 10, respectively, of Saccharomyces cerevisiae share a common ancestry; Melnick L et al.; The yeast Saccharomyces cerevisiae contains two clusters of eight genes each on chromosome 10 and 5, denoted, respectively, the COR and ARC regions . The genes in the COR region include TRS1 (a tRNA(Ser) gene), ANB1, CYC1, UTR1, UTR3, OSM1, tRNA(Gly) and RAD7 whereas the genes in the ARC region include TRS2 (a tRNA(Ser) gene), TIF51A, UTR5, ANP1, RAD23, UTR4, CYC7 and UTR2 . We have performed a physical analysis of the ARC region, including determining DNA sequence of the 7529 nucleotides; the open reading frames; the size and orientation of the transcripts; and the phenotypes resulting from deletions or gene disruptions . The ARC region was systematically compared to the COR region which was previously described . The gene pairs CYC1-CYC7 and ANB1-TIF51A were previously shown to be, respectively, approximately 80% and 90% identical . tRNA(Ser) genes, TRS1 and TRS2, are located in both clusters 953 nt and 344 nt downstream of ANB1 and TIF51A, respectively . Some of the other gene pairs of these clusters are related in function and share only short segments of similarity distributed within the regions . The best alignment based on amino acid and nucleotide sequences indicates that the ARC and COR regions are ancestrally related by a duplication, a transposition, and a single rearrangement, followed by extensive divergence . These comparisons allowed an evaluation of distantly related sequences not obviously revealed by standard computer analysis . Surprisingly, the alignment suggested that a translated region of the ARC ANP1 gene and the COR tRNA(Gly) gene are ancestrally related . Also translated regions of the COR gene RAD7 share similarities with both of the two adjacent ARC genes, ANP1 and RAD23 . Five examples of simple repeated amino acid and DNA sequences occurred in the ARC region but none in the COR region . We suggest that these repeated sequences played a role in the divergence of ARC genes. J Biol Chem, 1993 Oct 5, 268(28), 21416 - 24 Phosphatidylserine decarboxylase from Saccharomyces cerevisiae . Isolation of mutants, cloning of the gene, and creation of a null allele; Trotter PJ et al.; Phosphatidylserine decarboxylase plays a pivotal role in the synthesis of phospholipid by the mitochondria . The substrate phosphatidylserine is synthesized extramitochondrially and must be translocated to the mitochondria prior to decarboxylation . To understand the properties of the decarboxylase and exploit its unique topology to address basic questions of interorganelle cooperation in membrane assembly, we have begun to examine this enzyme in Saccharomyces cerevisiae . Strains of the yeast defective in enzyme activity were isolated by modified brute force using 1-acyl-2{N-(6-{7-nitrobenz-2- oxa-1,3-diazo-4-yl)}aminocaproyl} (NBD)-phosphatidyl{1'-14C}serine as substrate for permeabilized cells . Mutant strains with less than 5% wild type activity exhibited no defective growth phenotype . The gene for the phosphatidylserine decarboxylase (PSD) was cloned using an oligonucleotide probe degenerate for the sequence VGAT(I)/(N)VGSI, which is the longest stretch of sequence identity between the Escherichia coli sequence (I at position 5) and the putative CHO cell sequence (N at position 5) . The gene encodes a 500 amino acid protein with 28-43% identity to the bacterial and mammalian sequences . The yeast PSD gene maps to the long arm of chromosome 14 between the kex 2 and RAS 2 loci . Null mutations created by disrupting the PSD gene with TRP1 demonstrate that the gene is not essential for cell growth even when the engineered strains are deprived of choline and ethanolamine . Analysis of lipid synthesis and enzyme activity in null mutants indicates that there are two PSD genes. Toxicology, 1993 Oct 5, 82(1-3), 61 - 73 Saccharomyces cerevisiae: an alternative source for human microsomal liver enzymes and its use in drug interaction studies; Eugster HP et al.; Heterologous expression of human cDNAs in the yeast Saccharomyces cerevisiae represents an attractive alternative source of human enzymes and allows metabolic studies to be performed without the need of human tissue . Here we report on the functional expression of human microsomal epoxide hydrolase (hmEH) and cytochrome P450 1A1 and 1A2 in yeast . Microsomal fractions of corresponding yeast strains exhibited enzyme specific activities which allowed the characterization of the heterologous enzymes . The use of these microsomes enabled us to study drug interactions on the respective enzymes with pharmacologically relevant drugs such as carbamazepine epoxide, valpromide and ketoconazole. FEBS Lett, 1993 Oct 4, 331(3), 233 - 8 Interaction of Sec4 with GDI proteins from bovine brain, Drosophila melanogaster and Saccharomyces cerevisiae . Conservation of GDI membrane dissociation activity; Garrett MD et al.; Rab GDP dissociation inhibitor (Rab GDI), will induce the dissociation of GDP-bound rab3A from synaptic membranes and will inhibit GDP dissociation from Sec4, a member of the Rab subgroup of the Ras GTPase superfamily which is required for exocytosis in Saccharomyces cerevisiae . We report that Rab GDI releases GDP-bound Sec4 from yeast membranes . dGDI, a Drosophila homologue can similarly inhibit GDP dissociation from Sec4 and release GDP-bound Sec4 from yeast membranes . An activity partially purified from yeast cytosol dissociates GDP-bound Sec4 from yeast membranes, suggesting that yeast also possess a GDI protein that functions to recycle Sec4 from its target membrane. Mol Cell Biol, 1993 Oct, 13(10), 6274 - 82 Connections between the Ras-cyclic AMP pathway and G1 cyclin expression in the budding yeast Saccharomyces cerevisiae; Hubler L et al.; We have identified two processes in the G1 phase of the Saccharomyces cerevisiae cell cycle that are required before nutritionally arrested cells are able to return to proliferative growth . The first process requires protein synthesis and is associated with increased expression of the G1 cyclin gene CLN3 . This process requires nutrients but is independent of Ras and cyclic AMP (cAMP) . The second process requires cAMP . This second process is rapid, is independent of protein synthesis, and produces a rapid induction of START-specific transcripts, including CLN1 and CLN2 . The ability of a nutritionally arrested cell to respond to cAMP is dependent on completion of the first process, and this is delayed in cells carrying a CLN3 deletion . Mating pheromone blocks the cAMP response but does not alter the process upstream of Ras-cAMP . These results suggest a model linking the Ras-cAMP pathway with regulation of G1 cyclin expression. Mol Cell Biol, 1993 Oct, 13(10), 6102 - 13 PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae; Anderson JT et al.; Proteins that directly associate with nuclear polyadenylated RNAs, or heterogeneous nuclear RNA-binding proteins (hnRNPs), and those that associate with cytoplasmic mRNAs, or mRNA-binding proteins (mRNPs), play important roles in regulating gene expression at the posttranscriptional level . Previous work with a variety of eukaryotic cells has demonstrated that hnRNPs are localized predominantly within the nucleus whereas mRNPs are cytoplasmic . While studying proteins associated with polyadenylated RNAs in Saccharomyces cerevisiae, we discovered an abundant polyuridylate-binding protein, PUB1, which appears to be both an hnRNP and an mRNP . PUB1 and PAB1, the polyadenylate tail-binding protein, are the two major proteins cross-linked by UV light to polyadenylated RNAs in vivo . The deduced primary structure of PUB1 indicates that it is a member of the ribonucleoprotein consensus sequence family of RNA-binding proteins and is structurally related to the human hnRNP M proteins . Even though the PUB1 protein is a major cellular polyadenylated RNA-binding protein, it is nonessential for cell growth . Indirect cellular immunofluorescence combined with digital image processing allowed a detailed comparison of the intracellular distributions of PUB1 and PAB1 . While PAB1 is predominantly, and relatively uniformly, distributed within the cytoplasm, PUB1 is localized in a nonuniform pattern throughout both the nucleus and the cytoplasm . The cytoplasmic distribution of PUB1 is considerably more discontinuous than that of PAB1 . Furthermore, sucrose gradient sedimentation analysis demonstrates that PAB1 cofractionates with polyribosomes whereas PUB1 does not . These results suggest that PUB1 is both an hnRNP and an mRNP and that it may be stably bound to a translationally inactive subpopulation of mRNAs within the cytoplasm. Mol Cell Biol, 1993 Oct, 13(10), 6071 - 8 The Rox1 repressor of the Saccharomyces cerevisiae hypoxic genes is a specific DNA-binding protein with a high-mobility-group motif; Balasubramanian B et al.; The ROX1 gene encodes a repressor of the hypoxic functions of the yeast Saccharomyces cerevisiae . The DNA sequence of the gene was determined and found to encode a protein of 368 amino acids . The amino-terminal third of the protein contains a high-mobility-group motif characteristic of DNA-binding proteins . To determine whether the Rox1 repressor bound DNA, the gene was expressed in Escherichia coli cells as a fusion to the maltose-binding protein and this fusion was partially purified by amylose affinity chromatography . By using a gel retardation assay, both the fusion protein and Rox1 itself were found to bind specifically to a synthetic 32-bp DNA containing the hypoxic consensus sequence . We assessed the role of the general repressor Ssn6 in ANB1 repression . An ANB1-lacZ fusion was expressed constitutively in an ssn6 deletion strain, and deletion of the Rox1 binding sites in the ANB1 upstream region did not increase the level of derepression, suggesting that Ssn6 exerts its effect through Rox1 . Finally, ROX1 was mapped to yeast chromosome XVI, near the ARO7-OSM2 locus. J Cell Biol, 1993 Oct, 123(2), 405 - 16 The calcium-binding protein cell division cycle 31 of Saccharomyces cerevisiae is a component of the half bridge of the spindle pole body; Spang A et al.; Cdc31 mutants of Saccharomyces cerevisiae arrest at the nonpermissive temperature with large buds, G2 DNA content and, a single, abnormally large spindle pole body (SPB) (Byers, B . 