S. cerevisiae

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; meiosis; sporulation; and mating-type switching . We show here that pure BUF protein, in addition to binding to the negatively acting URS1 site, also binds to CAR1 sequences supporting transcriptional activation (upstream activation sequences) . To determine the BUF protein structure, we cloned and sequenced the BUF1 and BUF2 genes and found them to be identical to the RF-A (RP-A) gene whose products participate in yeast DNA replication as single-stranded DNA binding proteins . These data argue that BUF protein-binding sites serve multiple roles in transcription and replication.

Mol Cell Biol, 1993 Sep, 13(9), 5702 - 9
Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions; Sze JY et al.; The Leu3 protein of Saccharomyces cerevisiae binds to specific DNA sequences present in the 5' noncoding region of at least five RNA polymerase II-transcribed genes . Leu3 functions as a transcriptional activator only when the metabolic intermediate alpha-isopropylmalate is also present . In the absence of alpha-isopropylmalate, Leu3 causes transcription to be repressed below basal levels . We show here that different portions of the Leu3 protein are responsible for activation and repression . Fusion of the 30 C-terminal residues of Leu3 to the DNA-binding domain of the Gal4 protein created a strong cross-species activator, demonstrating that the short C-terminal region is not only required but also sufficient for transcriptional activation . Using a recently developed Leu3-responsive in vitro transcription assay as a test system for repression (J . Sze, M . Woontner, J . Jaehning, and G . B . Kohlhaw, Science 258:1143-1145, 1992), we show that mutant forms of the Leu3 protein that lack the activation domain still function as repressors . The shortest repressor thus identified had only about 15% of the mass of the full-length Leu3 protein and was centered on the DNA-binding region of Leu3 . Implications of this finding for the mechanism of repression are discussed.

Mol Cell Biol, 1993 Sep, 13(9), 5637 - 46
Saccharomyces cerevisiae HSP70 heat shock elements are functionally distinct; Young MR et al.; The Saccharomyces cerevisiae HSP70 gene SSA1 has multiple heat shock elements (HSEs) . To determine the significance of each of these sequences for expression of SSA1, we analyzed expression from a set of promoters containing point mutations in each of the HSEs, individually and in pairwise combinations . Of the three HSE-like sequences, two (HSE2 and HSE3) were active promoter elements; only one, HSE2, was active under basal growth conditions . Either HSE2 or HSE3 alone was able to drive SSA1 transcription at near-normal rates after heat shock . Both HSE2 and HSE3 were capable of driving basal transcription when placed in the context of the CYC1 promoter . Previous analysis had identified an upstream repressing sequence overlapping HSE2 that repressed basal transcription driven by HSE2 . Our analysis showed that basal transcription driven by HSE3 was repressed both by the distant upstream repressing sequence and by closer flanking sequences . The ability to drive basal transcription is not inherent in all natural HSEs, since the HSEs from the heat-inducible SSA3 and SSA4 genes showed no basal activity when placed in the CYC1 vector . Gel mobility shift experiments showed that the same population of heat shock transcription factor molecules bound to HSEs capable of driving basal activity and to HSEs having very low or undetectable basal activity.

Mol Cell Biol, 1993 Sep, 13(9), 5418 - 26
Inactivation of YME1, a member of the ftsH-SEC18-PAS1-CDC48 family of putative ATPase-encoding genes, causes increased escape of DNA from mitochondria in Saccharomyces cerevisiae; Thorsness PE et al.; The yeast nuclear gene YME1 was one of six genes recently identified in a screen for mutations that elevate the rate at which DNA escapes from mitochondria and migrates to the nucleus . yme1 mutations, including a deletion, cause four known recessive phenotypes: an elevation in the rate at which copies of TRP1 and ARS1, integrated into the mitochondrial genome, escape to the nucleus; a heat-sensitive respiratory-growth defect; a cold-sensitive growth defect on rich glucose medium; and synthetic lethality in rho- (cytoplasmic petite) cells . The cloned YME1 gene complements all of these phenotypes . The gene product, Yme1p, is immunologically detectable as an 82-kDa protein present in mitochondria . Yme1p is a member of a family of homologous putative ATPases, including Sec18p, Pas1p, Cdc48p, TBP-1, and the FtsH protein . Yme1p is most similar to the Escherichia coli FtsH protein, an essential protein involved in septum formation during cell division . This observation suggests the hypothesis that Yme1p may play a role in mitochondrial fusion and/or division.

Mol Cell Biol, 1993 Sep, 13(9), 5360 - 9
cis-acting components in the replication origin from ribosomal DNA of Saccharomyces cerevisiae; Miller CA et al.; The ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae contain an autonomously replicating sequence (ARS) that colocalizes with a chromosomal origin of replication . We show that a minimal sequence necessary for full ARS function corresponds to a 107-bp rDNA fragment which contains three 10-of-11-bp matches to the ARS consensus sequence . Point mutations in only one of the 10-of-11-bp matches, GTTTAT GTTTT, inactivate the rDNA ARS, indicating that this consensus sequence is essential . A perfect match to a revised ARS consensus is present but not essential . Sequences up to 9 bp 5' from the essential consensus are dispensable . A broad DNA region directly 3' to the essential consensus is required and is easily unwound as indicated by: (i) hypersensitivity to nicking of an approximately 100-bp region by mung bean nuclease in a negatively supercoiled plasmid and (ii) helical instability determined by thermodynamic analysis of the nucleotide sequence . A correlation between DNA helical instability and replication efficiency of wild-type and mutated ribosomal ARS derivatives suggests that a broad region 3' to the essential ARS consensus functions as a DNA unwinding element . Certain point mutations that do not stabilize the DNA helix in the 3' region but reduce ARS efficiency reveal an element distinct from, but overlapping, the DNA unwinding element . The nucleotide sequence of the functionally important constituents in the ARS appears to be conserved among the rDNA repeats in the chromosome.

Mol Cell Biol, 1993 Sep, 13(9), 5141 - 8
A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons; Caponigro G et al.; Differences in decay rates of eukaryotic transcripts can be determined by discrete sequence elements within mRNAs . Through the analysis of chimeric transcripts and internal deletions, we have identified a 65-nucleotide segment of the MAT alpha 1 mRNA coding region, termed the MAT alpha 1 instability element, that is sufficient to confer instability to a stable PGK1 reporter transcript and that accelerates turnover of the unstable MAT alpha 1 mRNA . This 65-nucleotide element is composed of two parts, one located within the 5' 33 nucleotides and the second located in the 3' 32 nucleotides . The first part, which can be functionally replaced by sequences containing rare codons, is unable to promote rapid decay by itself but can enhance the action of the 3' 32 nucleotides (positions 234 to 266 in the MAT alpha 1 mRNA) in accelerating turnover . A second portion of the MAT alpha 1 mRNA (nucleotides 265 to 290) is also sufficient to destabilize the PGK1 reporter transcript when positioned 3' of rare codons, suggesting that the 3' half of the MAT alpha 1 instability element is functionally reiterated within the MAT alpha 1 mRNA . The observation that rare codons are part of the 65-nucleotide MAT alpha 1 instability element suggests possible mechanisms through which translation and mRNA decay may be linked.

J Cell Sci, 1993 Sep, 106 ( Pt 1), 287 - 98
Prp20, the Saccharomyces cerevisiae homolog of the regulator of chromosome condensation, RCC1, interacts with double-stranded DNA through a multi-component complex containing GTP-binding proteins; Lee A et al.; Prp20, a homolog to the mammalian negative regulator of chromosome condensation, RCC1, is retained on double-stranded (ds) DNA-cellulose when extracts are prepared from asynchronously growing wild-type yeast strains . Conversely, neither Prp20 from ts mutant cell extracts nor wt yeast Prp20 produced in Escherichia coli, bind to dsDNA-cellulose . In vitro reconstitution assays using E . coli-expressed Prp20 and inactivated ts mutant extracts of prp20-1 reveal that the Prp20 protein requires the assistance of other proteins in the cell extract to promote its binding to dsDNA . Immunoprecipitations and sizing-column-chromatography indicate that the Prp20 protein binds to the dsDNA column through a multicomponent complex composed of six to seven proteins, which has a collective molecular mass greater than 150,000 Da . At least three of the members of this Prp20 complex will bind GTP in vitro . Moreover, the Prp20 complex is shown to specifically lose its ability to bind dsDNA during the DNA replication phase of the cell cycle . This loss of dsDNA binding during the S phase of the cell cycle does not affect the proper organization of the nucleoplasm and appears to be reversed before the cell enters mitosis.

Biol Chem Hoppe Seyler, 1993 Sep, 374(9), 903 - 8
Purification and characterization of the biologically active human truncated macrophage colony-stimulating factor expressed in Saccharomyces cerevisiae; Zhu DX et al.; A human truncated macrophage colony-stimulating factor (M-CSF) cDNA encoding amino acid residues from 3 to 149 of the native M-CSF was isolated by using the polymerase chain reaction . When introduced into Saccharomyces cerevisiae by a general secretion vector pVT 102u/alpha, it directs the expression of the biologically active dimeric form of M-CSF . Through the 3 stages of purification, i.e . concentration by DEAE-cellulose column chromatography, hydrophobic chromatography on phenyl-sepharose and Mono Q fast protein liquid chromatography, the recombinant truncated M-CSF was purified as to exhibit a specific activity of 1.02 x 10(7) units/mg of protein . SDS-PAGE of this purified truncated M-CSF showed that its apparent molecular mass is 22 kDa under reducing conditions.

EMBO J, 1993 Sep, 12(9), 3437 - 47
A new pair of B-type cyclins from Saccharomyces cerevisiae that function early in the cell cycle; Kuhne C et al.; Two new B-type cyclin genes from Saccharomyces cerevisiae, called CLB5 and CLB6, are located in a tail to tail arrangement adjacent to the G2/M phase promoting cyclins CLB2 and CLB1, respectively . These genomic cyclin arrays are flanked by tRNAs and repeated sequences of Ty elements suggesting an intrachromosomal gene duplication followed by an interchromosomal gene duplication . Based on their deduced protein sequence the CLB5 and CLB6 genes form a new pair of B-type cyclins . They are most related to each other and then to the deduced protein sequence of their adjacent genes CLB1 and CLB2 . Both genes are periodically expressed, peaking early in the cell cycle . Loss of function mutants are viable, but clb5- mutants exhibit a delay in S phase whereas clb6- mutants show a delay in late G1 and/or S phase . The clb5 mutant phenotype is somewhat more pronounced in a double null mutant . Both cyclins have the potential to interact with the p34CDC28 kinase in vivo.

EMBO J, 1993 Sep, 12(9), 3417 - 26
Properties of Saccharomyces cerevisiae wee1 and its differential regulation of p34CDC28 in response to G1 and G2 cyclins; Booher RN et al.; Wee1 is a protein kinase that negatively regulates p34cdc2 kinase activity . We have identified a Saccharomyces cerevisiae wee1 homolog encoded by the SWE1 gene . SWE1 overexpression arrests cells in G2 with short spindles whereas deletion of SWE1 did not alter the cell cycle but did eliminate the G2 delay observed in mih1- mutants . Swe1 immunoprecipitates were capable of tyrosine phosphorylating and inactivating p34CDC28 complexed with Clb2, a G2-type cyclin, but not p34CDC28 complexed with Cln2, a G1-type cyclin, consistent with the inability of Swe1 overexpression to inhibit the G1/S transition . These results suggest that specific cyclin subunits target p34CDC28 for distinct regulatory controls which may be important for ensuring proper p34CDC28 function during the cell cycle.

Mol Biol (Mosk), 1993 Sep-Oct, 27(5), 1094 - 9
{Heterologous expression of functionally active human cytochrome P-450 . I . Synthesis of cytochrome P-450IID6 by transformed Saccharomyces cerevisiae cells}; Krynetskii EIu et al.; Functionally active human cytochrome P-450IID6 was expressed in Saccharomyces cerevisiae cells transformed with yeast expression vectors containing a fragment of the 2 mu-plasmid and cytochrome P-450 cDNA . Transcription was regulated by yeast acid phosphatase PHO5 promoter or chimeric GAL10-CYC1 promoter . Induced yeast cells synthesized cytochrome P-450 that was revealed by the optical absorption of the reduced CO form . Intact yeast cells were capable to metabolize the drug sparteine forming biotransformation products usually found in human . Expression of the human cytochromes P-450 seems to be a promising route to investigate new drugs and to screen genotoxic compounds.