1981 . Molecular Genetics in Yeast . Alfred Benzon Symposium . 16:119-133) . In this report, we show that the CDC31 gene product is essential for cell viability . We demonstrate that purified CDC31 protein binds Ca2+ and that this binding is highly specific . Taken together, three lines of evidence indicate that CDC31 is a component of the SPB . First, CDC31 cofractionates with enriched preparations of SPBs . Second, immunofluorescence staining indicates that CDC31 colocalizes with a known SPB component . Third, immunoelectron microscopy with whole cells and with isolated SPBs reveals that CDC31 is localized to the half bridge of the SPB, which lies immediately adjacent to the SPB plaques . CDC31 was detected mainly at the cytoplasmic side of the half bridge and, therefore, defines a further substructure of the SPB . We suggest that CDC31 is a member of a family of calcium-binding, centrosome-associated proteins from a phylogenetically diverse group of organisms. J Cell Biol, 1993 Oct, 123(2), 387 - 403 MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae; Brown MT et al.; The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product . When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle . Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis . Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles . mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies . These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation . The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins. J Bacteriol, 1993 Oct, 175(20), 6562 - 70 A cdc-like autolytic Saccharomyces cerevisiae mutant altered in budding site selection is complemented by SPO12, a sporulation gene; Molero G et al.; LYT1 is an essential gene for the growth and morphogenesis of Saccharomyces cerevisiae . A detailed characterization of mutants carrying the lyt1-1 allele showed that this mutation was recessive and pleiotropic, affecting both mitotic and meiotic functions . At the nonpermissive temperature of 37 degrees C, lyt1 haploid strains budded at a distal position (instead of an axial one, as in wild-type haploid strains) and underwent autolysis when the buds were almost the size of the mother cells . These mitotic alterations in cell stability and budding topology were dependent on growth and protein synthesis . Autolysis was prevented by inhibiting DNA synthesis (with hydroxyurea) or by blocking the assembly of microtubules (with benomyl), suggesting that loss of cell viability must occur at a fixed mitotic cycle stage after DNA synthesis and mitotic spindle assembly . On the other hand, lyt1-1/lyt1-1 diploids failed to sporulate at both 24 and 37 degrees C . Taking into account these characteristics, the lyt1 mutant could be considered a cdc-like mutant . By genetic transformation of an appropriate lyt1 strain with a genomic library, ligated to the multicopy vector YEp13, we isolated a gene capable of complementing mitotic alterations but not the meiotic defect . This was the sporulation-specific gene SPO12, which is expressed under the control of the locus MAT in meiosis and is also expressed in the mitotic cycle (V . Parkes and L . H . Johnston, Nucleic Acids Res . 20:5617-5623, 1992) . A significant level of SPO12 mRNA can be detected when this gene is inserted in a multicopy plasmid. J Bacteriol, 1993 Oct, 175(19), 6345 - 7 Evidence that the Rad1 and Rad10 proteins of Saccharomyces cerevisiae participate as a complex in nucleotide excision repair of UV radiation damage; Siede W et al.; A newly characterized rad1 missense mutation (rad1-20) in the yeast Saccharomyces cerevisiae maps to a region of the Rad1 polypeptide known to be required for Rad1-Rad10 complex formation . The UV sensitivity of the rad1-20 mutant can be partially and specifically corrected by overexpression of wild-type Rad10 protein . These results suggest that complex formation between the Rad1 and Rad10 proteins is required for nucleotide excision repair. EMBO J, 1993 Oct, 12(10), 3997 - 4003 A Saccharomyces cerevisiae homologue of mammalian translation initiation factor 4B contributes to RNA helicase activity; Altmann M et al.; The TIF3 gene of Saccharomyces cerevisiae was cloned and sequenced . The deduced amino acid sequence shows 26% identity with the sequence of mammalian translation initiation factor eIF-4B . The TIF3 gene is not essential for growth; however, its disruption results in a slow growth and cold-sensitive phenotype . In vitro translation of total yeast RNA in an extract from a TIF3 gene-disrupted strain is reduced compared with a wild-type extract . The translational defect is more pronounced at lower temperatures and can be corrected by the addition of wild-type extract or mammalian eIF-4B, but not by addition of mutant extract . In vivo translation of beta-galactosidase reporter mRNA with varying degree of RNA secondary structure in the 5' leader region in a TIF3 gene-disrupted strain shows preferential inhibition of translation of mRNA with more stable secondary structure . This indicates that Tif3 protein is an RNA helicase or contributes to RNA helicase activity in vivo. Yeast, 1993 Oct, 9(10), 1139 - 47 The RHO4a and NUD1 genes on Saccharomyces cerevisiae chromosome XI; Van Vliet-Reedijk JC et al.; Sequence analysis of a 4 kb fragment from the right arm of Saccharomyces cerevisiae chromosome XI, in combination with Northern hybridization experiments revealed the presence of two genes, designated RHO4a and NUD1 . The first gene encodes a 32 kDa protein showing significant sequence similarity with members of the ras family . Its 3'-terminal sequence is virtually identical to a sequence published previously as the RHO4 gene {Matsui and Toh-e, Gene 114 (1992), 43-49}, which, however, appears to start at an internal ATG codon . The RHO4a sequence also overlaps the 5'-terminal sequence of the RNC1 gene {Chow et al., Nucl . Acids Res . 20 (1992), 5215-5221} proposed to encode the yeast yNucR endo/exonuclease . The remainder of this RNC1 gene overlaps with the 5'-end of the NUD1 gene . However, the RNC1 sequence lacks a portion of 276 bp that in our fragment is part of the intergenic region separating RHO4a and NUD1 . From these results we conclude that the proposed RNC1 gene is the result of a cloning artefact and that the yNucR protein is instead encoded by the NUD1 gene. Yeast, 1993 Oct, 9(10), 1121 - 30 GUT2, a gene for mitochondrial glycerol 3-phosphate dehydrogenase of Saccharomyces cerevisiae; Ronnow B et al.; A gut2 mutant of Saccharomyces cerevisiae is deficient in the mitochondrial glycerol 3-phosphate dehydrogenase and hence cannot utilize glycerol . Upon transformation of a gut2 mutant strain with a low-copy yeast genomic library, hybrid plasmids were isolated which complemented the gut2 mutation . The nucleotide sequence of a 3.2 kb PstI-XhoI fragment complementing a gut2 mutant strain is presented . The fragment reveals an open reading frame (ORF) encoding a polypeptide with a predicted molecular weight of 68.8 kDa . Disruption of the ORF leads to a glycerol non-utilizing phenotype . A putative flavin-binding domain, located at the amino terminus, was identified by comparison with the amino acid sequences of other flavoproteins . The cloned gene has been mapped both physically and genetically to the left arm of chromosome IX, where the original gut2 mutation also maps . We conclude that the presented ORF is the GUT2 gene and propose that it is the structural gene for the mitochondrial glycerol 3-phosphate dehydrogenase. Yeast, 1993 Oct, 9(10), 1111 - 20 Identification of two divergently transcribed genes centromere-proximal to the ARG4 locus on chromosome VIII of Saccharomyces cerevisiae; Rocco V et al.; We have sequenced a 3296 bp segment of the chromosome VIII adjacent to the 3' end of the ARG4 gene . This segment contains two divergently oriented open reading frames (YSC83 and YSC84) . Northern blot analysis showed the presence of transcripts corresponding to these two open reading frames in vegetative cells . Levels of these transcripts increase five to ten-fold during sporulation . These two genes are not essential for vegetative growth or sporulation . Analysis of the putative protein products on the SwissProt database revealed that the C-terminal region of the Ysc84 protein contains a putative SH3 domain. Yeast, 1993 Oct, 9(10), 1107 - 10 The nucleotide sequence of a 2.1 kb fragment from chromosome VI of Saccharomyces cerevisiae identifies a tRNA(Gly) gene, part of a delta element and a palindromic sequence; Van Heusden GP et al.; The nucleotide sequence was determined of a 2.1 kb DNA fragment located at approximately 35 kb to the right of the centromere of chromosome VI from Saccharomyces cerevisiae . Analysis revealed the presence of a tRNA(GLy) gene, part of a delta element and a remarkable palindromic sequence . The longest open reading frame found encodes a putative protein of 195 amino acids . Although the fragment was isolated by hybridization to a human diacylglycerol kinase cDNA, no evidence was obtained for the presence of a gene encoding diacylglycerol kinase. Yeast, 1993 Oct, 9(10), 1065 - 73 Regulation of the amino acid permeases in nitrogen-limited continuous cultures of the yeast Saccharomyces cerevisiae; Olivera H et al.; In the yeast Saccharomyces cerevisiae, there is a general amino acid permease, regulated by nitrogen catabolite repression, and several specific permeases whose nitrogen regulation is not well understood . In this study, we used continuous cultures to analyse the effect of nitrogen limitation and pH on the activity of general and several specific amino acid permeases . General permease activity was maximal in severe nitrogen limitation and diminished 400-fold in cells grown under nitrogen excess . For the specific permeases, the maximal uptake activity was found between mild limitation and nitrogen excess, while very small activity was detected under strict limitation . These results indicate that the nitrogen regulation of the general and the specific amino acid carriers is coordinated in such a way that no redundancy exists in amino acid transport . The regulation of the specific permeases was similar to that found for a system with anabolic function in nitrogen metabolism . All of these permeases are supposed to work through a proton symport mechanism, and thus rely on pH gradients to carry out their function . We studied the effect of pH on the kinetic constants of the general permease . Our results show that the effect of pH on the Km was different for acidic, neutral and basic amino acids, while the effect on Vmax was independent of the electrical charge of the amino acids. J Gen Microbiol, 1993 Oct, 139 ( Pt 10), 2323 - 7 Protection by sterols against the cytotoxicity of syringomycin in the yeast Saccharomyces cerevisiae; Julmanop C et al.; A brief exposure (ca 20 min) of the yeast Saccharomyces cerevisiae to the phytotoxin syringomycin was sufficient to kill the cell . The protective effect of sterols against this cytotoxicity of syringomycin was investigated . Syringomycin was much more toxic to growing cells than to stationary-phase cells . The cytotoxicity of syringomycin was reduced in an environment containing sterols . Cytotoxicity of syringomycin at 3 micrograms ml-1 (ca 2.5 microM) was completely abolished by the simultaneous presence of 10 microM-cholesterol in the medium . Cholesterol acetate had no protective effect . Ergosterol, sitosterol and stigmasterol also protected against syringomycin, but they were less effective than cholesterol . The protective effect of sterols against the action of syringomycin is consistent with our hypothesis that membrane ergosterol is a critical component for syringomycin-binding as suggested by recent genetic studies. Curr Genet, 1993 Oct, 24(4), 307 - 12 Molecular cloning of the PEL1 gene of Saccharomyces cerevisiae that is essential for the viability of petite mutants; Janitor M et al.; The PEL1 gene of Saccharomyces cerevisiae is essential for the cell viability of mitochondrial petite mutants, for the ability to utilize glycerol and ethanol on synthetic medium, and for cell growth at higher temperatures . By tetrad analysis the gene was assigned to chromosome III, centromere proximal of LEU2 . The PEL1 gene has been isolated and cloned by the complementation of a pel1 mutation . The molecular analysis of the chromosomal insert carrying PEL1 revealed that this gene corresponds to the YCL4W open reading frame on the complete DNA sequence of chromosome III . The putative Pel1 protein is characterized by a low molecular weight of approximately 17 kDa, a low codon adaptation index, and a high leucine content. Lipids, 1993 Oct, 28(10), 907 - 12 General resistance to sterol biosynthesis inhibitors in Saccharomyces cerevisiae; Ladeveze V et al.; Screening for resistance to fenpropimorph was undertaken in order to isolate yeast mutants affected in the regulation of the ergosterol pathway . Among the mutants isolated, one bearing the recessive fen1-1 mutation was characterized by a 1.5-fold increase in the ergosterol level and a general resistance to sterol biosynthesis inhibitors . The fen1-1 mutation was linked to MAT locus on chromosome III . The measurement of enzyme activities involved in the ergosterol pathway revealed that isopentenyl diphosphate (IPP) isomerase activity was specifically increased 1.5-fold as compared to the wild type strain . However, overexpression of IPP isomerase in the wild type strain was not by itself sufficient to lead to sterol increase or resistance to sterol biosynthesis inhibitors, showing that IPP isomerase is not a limiting step in the pathway . The fen1-1 mutation permits viability in aerobiosis of yeast disrupted for sterol-14 reductase in absence of exogenous ergosterol supplementation, whereas the corresponding strain bearing the wild type FEN1 allele grows only in anaerobiosis . This result shows that ignosterol is able to efficiently replace ergosterol as bulk membrane component and that the fen1-1 mutation eliminates the specific ergosterol requirement in yeast. Genetics, 1993 Oct, 135(2), 343 - 55 The Drosophila melanogaster suppressor of Hairy-wing zinc finger protein has minimal effects on gene expression in Saccharomyces cerevisiae; Kim J et al.; Many mutations in Drosophila melanogaster are gypsy retrotransposon insertions . Gypsy binds the protein (SUHW) encoded by the suppressor of Hairy-wing {su(Hw)} gene, and SUHW alters expression of surrounding genes . When gypsy is between an enhancer and promoter, SUHW blocks activation of transcription by the enhancer . Additionally, when gypsy is downstream of a promoter in a parallel orientation, SUHW increases truncation of transcripts at the poly(A) site in the gypsy 5' long terminal repeat, thereby decreasing the gene transcript levels . The effects of SUHW appear to involve fundamental and general mechanisms controlling gene expression because SUHW potentiates other poly(A) sites and blocks several enhancers in Drosophila . To investigate these mechanisms, SUHW was expressed in Saccharomyces cerevisiae . Although SUHW enters the nucleus and binds DNA in yeast, it has surprisingly minor effects on utilization of the CYC1 poly(A) site and transcription activation by a GAL upstream activation sequence . These observations indicate that the observed effects of SUHW on gene expression in Drosophila require specific interactions with other factors that are absent or unrecognizable in yeast. Genetics, 1993 Oct, 135(2), 309 - 20 A rare tRNA-Arg(CCU) that regulates Ty1 element ribosomal frameshifting is essential for Ty1 retrotransposition in Saccharomyces cerevisiae; Kawakami K et al.; Translation of the yeast retrotransposon Ty1 TYA1(gag)-TYB1(pol) gene occurs by a +1 ribosomal frameshifting event at the sequence CUU AGG C . Because overexpression of a low abundance tRNA-Arg(CCU) encoded by the HSX1 gene resulted in a reduction in Ty1 frameshifting, it was suggested that a translational pause at the AGG-Arg codon is required for optimum frameshifting . The present work shows that the absence of tRNA-Arg(CCU) affects Ty1 transposition, translational frameshifting, and accumulation of mature TYB1 proteins . Transposition of genetically tagged Ty1 elements decreases at least 50-fold and translational frameshifting increases 3-17-fold in cells lacking tRNA-Arg(CCU) . Accumulation of Ty1-integrase and Ty1-reverse transcriptase/ribonuclease H is defective in an hsx1 mutant . The defect in Ty1 transposition is complemented by the wild-type HSX1 gene or a mutant tRNA-Arg(UCU) gene containing a C for T substitution in the first position of the anticodon . Overexpression of TYA1 stimulates Ty1 transposition 50-fold above wild-type levels when the level of transposition is compared in isogenic hsx1 and HSX1 strains . Thus, the HSX1 gene determines the ratio of the TYA1 to TYA1-TYB1 precursors required for protein processing or stability, and keeps expression of TYB1 a rate-limiting step in the retrotransposition cycle. Genetics, 1993 Oct, 135(2), 297 - 308 Mixed segregation and recombination of chromosomes and YACs during single-division meiosis in spo13 strains of Saccharomyces cerevisiae; Hugerat Y et al.; Diploid yeast strains, homozygous for the mutation spo13, undergo a single-division meiosis and form dyads (two spores held together in one ascus) . Dyad analysis of spo13/spo13 strains with centromere-linked markers on five different chromosomes and on a pair of human DNA YACs shows that: (a) in spo13 meiosis, chromosomes undergo mixed segregation, namely some chromosomes segregate reductionally whereas others, in the same cell, segregate equationally; (b) different chromosomes exhibit different segregation tendencies; (c) recombination between homologous chromosomes might not determine that a bivalent undergoes reductional rather than equational segregation. Genetics, 1993 Oct, 135(2), 287 - 96 Point mutations that separate the role of Saccharomyces cerevisiae centromere binding factor 1 in chromosome segregation from its role in transcriptional activation; Foreman PK et al.; Centromere binding factor 1 (Cbf1p or CP1) binds to the CDEI region of Saccharomyces cerevisiae centromeres and is a member of the basic helix-loop-helix (bHLH) class of proteins . Deletion of the gene encoding Cbf1p results in an increased frequency of chromosome loss, hypersensitivity to low levels of microtubule disrupting drugs (such as thiabendazole and benomyl) and methionine auxotrophy . By polymerase chain reaction-based random mutagenesis of the CBF1 gene we have obtained a number of mutant alleles that make full-length protein with impaired function . The mutations in these alleles are clustered in or just downstream from the bHLH domain . Among the alleles obtained was a class that was more compromised for transcriptional activation and a class that was more compromised for chromosome loss and thiabendazole hypersensitivity . These results indicate that at least some aspects of the role of Cbf1p in chromosome segregation and transcriptional activation are distinct . In contrast, increased chromosome loss and thiabendazole hypersensitivity were not separated in any of the alleles, suggesting that these phenotypes reflect the same mechanistic defect . These observations are consistent with a model that suggests that one role of Cbf1p in chromosome segregation may be to improve the efficiency with which contact between the kinetochore and spindle microtubules is established or maintained. Genetics, 1993 Oct, 135(2), 275 - 86 Genetic evidence for functional interactions between actin noncomplementing (Anc) gene products and