J Gen Microbiol, 1993 Sep, 139 ( Pt 9), 2091 - 100
Inactivation of the CDC25 gene product in Saccharomyces cerevisiae leads to a decrease in glycolytic activity which is independent of cAMP levels; Oehlen LJ et al.; In the budding yeast Saccharomyces cerevisiae cyclic AMP (cAMP) can influence the activity of key enzymes in carbohydrate metabolism through modulation of the activity of cAMP-dependent protein kinase . One of the components involved in cAMP production is the CDC25 gene product, which can activate the RAS/adenylate cyclase pathway by promoting the exchange of guanine nucleotides bound to RAS . In two yeast strains carrying different thermosensitive alleles of the CDC25 gene, cAMP levels respond differently to an increase in growth temperature from 23 degrees C (permissive) to 36 degrees C (restrictive) . In strain OL86 (cdc25-5) the estimated intracellular concentration of cAMP dropped after transfer to restrictive temperature whereas in strain ts321 (cdc25-1) the cAMP level rose under the same conditions . Despite the differences in cAMP levels the glycolytic flux in the two mutants responded in a very similar way to the shift from permissive to restrictive temperature; after the increase in the incubation temperature, the specific glycolytic flux in both cdc25-1 and cdc25-5 initially increased from about 300 nmol min-1 (mg protein)-1 to about 500 nmol min-1 (mg protein)-1 (presumably mainly as a consequence of the increase in temperature), but then gradually fell to 100-200 nmol min-1 (mg protein)-1 . A similar pattern of CO2 production to that found in the two cdc25 mutants was also observed for several other thermosensitive mutants displaying a Start-II type of G1 arrest . In contrast, in a wild-type strain and in strains giving a Start-I type of G1 arrest, CO2 production did not drop after a temperature shift . The specific activities of glycolytic enzymes in the two cdc25 mutants did not show much change after the temperature shift, indicating that the decrease in glycolytic flux was not caused by a decrease in the activity of any of the glycolytic enzymes . Our data show that, at least in long-term regulation, the cAMP levels per se are not likely to be a prime factor controlling glycolytic flux.

Mutagenesis, 1993 Sep, 8(5), 467 - 71
Evidence for preferential repair of 3-carbethoxypsoralen plus UVA induced DNA lesions in the active MAT alpha locus in Saccharomyces cerevisiae using the UvrABC assay; Meniel V et al.; The occurrence of preferential repair in Saccharomyces cerevisiae of the active MAT alpha locus compared with the inactive HML alpha locus was confirmed after 254 nm UV irradiation . Experiments carried out using the UvrABC excinuclease assay with the monofunctional furocoumarin 3-carbethoxypsoralen (3-CPs) plus UVA radiation which induce mainly monoadducts in DNA demonstrated preferential repair of the active MAT alpha locus compared with the inactive HML alpha locus in a SIR+ strain . However, as after 254 nm UV irradiation, no difference in the rate of removal of 3-CPs plus UVA induced lesions was observed between the two loci in the sir-3 mutant in which both loci are active . Thus, it appears that 3-CPs plus UVA induced monoadducts as well as pyrimidine dimers are subject to preferential repair.

Mutagenesis, 1993 Sep, 8(5), 417 - 21
Mutagenic specificity of the base analog 6-N-hydroxylaminopurine in the URA3 gene of the yeast Saccharomyces cerevisiae; Shcherbakova PV et al.; The mutational specificity of the base analog 6-N-hydroxylaminopurine (HAP) was studied in the URA3 gene of the yeast Saccharomyces cerevisiae . Twenty-nine independent HAP-induced ura3 mutations were sequenced . GC-->AT transitions were found most frequently (21 out of 29) while AT-->GC transitions were less abundant (five out of 29) . Three GC-->TA transversions were also detected . Two interesting features of DNA context were revealed for transition mutations . One third of the transitions occurred at one site within short direct imperfect repeats converting them to perfect repeats . A model involving complementary interaction of imperfect repeats is proposed to explain the origin of these mutations . Nearly all of the rest of the GC-->AT as well as the AT-->GC transitions were found in the runs of several identical base pairs, predominantly in the middle or at the 3' template nucleotide of (G)n and (A)n runs.

Genetics, 1993 Sep, 135(1), 35 - 44
Loss of function of Saccharomyces cerevisiae kinesin-related CIN8 and KIP1 is suppressed by KAR3 motor domain mutations; Hoyt MA et al.; The kinesin-related products of the CIN8 and KIP1 genes of Saccharomyces cerevisiae redundantly perform an essential function in mitosis . The action of either gene-product is required for an outwardly directed force that acts upon the spindle poles . We have selected mutations that suppress the temperature-sensitivity of a cin8-temperature-sensitive kip1-delta strain . The extragenic suppressors analyzed were all found to be alleles of the KAR3 gene . KAR3 encodes a distinct kinesin-related protein whose action antagonizes Cin8p/Kip1p function . All seven alleles analyzed were altered within the region of KAR3 that encodes the putative force-generating (or "motor") domain . These mutations also suppressed the inviability associated with the cin8-delta kip1-delta genotype, a property not shared by a deletion of KAR3 . Other properties of the suppressing alleles revealed that they were not null for function . Six of the seven were unaffected for the essential karyogamy and meiosis properties of KAR3 and the seventh was dominant for the suppressing trait . Our findings suggest that despite an antagonistic relationship between Cin8p/Kip1p and Kar3p, aspects of their mitotic roles may be similar.

Genetics, 1993 Sep, 135(1), 25 - 34
The HIR4-1 mutation defines a new class of histone regulatory genes in Saccharomyces cerevisiae; Spector MS et al.; Both activation and repression have been implicated in the cell cycle-regulated transcription of the histone HTA1-HTB1 locus in Saccharomyces cerevisiae . Transcriptional repressors have been identified through the isolation of recessive mutations in the HIR1, HIR2 and HIR3 genes . These three regulatory genes encode proteins that act at a negative site in the HTA1-HTB1 promoter, and their inactivation results in cell cycle-independent transcription . We report here on the characterization of a fourth HIR mutant . The HIR4-1 mutation is dominant, and the phenotypes that it confers suggest that the mutant gene encodes an altered transcriptional activator . The function of this activator is very specific: it uniquely regulates transcription of the HTA1-HTB1 locus, and it may antagonize repressors that act through the HTA1-HTB1 negative site.

Genetics, 1993 Sep, 135(1), 17 - 23
Molecular and genetic analysis of the SNF7 gene in Saccharomyces cerevisiae; Tu J et al.; Mutations in the SNF7 gene of Saccharomyces cerevisiae prevent full derepression of the SUC2 (invertase) gene in response to glucose limitation . We report the molecular cloning of the SNF7 gene by complementation . Sequence analysis predicts that the gene product is a 27-kDa acidic protein . Disruption of the chromosomal locus causes a fewfold decrease in invertase derepression, a growth defect on raffinose, temperature-sensitive growth on glucose, and a sporulation defect in homozygous diploids . Genetic analysis of the interactions of the snf7 null mutation with ssn6 and spt6/ssn20 suppressor mutations distinguished SNF7 from the SNF2, SNF5 and SNF6 genes . The snf7 mutation also behaved differently from mutations in SNF1 and SNF4 in that snf7 ssn6 double mutants displayed a synthetic phenotype of severe temperature sensitivity for growth . We also mapped SNF7 to the right arm of chromosome XII near the centromere.

Curr Genet, 1993 Sep, 24(3), 268 - 70
Multicopy SUP35 gene induces de-novo appearance of psi-like factors in the yeast Saccharomyces cerevisiae; Chernoff YO et al.; Previously, we have shown that plasmid-mediated multiplication of Saccharomyces cerevisiae wild-type SUP35 gene leads to omnipotent suppression and is incompatible with psi-factor, which is an endogenous extrachromosomal suppressor . Here, we describe a frequent de-novo appearance of psi-like factors in mitotic progeny of yeast transformants containing multicopy SUP35 gene.

Appl Environ Microbiol, 1993 Sep, 59(9), 2851 - 6
Mechanism of adsorption of hard and soft metal ions to Saccharomyces cerevisiae and influence of hard and soft anions; Avery SV et al.; The applicability of the hard-and-soft principle of acids and bases in predicting metal adsorption characteristics in a biological context was investigated for metabolism-independent uptake of the metal ions Sr2+, Mn2+, Zn2+, Cu2+, Cd2+, and Tl+ by Saccharomyces cerevisiae . Metal adsorption increased with external metal concentration (5 to 50 microM), although some saturation of uptake of the harder ions examined, Sr2+, Mn2+, and Zn2+, was evident at the higher metal concentrations . Cation displacement experiments indicated that, with the exception of Tl+, relative covalent bonding (H+ displacement) of the metals was greater at low metal concentrations, while weaker electrostatic interactions (Mg2+ plus Ca2+ displacement) became increasingly important at higher concentrations . These results were correlated with curved Scatchard and reciprocal Langmuir plots of metal uptake data . Saturation of covalent binding sites was most marked for the hard metals, and consequently, although no relationship between metal hardness and ionic/covalent bonding ratios was evident at 10 microM metal, at 50 microM the ratio was generally higher for harder metals . Increasing inhibition of metal uptake at increasing external anion concentrations was partially attributed to the formation of metal-anion complexes . Inhibitory effects of the hard anion SO42(-) were most marked for uptake of the hard metals Sr2+ and Mn2+, whereas greater relative effects on adsorption of the softer cations Cu2+ and Cd2+ were correlated with complexation by the soft anion S2O32(-) . Inhibition of uptake of the borderline metal Zn2+ by SO42(-) and that by S2O32(-) were approximately equal.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutat Res, 1993 Sep, 289(1), 97 - 106
The mutator mut7-1 of Saccharomyces cerevisiae; von Borstel RC et al.; The mut7-1 mutant of Saccharomyces cerevisiae is a cell-division-cycle mutant, exhibiting temperature-sensitive lethality and enhancement of mutator activity with increases in temperature . The base-sequence alterations in mutants arising in a mut7-1 background differed from the control by there being a higher transversion/transition ratio and by the much increased production of multi-base deletions . The deletions were, in every instance, associated with repeated oligonucleotide sequences (3-8 bases in length), where one of the two sequences was removed during the deletion process . The mutant mut7-1 failed to complement with cdc2, the temperature-sensitive mutant of the locus which encodes DNA polymerase III (delta).

Mutat Res, 1993 Sep, 289(1), 91 - 6
Formaldehyde lacks genotoxicity in formaldehyde-hyperresistant strains of the yeast Saccharomyces cerevisiae; Wehner E et al.; Formaldehyde is a weak mutagen and recombinagen in wild type strains of Saccharomyces cerevisiae . Genotoxicity of formaldehyde is influenced by the activity of the SFA yeast gene . Yeast transformants containing multiple copies of the plasmid-contained SFA gene are hyperresistant to the chemical and grow in the presence of 5 mM formaldehyde, a concentration lethal for the wild type . The SFA-encoded protein mediates the degradation of formaldehyde and its activity is most probably responsible for the low or absent genotoxic effects in formaldehyde-treated cells . Multi-copy vectors containing the SFA gene are retained in yeast transformants growing in complex media supplemented with 5 mM formaldehyde . Cells harboring such multi-copy vectors may, therefore, be propagated in large batch cultures in undefined media in the presence of formaldehyde concentrations toxic to the wild type.

Mol Cell Biol, 1993 Sep, 13(9), 5290 - 300
Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae; Murphy SM et al.; The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527 . C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation . We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells . Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition . Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype . CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro . These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.

Biochemistry, 1993 Aug 31, 32(34), 8823 - 35
Purification and biochemical characterization of tubulin from the budding yeast Saccharomyces cerevisiae; Davis A et al.; We describe a method for isolating milligram quantities of assembly-competent tubulin from the budding yeast Saccharomyces cerevisiae . The tubulin is > 95% purified and free of contaminating enzyme activities . As a result, it has been possible to determine the yeast tubulin equilibrium-binding constant for Mg-GTP and the tubulin GTPase activity under nonassembling and assembling conditions . We also determined the critical concentration for yeast tubulin polymerization and found it to be significantly lower than that for bovine brain tubulin under identical conditions . Similarly, the dynamic properties both of individual yeast microtubules and of bulk microtubule suspensions were significantly different from those of bovine brain microtubules free of microtubule-associated proteins . The data suggest that the properties of the yeast tubulin may reflect the particular functional requirements of the yeast cell . With this method, it is now possible to introduce any desired tubulin gene mutation into the budding yeast and correlate the phenotypic effects of the mutation in cells with the effects of the mutation on the biochemical and polymerization properties of the tubulin.