actin cytoskeletal proteins in Saccharomyces cerevisiae; Vinh DB et al.; We describe here genetic interactions between mutant alleles of Actin-NonComplementing (ANC) genes and actin (ACT1) or actin-binding protein (SAC6, ABP1, TPM1) genes . The anc mutations were found to exhibit allele-specific noncomplementing interactions with different act1 mutations . In addition, mutant alleles of four ANC genes (ANC1, ANC2, ANC3 and ANC4) were tested for interactions with null alleles of actin-binding protein genes . An anc1 mutant allele failed to complement null alleles of the SAC6 and TPM1 genes that encode yeast fimbrin and tropomyosin, respectively . Also, synthetic lethality between anc3 and sac6 mutations, and between anc4 and tpm1 mutations was observed . Taken together, the above results strongly suggest that the ANC gene products contribute to diverse aspects of actin function . Finally, we report the results of tests of two models previously proposed to explain extragenic noncomplementation. Genetics, 1993 Oct, 135(2), 265 - 74 Screens for extragenic mutations that fail to complement act1 alleles identify genes that are important for actin function in Saccharomyces cerevisiae; Welch MD et al.; Null mutations in SAC6 and ABP1, genes that encode actin-binding proteins, failed to complement the temperature-sensitive phenotype caused by a mutation in the ACT1 gene . To identify novel genes whose protein products interact with actin, mutations that fail to complement act1-1 or act1-4, two temperature-sensitive alleles of ACT1, were isolated . A total of 14 extragenic noncomplementing mutations and 12 new alleles of ACT1 were identified in two independent screens . The 14 extragenic noncomplementing mutations represent alleles of at least four different genes, ANC1, ANC2, ANC3 and ANC4 (Actin NonComplementing) . Mutations in the ANC1 gene were shown to cause osmosensitivity and defects in actin organization; phenotypes that are similar to those caused by act1 mutations . We conclude that the ANC1 gene product plays an important role in actin cytoskeletal function . The 12 new alleles of ACT1 will be useful for further elucidation of the functions of actin in yeast. FEMS Microbiol Lett, 1993 Oct 1, 113(1), 35 - 41 A method for enucleation of Saccharomyces cerevisiae; Salek AT; The first method for enucleation of yeast Saccharomyces cerevisiae is reported . Various strains, including some killer strain and respiratory-deficient mutants of Saccharomyces cerevisiae were enucleated after treatment with cytochalasin B . Removal of nuclei from protruding sphaeroplasts was induced by centrifugation in a Percoll density gradient . The enucleation yield (which averaged about 80%) and the quality of the cytoplasts were best when the yeast culture had been synchronized with nocodazole before the preparation . The presence of 1 mM CaCl2 and ATP (10 microM) in the enucleation medium prevented the formation of fragile products or aggregation . Cytoplasts could be stored for at least 1 day without visible deterioration. FEMS Microbiol Lett, 1993 Oct 1, 113(1), 119 - 24 Identification of a nuclear pheromone-sensitive protein kinase not identical to p34CDC28 in Saccharomyces cerevisiae; Nientiedt M et al.; Nuclei of Saccharomyces cerevisiae cells contain a protein kinase, the activity of which is drastically reduced in response to an activation of the mating signal pathway by pheromone . Inhibition of this pheromone-sensitive kinase is also observed under conditions of constitutive activation of the signal pathway in a temperature-sensitive cdc70 mutant . The enzyme, which by SDS-PAGE has a molecular mass of 34,500 Da, is a protein serine kinase that phosphorylates several endogenous substrates in nuclear extracts . The activity of this kinase is temperature-resistant in a temperature-sensitive cdc28 mutant, indicating that it is not identical to p34CDC28, the catalytic component of the cell cycle protein kinase complex. Science, 1993 Oct 1, 262(5130), 110 - 4 Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping; Schwartz DC et al.; A light microscope-based technique for rapidly constructing ordered physical maps of chromosomes has been developed . Restriction enzyme digestion of elongated individual DNA molecules (about 0.2 to 1.0 megabases in size) was imaged by fluorescence microscopy after fixation in agarose gel . The size of the resulting individual restriction fragments was determined by relative fluorescence intensity and apparent molecular contour length . Ordered restriction maps were then created from genomic DNA without reliance on cloned or amplified sequences for hybridization or analytical gel electrophoresis . Initial application of optical mapping is described for Saccharomyces cerevisiae chromosomes. Eur J Biochem, 1993 Oct 1, 217(1), 487 - 92 TKL2, a second transketolase gene of Saccharomyces cerevisiae . Cloning, sequence and deletion analysis of the gene; Schaaff-Gerstenschlager I et al.; Transketolase activity is indispensable for the generation of erythrose 4-phosphate and therefore necessary for the biosynthesis of the aromatic amino acids . Yeast mutants with a deletion of the transketolase gene, TKL1, can grow without aromatic amino acid supplement indicating an additional source of erythrose 4-phosphate in the cells . Here we describe the cloning of TKL2, a gene coding for a second transketolase enzyme in Saccharomyces cerevisiae . The deduced protein sequence of TKL2 demonstrates 71% identity with TKL1 {Sundstrom, M., Lindqvist, Y., Schneider, G., Hellman, U . & Ronne, H . (1993) J . Biol . Chem., in the press} . Double mutants for both genes, TKL1 and TKL2, are auxotrophic for aromatic amino acids, indicating a complete block in the transketolase activity . Deletion of TKL2 alone does not lead to a significant phenotype, and transketolase activity is not reduced in these mutants . Overexpression of TKL2 on a multi-copy plasmid in a tkl1 background showed that TKL2 is functionally expressed: transketolase enzyme activity was detectable in the transformants and the protein reacts with anti-transketolase serum in Western blot analysis . In addition, transformation of the tkl1 tkl2 double mutant with the TKL2 plasmid can compensate the growth defect on a medium without aromatic amino acids. Biochem Med Metab Biol, 1993 Oct, 50(2), 135 - 44 Expression of recombinant human methylmalonyl-CoA mutase: in primary mut fibroblasts and Saccharomyces cerevisiae; Andrews E et al.; Methylmalonyl-CoA mutase is an adenosylcobalamin-dependent enzyme which catalyzes isomerization of methylmalonyl-CoA to succinyl-CoA . Previous reports have described cloning and sequencing of a cDNA for human methylmalonyl-CoA mutase . This clone does not express an active apoenzyme after gene transfer into primary MCM-deficient fibroblasts and contains several sequences which differ from the consensus sequence of other cDNA clones . We describe reconstruction of a functional MCM cDNA and expression of recombinant enzyme activity in primary fibroblasts and Saccharomyces cerevisiae . This consensus human MCM cDNA is capable of complementing the inherited defect in mut MMA and overexpressing an enzyme in yeast with kinetic properties indistinguishable from the enzyme in murine or human tissues. Eur J Biochem, 1993 Oct 1, 217(1), 469 - 77 The role of the NAD-dependent glutamate dehydrogenase in restoring growth on glucose of a Saccharomyces cerevisiae phosphoglucose isomerase mutant; Boles E et al.; Phosphoglucose isomerase pgi1-deletion mutants of Saccharomyces cerevisiae cannot grow on glucose as the sole carbon source and are even inhibited by glucose . These growth defects could be suppressed by an over-expression on a multi-copy plasmid of the structural gene GDH2 coding for the NAD-dependent glutamate dehydrogenase . GDH2 codes for a protein with 1092 amino acids which is located on chromosome XII and shows high sequence similarity to the Neurospora crassa NAD-glutamate dehydrogenase . Suppression of the pgi1 deletion by over-expression of GDH2 was abolished in strains with a deletion of the glucose-6-phosphate dehydrogenase gene ZWF1 or gene GDH1 coding for the NADPH-dependent glutamate dehydrogenase . Moreover, this suppression required functional mitochondria . It is proposed that the growth defect of pgi1 deletion mutants on glucose is due to a rapid depletion of NADP which is needed as a cofactor in the oxidative reactions of the pentose phosphate pathway . Over-expression of the NAD-dependent glutamate dehydrogenase leads to a very efficient conversion of glutamate with NADH generation to 2-oxoglutarate which can be converted back to glutamate by the NADPH-dependent glutamate dehydrogenase with the consumption of NADPH . Consequently, over-expression of the NAD-dependent glutamate dehydrogenase causes a substrate cycling between 2-oxoglutarate and glutamate which restores NADP from NADPH through the coupled conversion of NAD to NADH which can be oxidized in the mitochondria . Furthermore, the requirement for an increase in NADPH consumption for the suppression of the phosphoglucose isomerase defect could be met by addition of oxidizing agents which are known to reduce the level of NADPH. Biosci Biotechnol Biochem, 1993 Oct, 57(10), 1686 - 90 Secretion and overproduction of carboxypeptidase Y by a Saccharomyces cerevisiae ssl1 mutant strain; Ichikawa K et al.; Carboxypeptidase Y (CPY; EC 3.4.16.1) is the yeast vacuolar protease . To have CPY secreted and to increase its secretion level, we tried to express the prepro-CPY gene under the control of the inducible GAL10 promoter or constitutive ENO1 promoter on a multicopy plasmid . In the strains KK4, PEP4, and A2-1-1A, carrying the CPY expression plasmid, active CPY was not detected in the culture broth although the CPY activity was greatly increased inside the cells . In contrast, when we used a strain that contained the ssl1 (super-secretion of lysozyme) mutation, a large amount of active CPY (about 10-50 mg/liter) was