Nucleic Acids Res, 1993 Aug 25, 21(17), 4133 - 41
Mutational analysis of the Saccharomyces cerevisiae general regulatory factor CP1; Masison DC et al.; The Saccharomyces cerevisiae general regulatory factor CP1, a helix-loop-helix protein that binds the centromere DNA element I (CDEI) of yeast centromeres, is required in yeast for optimal centromere function and for methionine prototrophy . Mutant alleles of CEP1, the gene encoding CP1, were generated by linker insertion, 5'- and 3'-deletion, and random mutagenesis and assayed for DNA binding activity and their ability to confer CP1 function when expressed in yeast . A heterologous CDEI-binding protein, TFEB, was also tested for CP1 function . The results suggested that DNA binding is required for both biological functions of CP1 but is not sufficient . A direct and quantitative correlation was observed between the chromosome loss and nutritional (i.e., Met) phenotypes of strains carrying loss of function alleles, but qualitatively the chromosome loss phenotype was more sensitive to decreased CP1 expression . The data are consistent with a model in which CP1 performs the same general chromatin-related function at centromeres and MET gene promoters and is normally present in functional excess.

J Biol Chem, 1993 Aug 25, 268(24), 18286 - 92
VMA13 encodes a 54-kDa vacuolar H(+)-ATPase subunit required for activity but not assembly of the enzyme complex in Saccharomyces cerevisiae; Ho MN et al.; Previous purifications and characterizations of the Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) have indicated that this enzyme is a multisubunit complex composed of at least eight subunits of 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa (Kane, P . M., Yamashiro, C . T., and Stevens, T . H . (1989) J . Biol . Chem . 264, 19236-19244) . We report the cloning and characterization of an additional V-ATPase subunit, the 54-kDa subunit, which is encoded by the VMA13 gene . VMA13 was isolated by complementation of the growth phenotypes associated with the vma13 mutation, which was originally described as cls11 (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y . (1991) J . Biol . Chem . 266, 13971-13977) . The nucleotide sequence of the VMA13 gene predicted a hydrophilic polypeptide with a calculated molecular mass of 54,415 daltons . The VMA13 54-kDa gene product resides on the vacuolar membrane and co-purified with the active V-ATPase complex . Characterization of a null vma13 mutant (delta vma13) revealed that the Vma13 polypeptide is essential for V-ATPase activity . However, the Vma13 polypeptide is not required for targeting of the other V-ATPase subunits (100-, 69-, 60-, 42-, 27-, or 17-kDa subunits) to the vacuolar membrane as shown by the association of these subunits with vacuolar membranes isolated from delta vma13 cells . The nature of the V-ATPase "complex" in delta vma13 mutant is, nevertheless, fundamentally different from the wild-type enzyme . This is evidenced by the fact that the inactive V-ATPase complex from delta vma13 cells is less stable than the wild-type enzyme . Taken together, these results indicate that VMA13 encodes the 54-kDa subunit of the V-ATPase and that this subunit is essential for activity, but not assembly, of the enzyme complex.

Biochemistry, 1993 Aug 17, 32(32), 8199 - 206
Histidine2 of the alpha-factor of Saccharomyces cerevisiae is not essential for binding to its receptor or for biological activity; Levin Y et al.; Seven His2 analogs of the Saccharomyces cerevisiae {Nle12}alpha-factor, WXWLQLKPGQP(Nle)Y, where X = beta-D-thienylalanine, beta-L-thienylalanine, 1-D-methylhistidine, 1-L-methylhistidine, 3-D-methylhistidine, 3-L-methylhistidine, and beta-3-L-pyridylalanine, were synthesized and purified to homogeneity . Assays were carried out on binding to the alpha-factor receptor and of biological activity determined by either growth arrest or morphological changes in target cells . In the L-isomer, replacement of the imidazole of histidine by thiophene or 3-pyridyl groups or derivatization of either nitrogen of the imidazole ring by methylation resulted in a 2-100-fold decrease in bioactivity . D-Isomers of the beta-thienylalanyl-, 1-methylhistidinyl-, or 3-methylhistidinyl-alpha-factors did not possess measurable bioactivity with the exception of comparatively low activity of the 3-D-methylhistidinyl and 1-D-methylhistidinyl-alpha-factors in the morphogenesis assay . In contrast, both active and inactive analogs demonstrated binding affinities 10-20-fold less than that of {Nle12}alpha-factor . These results indicate that the histidine residue of alpha-factor is not required for binding to the receptor or for biological activity and that bioactivity and binding can be dissociated through the use of pheromone analogs.

FEMS Microbiol Lett, 1993 Aug 15, 112(1), 25 - 9
Genetic evidence of a new flocculation suppressor gene in Saccharomyces cerevisiae; Sieiro C et al.; The flocculation character in strain IM1-8b of Saccharomyces cerevisiae is controlled by a single and dominant gene shown to be allelic to FLO1 . Such a gene has been both mitotically and meiotically mapped on the right arm of chromosome I at 4.7 cM from PHO11 . The phenotype was suppressed by a single gene of wide distribution among non-flocculent strains (proposed as fsu3) that, however, was unable to suppress other FLO1 genes in other flocculent strains.

FEMS Microbiol Lett, 1993 Aug 15, 112(1), 119 - 23
Expression of Aspergillus oryzae alpha-amylase gene in Saccharomyces cerevisiae; Randez-Gil F et al.; A fragment containing the full length cDNA from Aspergillus oryzae alpha-amylase has been amplified by PCR using specific synthetic oligonucleotides . The amplified cDNA was designed to favour its expression in yeast by modifying its upstream untranslated region . It was subcloned in the expression vector pYEX alpha 1, placed under the control of the yeast CYC1-GAL10 promoter and used to transform Saccharomyces cerevisiae . Cells were then able to express and secrete active alpha-amylase to the medium in a regulated fashion . The recombinant enzyme had similar electrophoretic mobility and catalytic properties to the original A . oryzae alpha-amylase.

Proc Natl Acad Sci U S A, 1993 Aug 15, 90(16), 7637 - 41
The rRNA-encoding DNA array has an altered structure in topoisomerase I mutants of Saccharomyces cerevisiae; Christman MF et al.; All the chromosomes from isogenic TOP1 and top1 strains have similar mobility on pulsed-field gels except for chromosome XII, which fails to migrate into the gels in top1 mutants . Chromosome XII contains the tandem repeats of rRNA-encoding DNA (rDNA) . When a segment of chromosome XII containing only rDNA is transferred to chromosome III by a recombination event, chromosome III fails to enter a pulsed-field gel in extracts from top1 strains, indicating that the aberrant migration of chromosome XII in top1 mutants is caused by the presence of rDNA . Failure of chromosome XII to migrate into a pulsed-field gel occurs only in preparations from exponentially growing top1 cultures and not in preparations from stationary-phase top1 cultures . rDNA from a top1 strain does enter the gel if it is cut with an enzyme (Pst I) that cuts the tandem rDNA array into single 9-kb repeat units, indicating that more than a single repeat unit is required to maintain the aberrant structure.

Arch Biochem Biophys, 1993 Aug 15, 305(1), 47 - 53
Isolation and properties of YCK2, a Saccharomyces cerevisiae homolog of casein kinase-1; Vancura A et al.; A soluble fragment of YCK2, a casein kinase-1 isoform from Saccharomyces cerevisiae, has been purified and characterized in vitro . The procedure enriches enzyme activity to a final specific activity of 4.7 mumol min-1 mg-1 (when assayed with casein as substrate) . Structural analysis reveals that the preparation arises from N-terminal modification and C-terminal proteolysis of the initially synthesized 546-residue protein, consisting of residues 2-495 +/- 1 . Kinetic analysis demonstrates that YCK2 is similar to casein kinase-1 isolated from other organisms in its inability to use GTP as nucleotide substrate, in its sensitivity to heparin and ribofuranosyl-benzimidazole inhibitors, and in its peptide substrate selectivity . The enzyme is unusual, however, in that it is insensitive to the potent mammalian casein kinase-1 inhibitor N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide.

J Biol Chem, 1993 Aug 5, 268(22), 16815 - 21
Cloning, sequencing, and mutation of thiol-specific antioxidant gene of Saccharomyces cerevisiae; Chae HZ et al.; We have previously shown that the yeast Saccharomyces cerevisiae contains an antioxidant enzyme that can provide protection against a thiol-containing oxidation system but not against an oxidation system without thiol . This 25-kDa enzyme was thus named thiol-specific antioxidant (TSA) . We have now isolated and sequenced a yeast genomic DNA fragment that encodes TSA . Comparison of the predicted amino acid sequence of TSA with those of conventional antioxidant enzymes, including catalases, peroxidases, and superoxide dismutases, revealed no sequence homology . The 195-amino acid TSA sequence contains 2 cysteine residues . Southern blot analysis of petite yeast DNA, studies with protein synthesis inhibitors, and protein immunoblot analyses of cytosolic and mitochondrial proteins suggest that TSA is a cytosolic protein encoded by nuclear DNA (chromosome XIII) . The yeast TSA gene was selectively disrupted by homologous recombination . The haploid tsa mutant was viable under air, suggesting that TSA is not essential for cell viability . The growth rates of the tsa mutant and wild-type strains were identical under anaerobic conditions . However, under aerobic conditions, especially in the presence of methyl viologen or a peroxide (t-butyl hydroperoxide or H2O2), the growth rate of the mutant was significantly less than that of wild-type cells . This result suggests that TSA is a physiologically important antioxidant.

Mol Microbiol, 1993 Aug, 9(3), 545 - 55
Identification of a new nuclear gene (CEM1) encoding a protein homologous to a beta-keto-acyl synthase which is essential for mitochondrial respiration in Saccharomyces cerevisiae; Harington A et al.; We have analysed a new gene, CEM1, from Saccharomyces cerevisiae . Inactivation of this gene leads to a respiratory-deficient phenotype . The deduced protein sequence shows strong similarities with beta-keto-acyl synthases or condensing enzymes . Typically, enzymes of this class are involved in the synthesis of fatty acids or similar molecules . An analysis of the mitochondrial lipids and fatty acids shows no major difference between the wild type and deleted strains, implying that the CEM1 gene product is not involved in the synthesis of the bulk fatty acids . Thus it is possible that the CEM1 protein is involved in the synthesis of a specialized molecule, probably related to a fatty acid, which is essential for mitochondrial respiration.

Mol Microbiol, 1993 Aug, 9(3), 521 - 32
Inhibition of translational initiation in Saccharomyces cerevisiae by secondary structure: the roles of the stability and position of stem-loops in the mRNA leader; Oliveira CC et al.; A new modular gene-expression system for use in studies of translational control in Saccharomyces cerevisiae was constructed . A GAL::PGK fusion promoter (GPF) directed the inducible synthesis of mRNAs initiated at a single major site . A series of leader sequences were tested in combination with each of two reporter genes (encoding chloramphenicol acetyl transferase (cat) and luciferase (luc)) . Stem-loop structures of three different sizes and predicted stabilities were inserted into each of two different unique restriction sites in the leader . After correction for relative mRNA abundance, a stem-loop of predicted stability equivalent to approximately -18 kcal mol-1 inhibited translation by up to 89% . The degree of inhibition exerted by the other stem-loops correlated positively with their predicted stabilities . Combinations of two stem-loops at different sites yielded an inhibitory effect greater than that of either individual stem-loop alone . Similar inhibitory effects were observed with both reporter genes . However, inhibition of translation, particularly of the cat gene, was more effective when the stem-loop was positioned close to the start codon rather than at the 5' end of the leader . The observed results reflect an important form of post-transcriptional control that is expected to act on a large number of genes in yeast.

FEMS Microbiol Lett, 1993 Aug 1, 111(2-3), 165 - 70
Retention of a co-translational translocated mutant protein of carboxypeptidase Y of Saccharomyces cerevisiae in endoplasmic reticulum; Ramezani Rad M et al.; Co-translational translocation of Saccharomyces cerevisiae vacuolar glycoprotein carboxypeptidase Y (CpY) was highly efficient when studied with an in vivo and in vitro homologous system, comparison of limited proteolytic cleavage of immunoprecipitated translational products of CpY and subcellular localisation of a mutant CpY . The efficient segregation of CpY mRNA in highly purified fractions of rough microsomes was characterised . CpY1 mutant showed retention of core glycosylated material (proCpY1) in the rough and smooth endoplasmic reticulum fractions . It is suggested that the presence of structures that are incompatible with intercompartmental transport of vacuolar protein leads to retention of the mutated CpY by the endoplasmic reticulum.