detected in the culture broth . The ssl1 mutants secreted active CPY when the CPY level was increased by expressing it under the control of a strong promoter on a multicopy plasmid, while the endogenous expression of chromosomal CPY gene in the same ssl1 mutant caused a deficiency in the processing of pro-CPY to mature CPY. Mutat Res, 1993 Oct, 289(2), 157 - 63 Induction of mitotic crossing-over by the topoisomerase II poison DACA (N-{2-dimethylamino)ethyl}acridine-4-carboxamide) in Saccharomyces cerevisiae; Ferguson LR et al.; The antitumor agent DACA (N-{2-dimethylamino)ethyl}acridine-4-carboxamide) a new DNA intercalating topoisomerase II poison, was distinguishable from clinical topoisomerase poisons (amsacrine, daunorubicin, doxorubicin and etoposide) in its induction of aberrant colonies in the yeast Saccharomyces cerevisiae D5 . It was not only more recombinogenic, but was recombinogenic at non-toxic drug concentrations . DACA at 680 microM (2-h exposure time), induced 1.2% aberrant colonies of which 0.32% were mitotic crossing-over events . The presence of the rad52 mutation abolished mitotic crossing-over and greatly increased drug toxicity . The concentration for 50% inhibition of survival of the rad52 mutant was 100 microM, as compared with 4900 microM for the wild-type . Drug toxicity was marginally increased by the presence of rad3 and rad18 mutations . Rad3 mutations increased the incidence of crossing-over events but had little effect on other mutagenic or recombinogenic events . In contrast, the rad18 mutation increased the incidence of all types of aberrant colonies . The inclusion of hydroxyurea and caffeine, as non-specific repair inhibitors, caused weak and strong inhibition, respectively, of all types of aberrant colonies . Inclusion of the protein-synthesis inhibitor cycloheximide reduced mitotic cross-over but had little effect on the incidence of other aberrations . It is concluded that DACA induces lesions which are repaired by a recombinational repair pathway involving the RAD52 product, and that RAD3 and RAD18 products are each involved in the generation of recombinational events. Gene, 1993 Sep 30, 132(1), 41 - 7 Protein farnesyltransferase: production in Escherichia coli and immunoaffinity purification of the heterodimer from Saccharomyces cerevisiae; Mayer MP et al.; Protein farnesylation in Saccharomyces cerevisiae is mediated by a heterodimeric enzyme, protein farnesyltransferase (PFTase), encoded by the genes RAM1 and RAM2 . A series of plasmids for the expression of RAM1 and RAM2 in Escherichia coli was prepared and evaluated . Maximal production of functional PFTase was seen in strains containing a multicopy plasmid with a synthetic operon in which the RAM1 and RAM2 structural genes were translationally coupled by overlapping TAATG stop-start codons and by locating a ribosome-binding site near the 3' end of the upstream gene . This was accomplished by an insertional mutation at the 3'-end of RAM1 that embedded an AGGAGGAG sequence within codons for the tetrapeptide, QEEF, added to the end of the Ram1 protein . The QEEF C-terminal motif in the Ram1 subunit of PFTase facilitated purification of the enzyme by immunoaffinity chromatography on an anti-alpha-tubulin column prepared using monoclonal antibodies that recognized a tripeptide EEF epitope . Heterodimeric recombinant yeast PFTase::QEEF (re-PFTase::QEEF) constituted approximately 4% of total soluble protein in induced cells and was readily purified 25-fold in two steps by ion exchange and immunoaffinity chromatography in an overall 25% yield . Michaelis constants for farnesyl diphosphate (FPP) and Hras protein (modified to contain a yeast a-mating factor PACVIA sequence at the C terminus) were 5.5 and 15 microM, respectively; the kcat was 0.7 s-1. Gene, 1993 Sep 30, 132(1), 33 - 40 High-level heterologous gene expression in Saccharomyces cerevisiae from a stable 2 microns plasmid system; Ludwig DL et al.; The best candidate for a high-copy-number and mitotic stability expression system in yeast is the endogenous 2 microns plasmid . Nevertheless, derivatives of the 2 microns plasmid typically exhibit lower copy numbers and require selection for adequate maintenance within cells . We report the construction and utilization of an efficient heterologous gene expression system containing a 4.5-kb inducible expression cassette inserted into the 2 microns plasmid and selected in cells utilizing a carrier plasmid which is subsequently lost via FRT/Flp recombination . The non-selectable 2 micron plasmid, containing the cassette, was found to be stably maintained in cells, without selection, at high copy number . The dynamics of resolution and partitioning of this plasmid were analyzed during the course of 50 generations of growth under non-selective conditions . The heterologous lacZ reporter gene coding for beta-galactosidase (beta Gal) is driven by the hybrid, galactose-inducible promoter GAL10::pMF alpha 1 . Upon induction, beta Gal was secreted into the periplasm and culture supernatant at levels which could be detected directly from Coomassie blue-stained SDS-PAGE . Furthermore, plasmid-containing cells could be maintained directly on rich YPD medium and identified either by utilizing XGal or by observing inhibition of colony growth on YPGal solid medium . The cassette was designed for direct, high-level, inducible expression of cloned genes downstream from the MF alpha 1 signal sequence, with or without a C-terminal lacZ fusion . This vector represents the first demonstration of a non-selectable, mitotically stable, episomal plasmid system capable of expressing recombinant proteins at high levels.(ABSTRACT TRUNCATED AT 250 WORDS) Gene, 1993 Sep 30, 132(1), 149 - 54 Diphthamide synthesis in Saccharomyces cerevisiae: structure of the DPH2 gene; Mattheakis LC et al.; A gene involved in diphthamide biosynthesis, DPH2, was cloned from Saccharomyces cerevisiae by complementation of a diphthamide mutant . DPH2 exists as a single-copy gene in the yeast genome and is located on the left arm of chromosome XI . Sequence analysis of the DPH2 locus predicts that the DPH2 gene product is a 534-amino acid (aa) protein, with a calculated M(r) of 59,772 . This conclusion was supported by Northern blot analysis of the DPH2 transcript and gel analysis of the DPH2 protein overproduced in Escherichia coli . Gene disruption studies indicate that the DPH2 gene is not essential for viability of yeast . The role of DPH2 in diphthamide biosynthesis is discussed. J Biol Chem, 1993 Sep 25, 268(27), 20191 - 7 Characterization of two novel single-stranded DNA-specific autonomously replicating sequence-binding proteins from Saccharomyces cerevisiae, one of which is adenylosuccinate synthetase; Zeidler R et al.; We report here the identification and characterization of two novel proteis from Saccharomyces cerevisiae that bind to the T-rich strand of the core consensus autonomously replicating sequence (ARS) in a highly specific manner . The two proteins, 40 and 45 kDa in size, can be distinguished by multiple criteria from each other and from the 65-kDa ssArS-T-binding protein identified recently in our laboratory (Schmidt, A . M . A., Herterich, S . U., and Krauss, G . (1991) EMBO J . 10, 981-985) . The specificity of binding is inferred from gel shift and nuclease-footprinting experiments using single-stranded probes containing the core consensus ARS . With a 321-nucleotide single-stranded ARS1 fragment, specific protection of the A and B1 domain against DNase I digestion is observed . Partial amino acid sequencing and enzymatic assays identify the 45-kDa protein as adenylosuccinate synthetase, an enzyme necessary for the de novo synthesis of adenylate. J Biol Chem, 1993 Sep 25, 268(27), 20533 - 9 Intracellular localization of the Apn1 DNA repair enzyme of Saccharomyces cerevisiae . Nuclear transport signals and biological role; Ramotar D et al.; The Apn1 DNA repair enzyme of Saccharomyces cerevisiae acts on abasic sites and oxygen radical damages . Apn1 is homologous to the repair endonuclease IV of Escherichia coli, but the yeast protein is approximately 80 residues longer at the C terminus . The Apn1 C terminus is rich in basic amino acids and includes two lysine/arginine clusters related to the nuclear transport signals of some other proteins . We show here by indirect immunofluorescence that Apn1 is localized to the yeast nucleus . Mutant Apn1 proteins were engineered with progressive deletions inward from the C terminus . Elimination of just the last 12 residues from Apn1 (to yield Apn355) did not alter the stability in yeast cells or the in vitro activity of the enzyme . Greater truncation of Apn1 produced proteins of apparently lower (Apn334) or much lower (Apn315 and Apn293) in vivo stability . Both Apn355 and Apn334 failed to concentrate in the yeast nucleus and remained in the cytoplasm . These delocalized derivatives also failed to restore wild-type resistance to oxidative or alkylating agents in a delta apn1 strain . Apn355 and Apn334 complemented repair-deficient E . coli as effectively as did wild-type Apn1 . Resistance to these DNA-damaging agents in yeast was restored if Apn355 and Apn334 (but not Apn315 or Apn293) were overproduced approximately 20-fold, which suggests either weak active transport or passive diffusion of these derivatives into the nucleus . Replacement of the C-terminal 12 residues of Apn1 with the nuclear targeting sequence of SV40 T-antigen did not restore effective function or nuclear localization in yeast. Biochemistry, 1993 Sep 21, 32(37), 9563 - 9 Chicken progesterone receptor expressed in Saccharomyces cerevisiae is correctly phosphorylated at all four Ser-Pro phosphorylation sites; Poletti A et al.; This study describes the phosphorylation of chicken progesterone receptor (cPR) produced in yeast, Saccharomyces cerevisiae, and examines the dependence of specific phosphorylations on hormone and DNA binding . The chicken progesterone receptor is expressed in vivo as two forms, cPRB and a smaller form, cPRA . Characterization of the phosphorylation sites in the cPRB form expressed in yeast shows that progesterone receptor is phosphorylated on the three serines (Ser211, Ser260, and Ser530) reported previously in chicken oviduct . An additional site which was phosphorylated in response to hormone was also detected and was subsequently identified as Ser367 . Although cPRB and cPRA are phosphorylated identically in chicken oviduct, cPRA expressed in yeast is phosphorylated on Ser211, Ser260, and Ser367, but phosphorylation of Ser530 is almost undetectable . In contrast, cPRB expressed in yeast is phosphorylated on all four sites . No phosphorylations were found in or near the region required for hormone binding, indicating that phosphorylation is not required for hormone binding . In order to determine whether any of the phosphorylations were DNA-dependent, phosphorylation was also studied using cPRA containing a partial deletion of the DNA binding domain . Two of the sites, Ser211 and Ser367, showed reduced phosphorylation in this mutant, suggesting a possible requirement for DNA binding activity for the phosphorylation of these sites . To our knowledge, this is one of the first demonstrations that a eucaryotic protein expressed in yeast is correctly phosphorylated. Eur J Biochem, 1993 Sep 15, 216(3), 849 - 61 Cloning of two related genes encoding the 56-kDa and 123-kDa subunits of trehalose synthase from the yeast Saccharomyces cerevisiae; Vuorio OE et al.; Preparations of intact trehalose synthase contain three polypeptides with molecular masses of 56, 102 and 123 kDa . We have cloned the genes TSS1 and TSL1 coding for the 56- and 123-kDa subunits, respectively . These genes are located on chromosomes II (TSS1) and XIII (TSL1) . The TSS1 gene was found to be identical with CIF1, a gene required for normal growth on glucose . The product of the entire TSS1 gene exhibits 37% identity with a 502-amino-acid stretch from the middle of the TSL1 product . Disruption of the TSS1 gene in yeast eliminates both trehalose 6-phosphate synthase (Tre6P synthase) and trehalose 6-phosphate phosphatase (Tre6Pase) activities, and reintroduction of this gene restores these activities . Transformation of Escherichia coli with TSS1 increases its Tre6P synthase activity . Specific proteolytic degradation of the 123-kDa polypeptide from the N-terminus greatly influences the Tre6P synthase activity, decreasing its inhibition by phosphate and activatability by fructose 6-phosphate but has little effect on the Tre6Pase activity . These results suggest that this N-terminal part confers regulatory properties upon the Tre6P synthase activity. J Biol Chem, 1993 Sep 15, 268(26), 19753 - 9 Enzymatic properties of the PMA2 plasma membrane-bound H(+)-ATPase of Saccharomyces cerevisiae; Supply P et al.; The PMA1 H(+)-ATPase can be functionally replaced by its isoform PMA2 in the plasma membrane from Saccharomyces cerevisiae (Supply, P., Wach, A., Thines-Sempoux, D., and Goffeau, A . (1993) J . Biol . Chem . 268, 19744-19752) . From strains expressing either only PMA1 or PMA2, plasma membranes were isolated and their ATPase activities compared . Despite their 89% identity, the two enzymes differ as to the following parameters: activation by glucose and by Triton X-100, pH optimum, requirement for divalent cations, and inhibition by vanadate and by erythrosin B . More striking, the glucose-activated PMA2 enzyme displays a three to four times higher apparent affinity for MgATP, and maximal activity is reached with a 10-fold lower free Mg2+ concentration . These results suggest that the difference in PMA1 and PMA2 expression level is correlated with different H(+)-ATPase functions . The analysis of the PMA1 and PMA2 sequence alignment, compared with reported PMA1 mutations, points to a few residue substitutions as putative contributors to the observed kinetic changes. J Biol Chem, 1993 Sep 15, 268(26), 19744 - 52 Proliferation of intracellular structures upon overexpression of the PMA2 ATPase in Saccharomyces cerevisiae; Supply P et al.; The PMA2 gene is a presumed isogene of the PMA1 gene, encoding the major yeast plasma membrane H(+)-ATPase . When controlled by its own promoter, PMA2 in multiple copies does not complement a deficient PMA1 gene . Under the control of the PMA1 promoter, however, and expressed on a centromeric plasmid in yeast strains specially designed for stable expression, the PMA2 gene replaces the PMA1 gene to some extent, allowing growth on standard medium but not on acidic media . Plasma membranes of cells expressing only the PMA2 enzyme display low ATPase activity correlating with low amounts of PMA2 protein . This low activity is maintained throughout growth and does not increase when overexpression is favored by increased gene dosage . Immunoelectron microscopy reveals a dramatic proliferation of intracellular structures (probably endoplasmic reticulum) in which overexpressed PMA2 protein accumulates . Overexpression of PMA1 ATPase causes a similar phenomenon, but quantitative effects are lower compared to PMA2 . These results indicate that the PMA2 gene encodes a functional plasma membrane H(+)-ATPase and suggest a specific control of the intracellular traffic of plasma membrane ATPase. J Biol Chem, 1993 Sep 15, 268(26), 19669 - 74 Ribosomal protein L32 of Saccharomyces cerevisiae regulates both splicing and translation of its own transcript; Dabeva MD et al.; Ribosomal protein L32 of Saccharomyces cerevisiae regulates the splicing of its own transcript (1, 2) apparently by interacting with a structure composed largely of the 5' exon . However, even in strains overproducing L32 mRNA, e.g . from a cDNA copy of the gene, little accumulation of L32 is observed after a brief pulse label . When the 5' leader of the RPL32 mRNA is replaced by an exogenous leader, the amount of pulse-labeled L32 increases severalfold, suggesting that L32 regulates the translation of its own mRNA, acting through sequences in the 5' region . This conclusion was confirmed by the observation that in cells carrying a chimeric gene in which the L32 leader is fused to LacZ coding sequences, the presence of a second gene that overexpresses L32 itself reduces the level of beta-galactosidase by 50%, in spite of a doubling of L32-lacZ fusion mRNA, presumably due to stabilization of the message . Mutations within the 5' leader that abolish the regulation of splicing also abolish the regulation of translation, suggesting that the regulation of translation by L32 involves a structure similar to that proposed for the regulation of splicing . In cells overproducing L32-mRNA about half the excess mRNA was found in ribonucleoproteins of < 25 S, unassociated with ribosomal particles . Much of the rest was found in ribonucleoproteins of 80-120 S. J Biol Chem, 1993 Sep 15, 268(26), 19436 - 44 The Saccharomyces cerevisiae LOS1 gene involved in pre-tRNA splicing encodes a nuclear protein that behaves as a component of the nuclear matrix; Shen WC et al.; Mutations of the Saccharomyces cerevisiae LOS1 gene cause the accumulation of end matured intron-containing pre-tRNAs at elevated temperatures . In an effort to decipher the role of the LOS1 protein in pre-tRNA splicing, we have analyzed the LOS1 gene and its protein product . The LOS1 gene is located on the left arm of chromosome XI and the order of genes in this area of the chromosome is ... . URA1 .. . SAC1 TRP3 UBA1 STE6 LOS1 ... . FAS1.... . The LOS1 open reading frame encodes a putative protein of 1100 amino acids that shows no significant homology to other genes . The LOS1 open reading frame was tagged with the influenza virus hemagglutinin epitope recognized by the 12CA5 antibody . The 12CA5 antibody recognizes an epitope-tagged protein of the size predicted by the LOS1 open reading frame . Using this antibody for indirect immunofluorescence and cell fractionation studies we show that the LOS1 protein is located in nuclei . Los1p cannot be extracted from nuclei by treatment with nucleases, salts, or Triton X-100 . This insolubility suggests that Los1p is a component of the nucleoskeleton . We propose that LOS1 mutations may affect pre-tRNA processing via alteration of the nuclear matrix. FEMS Microbiol Lett, 1993 Sep 15, 112(3), 325 - 8 The deficiency of sterol biosynthesis in Saccharomyces cerevisiae affects the synthesis of glycosyl derivatives of dolichyl phosphates; Szkopinska A et al.; Mutants deficient in sterol (thermosensitive ergosterol auxotrophs) erg 8, 9, 12 and heme synthesis hem 1, 12 were screened for the level of free dolichol and dolichyl phosphate synthesized in the mevalonate pathway as well as for the activity of dolichyl phosphate-dependent glycosyl transferases . The amount of DolP synthesized via CTP-dependent phosphorylation was the same in mutants and parental strains . However, mannosylation and glucosylation of endogenous dolichyl phosphates in ergosterol mutants was about four times lower compared to parental strains, while the same reactions carried out with exogenous Dol24P reached 80% of the level observed in parental strains indicating that activities of DolPMan and DolPGlc synthases are not the rate-limiting factors . It is postulated that the de novo synthesis of DolP is impaired in the ergosterol mutants . Moreover, a block in the ergosterol branch of the metabolic pathway (erg 9) causes an increase in the de novo synthesis of dolichyl phosphate. J Mol Biol, 1993 Sep 5, 233(1), 183 - 8 A Saccharomyces cerevisiae cyclophilin resident in the endoplasmic reticulum; Frigerio G et al.; Cyclophilins are cyclosporin A sensitive peptidyl-prolyl cis-trans isomerases found in the cytoplasm of a wide range of species from Escherichia coli to man . Cyclophilin homologues are translocated into mitochondria, the endoplasmic reticulum and the bacterial periplasmic space . Here, the nucleotide sequence of a non-essential fifth Saccharomyces cerevisiae cyclophilin homologue encoded