Glycobiology, 1993 Aug, 3(4), 357 - 64
Biosynthesis of asparagine-linked oligosaccharides in Saccharomyces cerevisiae: the alg2 mutation; Jackson BJ et al.; In the yeast Saccharomyces cerevisiae, the alg2 mutation causes temperature-sensitive growth and abnormal accumulation of the lipid-linked oligosaccharide Man2GlcNAc2-PP-Dol (Jackson et al., Arch . Biochem . Biophys., 272, 203-209, 1989; Huffaker and Robbins, Proc . Natl . Acad . Sci . USA, 80, 7466-7470, 1983) . A gene having the function and genomic location of ALG2 was cloned from libraries based on the multicopy plasmid YEp24 and on the centromere plasmid YCp50 . Alg2 mutants transformed with plasmids containing ALG2 regained the capacity to grow and to synthesize lipid-linked oligosaccharides normally at the previously non-permissive temperature . ALG2 was essential for viability in haploid and diploid yeast . The ALG2 gene was transcribed into a single mRNA of 1.7 kb in size . The stability of ALG2 mRNA, assessed after thermal inactivation of RNA polymerase II in an rpb1-1 mutant (Herrick et al., Mol . Cell . Biol., 10, 2269-2284, 1990) was very low, with a t1/2 of < 5 min . The ALG2 transcript accumulation was growth dependent, and it was at least an order of magnitude lower in stationary phase cells compared to exponentially growing cells . The putative translation product of ALG2 contained a potential dolichol recognition domain similar to that found in all three glycosyltransferases of the lipid-linked pathway that have been sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1993 Aug 1, 215(3), 601 - 9
A region of the C-terminal part of the 11-kDa subunit of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae contributes to the structure of the Qout reaction domain; Hemrika W et al.; QCR8, the gene encoding the 11-kDa subunit of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae has been resequenced in the course of a search for mutants disturbed in subunit function . Resequencing shows that the previously published sequence {Maarse A.C . & Grivell L.A . (1987) Eur . J . Biochem 155, 419-425} lacks a C at position 185 of the coding sequence . As a result of this extra nucleotide, the reading frame now contains 285 base pairs and it codes for a protein of 94 amino acids with a calculated molecular mass of 11.0 kDa . Despite the altered C-terminus, similarity to the corresponding beef heart subunit is not significantly altered . One mutant (LTN1), arising from hydroxylamine mutagenesis, has been studied in detail: Assembly of the enzyme appears to be normal, as judged from the levels of the subunits observed in Western blots, while spectral analysis showed that only holo-cytochrome b was lowered to 70% of that of the wildtype . Measurement of the specific activity and calculation of the turnover number of the enzyme showed that these were 45% and 56% of that of the wild type, respectively . Further analysis of the mutant showed that the affinity for the inhibitor myxothiazol was decreased, that the 11-kDa subunit stabilises the enzyme once assembly has occurred, and that the reduction of cytochrome b via the Qout site is impaired . Sequence analysis showed that this mutant carries a deletion of 12 nucleotides at position 206-217 of the coding sequence, resulting in the replacement of residues 69-73 (WWKNG) by a cysteine . These results are discussed in terms of the 11-kDa subunit contributing to the conformation of the Qout binding domain.

Proc Natl Acad Sci U S A, 1993 Aug 1, 90(15), 6929 - 33
Reconstitution of retinoid X receptor function and combinatorial regulation of other nuclear hormone receptors in the yeast Saccharomyces cerevisiae; Hall BL et al.; The nuclear hormone receptor family of transcription factors regulates gene expression via a complex combinatorial network of interactions . Of particular interest is the ability of retinoid X receptors (RXRs) to form heterodimers with retinoic acid receptors (RARs) and thyroid hormone receptors (TRs), thereby modifying their activities . We report here that RXR, RAR, and TR function can be reconstituted in the yeast Saccharomyces cerevisiae and demonstrate that the combinatorial regulation seen in vertebrate cells can be reproduced in the yeast background . Using this system, we have shown that RARs respond to a wide variety of retinoid ligands but that RXRs are specific for the 9-cis isomer of retinoic acid . RXR enhanced the activity of RARs and TRs on a variety of hormone response elements without demonstrably altering their DNA specificity . Interestingly, the ability of RXR to potentiate gene activation by RARs and by TRs varied for different receptor isoforms.

EMBO J, 1993 Aug, 12(8), 3105 - 10
Salt tolerance and methionine biosynthesis in Saccharomyces cerevisiae involve a putative phosphatase gene; Glaser HU et al.; The progressive salinization of irrigated land poses a threat to the future of agriculture in arid regions . The identification of crucial metabolic steps in salt tolerance is important for the understanding of stress physiology and may provide the tools for its genetic engineering . In the yeast Saccharomyces cerevisiae we have isolated a gene, HAL2, which upon increase in gene dosage improves growth under NaCl and LiCl stresses . The HAL2 protein is homologous to inositol phosphatases, enzymes known to be inhibited by lithium salts . Complementation analysis demonstrated that HAL2 is identical to MET22, a gene involved in methionine biosynthesis . Accordingly, methionine supplementation improves the tolerance of yeast to NaCl and LiCl . These results demonstrate an unsuspected interplay between methionine biosynthesis and salt tolerance.

Mol Cell Biol, 1993 Aug, 13(8), 4999 - 5009
Promoter elements determining weak expression of the GAL4 regulatory gene of Saccharomyces cerevisiae; Griggs DW et al.; The GAL4 gene of Saccharomyces cerevisiae (encoding the activator of transcription of the GAL genes) is poorly expressed and is repressed during growth on glucose . To determine the basis for its weak expression and to identify DNA sequences recognized by proteins that activate transcription of a gene that itself encodes an activator of transcription, we have analyzed GAL4 promoter structure . We show that the GAL4 promoter is about 90-fold weaker than the strong GAL1 promoter and at least 7-fold weaker than the feeble URA3 promoter and that this low level of GAL4 expression is primarily due to a weak promoter . By deletion mapping, the GAL4 promoter can be divided into three functional regions . Two of these regions contain positive elements; a distal region termed the UASGAL4 (upstream activation sequence) contains redundant elements that increase promoter function, and a central region termed the UESGAL4 (upstream essential sequence) is essential for even basal levels of GAL4 expression . The third element, an upstream repression sequence, mediates glucose repression of GAL4 expression and is located between the UES and the transcriptional start site . The UASGAL4 is unusual because it is not interchangable with UAS elements in other yeast promoters; it does not function as a UAS element when inserted in a CYC1 promoter, and a normally strong UAS functions poorly in place of UASGAL4 in the GAL4 promoter . Similarly, the UES element of GAL4 does not function as a TATA element in a test promoter, and consensus TATA elements do not function in place of UES elements in the GAL4 promoter . These results suggest that GAL4 contains a weak TATA-less promoter and that the proteins regulating expression of this regulatory gene may be novel and context specific.