by chromosome XII is presented . As expected from its hydrophobic signal sequence and a hydrophilic carboxy terminus ending in the tetrapeptide HDEL, epitope-tagged cyclophilin D was found associated with the endoplasmic reticulum. Mol Gen Genet, 1993 Sep, 240(3), 419 - 27 The NAM1/MTF2 nuclear gene product is selectively required for the stability and/or processing of mitochondrial transcripts of the atp6 and of the mosaic, cox1 and cytb genes in Saccharomyces cerevisiae; Groudinsky O et al.; The NAM1/MTF2 gene was firstly isolated as a multicopy suppressor of mitochondrial splicing deficiencies and independently as a gene of which a thermosensitive allele affects mitochondrial transcription in organello . To determine which step in mitochondrial RNA metabolism is controlled in vivo by the NAM1 gene, mitochondrial transcripts of seven transcription units from strains carrying an inactive nam1::URA3 gene disruption in various mitochondrial genetic backgrounds were analysed by Northern blot hybridisations . In a strain carrying an intron-containing mitochondrial genome, the inactivation of the NAM1 gene led to a strong decrease in (or total absence of) the mosaic cytb and cox1 mRNAs and in transcripts of the atp6-rf3/ens2 genes, which are co-transcribed with cox1 . Neither the accumulation of unspliced cytb or cox1 pre-mRNAs, nor that of excised circular intron molecules of ai1 or ai2 were observed, but the abundance of the bi1 and ai7 lariats was comparable to that observed in the wild-type strain, thus demonstrating that transcription of the cytb and cox1 genes does occur . In strains carrying the intron-less mitochondrial genome with or without the rf3/ens2 sequence, wild-type amounts of cytb and cox1 mRNAs were detected while the amount of the atp6 mRNA was always strongly decreased . The abundance of transcripts from five other genes was either slightly (21S rRNA) or not at all (cox2, cox3, atp9 and 15S rRNA) affected by the nam1 inactivation.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1993 Sep, 240(3), 374 - 86 The centromere and promoter factor, 1, CPF1, of Saccharomyces cerevisiae modulates gene activity through a family of factors including SPT21, RPD1 (SIN3), RPD3 and CCR4; McKenzie EA et al.; In Saccharomyces cerevisiae, the CPF1 gene encodes a centromere binding protein that also plays a role in transcription; cpf1 strains are methionine auxotrophs . In this paper we describe four strains that are methionine prototrophs despite containing a defective CPF1 gene . These strains, which contain mutations at either the SPT21, RPD1 (SIN3), RPD3 or CCR4 loci, have defective centromere function and a chromatin structure around the CDEI elements in the MET25 promoter characteristic of strains lacking CPF1 . This indicates that the roles of CPF1 in transcription, centromere function and chromatin modulation around CDEI sites are different . We propose that CPF1 functions to overcome the repressing action, mediated via inactive chromatin, of proteins such as SPT21 or RPD1 (SIN3) on gene expression . The absence of proteins such as SPT21 or RPD1 (SIN3) relieves this repression and explains how methionine prototrophy is restored in the absence of CPF1. Mol Gen Genet, 1993 Sep, 240(3), 323 - 32 MSI3, a multicopy suppressor of mutants hyperactivated in the RAS-cAMP pathway, encodes a novel HSP70 protein of Saccharomyces cerevisiae; Shirayama M et al.; The MSI3 gene was isolated as a multicopy suppressor of the heat shock-sensitive phenotype of the ira1 mutation, which causes hyperactivation of the RAS-cAMP pathway . Overexpression of MSI3 also suppresses the heat shock-sensitive phenotype of the bcy1 mutant . Determination of the DNA sequence of MSI3 revealed that MSI3 can encode a 77.4 kDa protein related to the HSP70 family . The amino acid sequence of Msi3p is about 30% identical to that of the Ssa1p of Saccharomyces cerevisiae . This contrasts with the finding that members of the HSP70 family generally show at least 50% amino acid identity . The consensus nucleotide sequence of the heat shock element (HSE) was found in the upstream region of MSI3 . Moreover, the steady-state levels of the MSI3 mRNA and protein were increased upon heat shock . These results indicate that the MSI3 gene encodes a novel HSP70-like heat shock protein . Disruption of the MSI3 gene was associated with a temperature sensitive growth phenotype but unexpectedly, thermotolerance was enhanced in the disruptant. Eur J Biochem, 1993 Sep 1, 216(2), 573 - 7 A novel primary Ca(2+)-transport system from Saccharomyces cerevisiae; Okorokov LA et al.; A novel primary Ca(2+)-transport system in membranes from Saccharomyces cerevisiae is described . Ca2+ transport is strictly dependent on the presence of ATP; other nucleotides like GTP, UTP and CTP do not efficiently (< 10% of the rate of ATP) drive uptake . Transport is inhibited by sodium vanadate with an IC50 of 130 microM, but is insensitive to carbonylcyanide p-trifluoromethoxy-phenylhydrazone, valinomycin, gramicidin or calmodulin . Ca2+ accumulates in a free form and can be readily released by the Ca2+ ionophore A-23187 or by osmotic shock . The apparent Km values of transport activity for free Ca2+ was determined to be 0.11 microM and 5 microM for Mg.ATP, respectively . Taken together the results indicate that the Ca2+ transport described here does not belong to the plasma-membrane-type Ca(2+)-ATPase family but rather to the family of endomembrane-type ATPases . Cell-fractionation studies of crude membranes on sucrose gradient centrifugation have shown that the Ca(2+)-transport activity separates from marker enzymes for endoplasmic reticulum, vacuole, or plasma membrane and migrates with GDPase activity, a marker for the yeast Golgi complex. Mol Cell Biol, 1993 Sep, 13(9), 5408 - 17 Mutations in a protein tyrosine phosphatase gene (PTP2) and a protein serine/threonine phosphatase gene (PTC1) cause a synthetic growth defect in Saccharomyces cerevisiae; Maeda T et al.; Two protein tyrosine phosphatase genes, PTP1 and PTP2, are known in Saccharomyces cerevisiae . However, the functions of these tyrosine phosphatases are unknown, because mutations in either or both phosphatase genes have no clear phenotypic effects . In this report, we demonstrate that although ptp2 has no obvious phenotype by itself, it has a profound effect on cell growth when combined with mutations in a novel protein phosphatase gene . Using a colony color sectoring assay, we isolated 25 mutants in which the expression of PTP1 or PTP2 is required for growth . Complementation tests of the mutants showed that they have a mutation in one of three genes . Cloning and sequence determination of one of these gene, PTC1, indicated that it encodes a homolog of the mammalian protein serine/threonine phosphatase 2C (PP2C) . The amino acid sequence of the PTC1 product is approximately 35% identical to PP2C . Disruption of PTC1 indicated that the PTC1 function is nonessential . In contrast, ptc1 ptp2 double mutants showed a marked growth defect . To examine whether PTC1 encodes an active protein phosphatase, a glutathione S-transferase (GST)-PTC1 fusion gene was constructed and expressed in Escherichia coli . Purified GST-PTC1 fusion protein hydrolyzed a serine phosphorylated substrate in the presence of the divalent cation Mg2+ or Mn2+ . GST-PTC1 also had weak (approximately 0.5% of its serine phosphatase activity) protein tyrosine phosphatase activity. Mol Cell Biol, 1993 Sep, 13(9), 5393 - 407 Mutational and functional analysis of dominant SPT2 (SIN1) suppressor alleles in Saccharomyces cerevisiae; Lefebvre L et al.; The Saccharomyces cerevisiae SPT2 gene was identified by genetic screens for mutations which are suppressors of Ty and delta insertional mutations at the HIS4 locus . The ability of spt2 mutations to suppress the transcriptional interference caused by the delta promoter insertion his-4-912 delta correlates with an increase in wild-type HIS4 mRNA levels . The SPT2 gene is identical to SIN1, which codes for a factor genetically defined as a negative regulator of HO transcription . Mutations in SPT2/SIN1 suppress the effects of trans-acting mutations in SWI genes and of partial deletions in the C-terminal domain of the largest subunit of RNA polymerase II . Nuclear localization and protein sequence similarities suggested that the SPT2/SIN1 protein may be related to the nonhistone chromosomal protein HMG1 . To assess the significance of this structural similarity and identify domains of SPT2 functionally important in the regulation of his4-912 delta, we have studied recessive and dominant spt2 mutations created by in vitro mutagenesis . We show here that several alleles carrying C-terminal deletions as well as point mutations in the C-terminal domain of the SPT2 protein exhibit a dominant suppressor phenotype . C-terminal basic residues necessary for wild-type SPT2 protein function which are absent from HMG1 have been identified . The competence of these mutant SPT2 proteins to interfere with the maintenance of the His- (Spt+) phenotype of a his4-912 delta SPT2+ strain is lost by deletion of internal HMG1-like sequences and is sensitive to the wild-type SPT2+ gene dosage . Using cross-reacting antipeptide polyclonal antibodies, we demonstrate that the intracellular level of the wild-type SPT2 protein is not affected in presence of dominant mutations and furthermore that the reversion of the dominance by internal deletion of HMG1-like sequences is not mediated by altered production or stability of the mutant polypeptides . Our results suggest that the products of dominant alleles directly compete with the wild-type protein . On the basis of primary sequence similarities, we propose that an HMG-box-like motif is required for SPT2 function in vivo and that this motif also is necessary for the dominant suppressor phenotype exhibited by some mutant SPT2 alleles. J Bacteriol, 1993 Sep, 175(18), 5851 - 61 The Saccharomyces cerevisiae DAL80 repressor protein binds to multiple copies of GATAA-containing sequences (URSGATA); Cunningham TS et al.; Induced expression of the allantoin (DAL) catabolic genes in Saccharomyces cerevisiae has been suggested to be mediated by interaction of three different types of promoter elements . First is an inducer-independent upstream activation sequence, UASNTR, whose operation is sensitive to nitrogen catabolite repression . The GLN3 product is required for UASNTR-mediated transcriptional activation . This site consists of two separated elements, each of which has a GATAA sequence at its core . Response of the DAL genes to inducer is mediated by a second type of cis-acting element, DAL UIS . The DAL82 and DAL81 genes are required for response to inducer; DAL82 protein is the UIS-binding protein . When only the UASNTR and UIS elements are present, DAL gene expression occurs at high levels in the absence of inducer . We, therefore, hypothesized that a third element, an upstream repressor sequence (URS) mediates maintenance of DAL gene expression at a low level when inducer is absent . Since the DAL and UGA genes are overexpressed and largely inducer independent in dal80 deletion mutants, we have suggested DAL80 protein negatively regulates a wide spectrum of nitrogen-catabolic gene expression, likely in conjunction with a URS element . Here we show that DAL80 protein binds to DAL3 and UGA4 upstream DNA sequences, designated URSGATA, consisting of two GATAA-containing sites separated by at least 15 bp . The preferred orientation of the sites is tail to tail, but reasonable binding activity is also observed with a head-to-tail configuration . URSGATA elements contain the sequence GATAA at their core and hence share sequence homology with UASNTR elements. Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 8164 - 8 PMT1, the gene for a key enzyme of protein O-glycosylation in Saccharomyces cerevisiae; Strahl-Bolsinger S et al.; The integral endoplasmic reticulum membrane protein catalyzing the initial reaction of protein O-glycosylation in Saccharomyces cerevisiae has been purified to homogeneity . The 92-kDa N-glycosylated protein transfers mannose residues from dolichyl phosphate-D-mannose to specific serine/threonine residues of proteins entering the secretory pathway . This type of mannosyl transfer reaction has so far been observed only in fungal cells . Oligonucleotides derived from peptide sequences of the transferase were used to screen a genomic yeast library . A clone was isolated which contains an open reading frame of 2451 bp corresponding to an mRNA transcript of 3 kb . The predicted protein consists of 817 amino acids including three potential N-glycosylation sites . The hydropathy plot indicates a tripartite structure of the protein: an amino-terminal third and a carboxyl-terminal third, both with multiple potential transmembrane helices, and a central hydrophilic part . Expression of the clone in Escherichia coli resulted in mannosyltransferase activity . Gene disruption led to a complete loss of in vitro mannosyltransferase activity from dolichyl phosphate-D-mannose to a peptide used as acceptor in the enzymatic assay . In vivo it was observed, however, that protein O-mannosylation in the disruptant had decreased only to about 40-50%, indicating the existence of an additional transferase which had not been measured by the in vitro enzyme assay. Proc Natl Acad Sci U S A, 1993 Sep 1, 90(17), 7985 - 9 RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae; Siede W et al.; Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or gamma radiation after release from G1 arrest induced by alpha factor results in delayed resumption of the cell cycle . As is the case with G2 arrest following ionizing radiation damage {Weinert, T . A . & Hartwell, L . H . (1988) Science 241, 317-322}, the normal execution of DNA damage-induced G1 arrest depends on a functional yeast RAD9 gene . We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast . These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication . This checkpoint apparently operates after the mating pheromone-induced G1 arrest point but prior to replicative DNA synthesis, S phase-associated maximal induction of histone H2A mRNA, and bud emergence. J Bacteriol, 1993 Sep, 175(17), 5714 - 6 Density fluctuation during the cell cycle in the defective vacuolar morphology mutants of Saccharomyces cerevisiae; Ohsumi M et al.; The buoyant densities of the yeast cells of defective vacuolar morphology mutants were examined by equilibrium sedimentation centrifugation in a Percoll density gradient . These vacuoleless mutants also show density fluctuation as wild-type cells during the cell cycle . This suggests that morphological changes of the vacuole are not related to cyclic density fluctuation in Saccharomyces cerevisiae. J Bacteriol, 1993 Sep, 175(17), 5520 - 8 Glucose uptake and catabolite repression in dominant HTR1 mutants of Saccharomyces cerevisiae; Ozcan S et al.; Growth and carbon metabolism in triosephosphate isomerase (delta tpi1) mutants of Saccharomyces cerevisiae are severely inhibited by glucose . By using this feature, we selected for secondary site revertants on glucose . We defined five complementation groups, some of which have previously been identified as glucose repression mutants . The predominant mutant type, HTR1 (hexose transport regulation), is dominant and causes various glucose-specific metabolic and regulatory defects in TPI1 wild-type cells . HTR1 mutants are deficient in high-affinity glucose uptake and have reduced low-affinity transport . Transcription of various known glucose transporter genes (HXT1, HXT3, and HXT4) was defective in HTR1 mutants, leading us to suggest that HTR mutations affect a negative factor of HXT gene expression . By contrast, transcript levels for SNF3, which encodes a component of high-affinity glucose uptake, were unaffected . We presume that HTR1 mutations affect a negative factor of HXT gene expression . Multicopy expression of HXT genes or parts of their regulatory sequences suppresses the metabolic defects of HTR1 mutants but not their derepressed phenotype at high glucose concentrations . This suggests that the glucose repression defect is not a direct result of the metabolically relevant defect in glucose transport . Alternatively, some unidentified regulatory components of the glucose transport system may be involved in the generation or transmission of signals for glucose repression. J Bacteriol, 1993 Sep, 175(17), 5366 - 74 Cysteine biosynthesis in Saccharomyces cerevisiae occurs through the transsulfuration pathway which has been built up by enzyme recruitment; Cherest H et al.; The transsulfuration pathways allow the interconversion of homocysteine and cysteine with the intermediary formation of cystathionine . The various organisms studied up to now incorporate reduced sulfur into a three- or a four-carbon chain and use differently the transsulfuration pathways to synthesize sulfur amino acids . In enteric bacteria, the synthesis of cysteine is the first step of organic sulfur metabolism and homocysteine is derived from cysteine . Fungi are capable of incorporating reduced sulfur into a four-carbon chain, and they possess two operating transsulfuration pathways . By contrast, synthesis of cysteine from homocysteine is the only existing transsulfuration pathway in mammals . In Saccharomyces cerevisiae, genetic, phenotypic, and enzymatic study of mutants has allowed us to demonstrate that homocysteine is the first sulfur amino acid to be synthesized and cysteine is derived only from homocysteine (H . Cherest and Y . Surdin-Kerjan, Genetics 130:51-58, 1992) . We report here the cloning of genes STR4 and STR1, encoding cystathionine beta-synthase and cystathionine gamma-lyase, respectively . The only phenotypic consequence of the inactivation of STR1 or STR4 is cysteine auxotrophy . The sequencing of gene STR4 has allowed us to compare all of the known sequences of transsulfuration enzymes and enzymes catalyzing the incorporation of reduced sulfur in carbon chains . These comparisons reveal a partition into two families based on sequence motifs . This partition mainly correlates with similarities in the catalytic mechanisms of these enzymes. Mol Cell Biol, 1993 Sep, 13(9), 5829 - 42 SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase; Zheng P et al.; SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae . We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein . Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L) . Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated . These colonies were greatly enriched for budded cells, especially those with large buds . Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency . Spk1 protein was identified by immunoprecipitation and immunoblotting . It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays . Spk1 was localized to the nucleus by immunofluorescence . The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements . These elements confer S-phase-specific transcription to many genes involved in DNA synthesis . Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes . The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage . spk1 strains were hypersensitive to UV irradiation . Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability . The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis. Mol Cell Biol, 1993 Sep, 13(9), 5749 - 61 Saccharomyces cerevisiae BUF protein binds to sequences participating in DNA replication in addition to those mediating transcriptional repression (URS1) and activation; Luche RM et al.; The heteromeric BUF protein was originally shown to bind to URS1 elements which are situated upstream of many genes in Saccharomyces cerevisiae and mediate negative control of their transcription . Among the genes regulated through the URS1 site and the proteins interacting with it are those participating in carbon, nitrogen, and inositol metabolism; electron transport; meio