EMBO J, 1993 Aug, 12(8), 3049 - 59
Vps1p, a member of the dynamin GTPase family, is necessary for Golgi membrane protein retention in Saccharomyces cerevisiae; Wilsbach K et al.; The KEX2-encoded endoprotease of Saccharomyces cerevisiae resides in the Golgi complex where it participates in the maturation of alpha-factor mating pheromone precursor . Clathrin heavy chain gene disruptions cause mislocalization of Kex2p to the cell surface and reduce maturation of the alpha-factor precursor . Based on these findings, a genetic screen has been devised to isolate mutations that affect retention of Kex2p in the Golgi complex . Two alleles of a single genetic locus, lam1 (lowered alpha-factor maturation), have been isolated, which result in inefficient maturation of alpha-factor precursor . In lam1 cells, Kex2p is not mislocalized to the cell surface but is abnormally unstable . Normal stability is restored by the pep4 mutation which reduces the activity of vacuolar proteases . In contrast, the pheromone maturation defect is not corrected by pep4 . Organelle fractionation by sucrose density gradient centrifugation shows that Kex2p is not retained in the Golgi complex of lam1 cells . Vacuolar protein precursors are secreted by lam1 mutants, revealing another sorting defect in the Golgi complex . Genetic complementation reveals that lam1 is allelic to the VPS1 gene, which encodes a dynamin-related GTPase . These results indicate that Vps1p is necessary for membrane protein retention in a late Golgi compartment.

Mol Cell Biol, 1993 Aug, 13(8), 5043 - 56
Location and characterization of autonomously replicating sequences from chromosome VI of Saccharomyces cerevisiae; Shirahige K et al.; We have reported the isolation of linking clones of HindIII and EcoRI fragments, altogether spanning a 230-kb continuous stretch of chromosome VI . The presence or absence of autonomously replicating sequence (ARS) activities in all of these fragments has been determined by using ARS searching vectors containing CEN4 . Nine ARS fragments were identified, and their positions were mapped on the chromosome . Structures essential for and/or stimulative to ARS activity were determined for the ARS fragments by deletions and mutations . The organization of functional elements composed of core and stimulative sequences was found to be variable . Single core sequences were identified in eight of nine ARSs . The remaining ARS (ARS603) essential element is composed of two core-like sequences . The lengths of 3'- and 5'-flanking stimulative sequences required for the full activity of ARSs varied from ARS to ARS . Five ARSs required more than 100 bp of the 3'-flanking sequence as stimulative sequences, while not more than 79 bp of the 3' sequence was required by the other three ARSs . In addition, five ARSs had stimulative sequences varying from 127 to 312 bp in the 5'-flanking region of the core sequence . In general, these stimulative activities were correlated with low local delta Gs of unwinding, suggesting that the low local delta G of an ARS is an important element for determining the efficiency of initiation of replication of ARS plasmids.

Mol Cell Biol, 1993 Aug, 13(8), 4691 - 702
CSE1 and CSE2, two new genes required for accurate mitotic chromosome segregation in Saccharomyces cerevisiae; Xiao Z et al.; By monitoring the mitotic transmission of a marked chromosome bearing a defective centromere, we have identified conditional alleles of two genes involved in chromosome segregation (cse) . Mutations in CSE1 and CSE2 have a greater effect on the segregation of chromosomes carrying mutant centromeres than on the segregation of chromosomes with wild-type centromeres . In addition, the cse mutations cause predominantly nondisjunction rather than loss events but do not cause a detectable increase in mitotic recombination . At the restrictive temperature, cse1 and cse2 mutants accumulate large-budded cells, with a significant fraction exhibiting aberrant binucleate morphologies . We cloned the CSE1 and CSE2 genes by complementation of the cold-sensitive phenotypes . Physical and genetic mapping data indicate that CSE1 is linked to HAP2 on the left arm of chromosome VII and CSE2 is adjacent to PRP2 on chromosome XIV . CSE1 is essential and encodes a novel 109-kDa protein . CSE2 encodes a 17-kDa protein with a putative basic-region leucine zipper motif . Disruption of CSE2 causes chromosome missegregation, conditional lethality, and slow growth at the permissive temperature.

Mol Cell Biol, 1993 Aug, 13(8), 4618 - 31
Guanine nucleotide exchange factor for eukaryotic translation initiation factor 2 in Saccharomyces cerevisiae: interactions between the essential subunits GCD2, GCD6, and GCD7 and the regulatory subunit GCN3; Bushman JL et al.; Phosphorylation of eukaryotic translation initiation factor 2 (eIF-2) in amino acid-starved cells of the yeast Saccharomyces cerevisiae reduces general protein synthesis but specifically stimulates translation of GCN4 mRNA . This regulatory mechanism is dependent on the nonessential GCN3 protein and multiple essential proteins encoded by GCD genes . Previous genetic and biochemical experiments led to the conclusion that GCD1, GCD2, and GCN3 are components of the GCD complex, recently shown to be the yeast equivalent of the mammalian guanine nucleotide exchange factor for eIF-2, known as eIF-2B . In this report, we identify new constituents of the GCD-eIF-2B complex and probe interactions between its different subunits . Biochemical evidence is presented that GCN3 is an integral component of the GCD-eIF-2B complex that, while dispensable, can be mutationally altered to have a substantial inhibitory effect on general translation initiation . The amino acid sequence changes for three gcd2 mutations have been determined, and we describe several examples of mutual suppression involving the gcd2 mutations and particular alleles of GCN3 . These allele-specific interactions have led us to propose that GCN3 and GCD2 directly interact in the GCD-eIF-2B complex . Genetic evidence that GCD6 and GCD7 encode additional subunits of the GCD-eIF-2B complex was provided by the fact that reduced-function mutations in these genes are lethal in strains deleted for GCN3, the same interaction described previously for mutations in GCD1 and GCD2 . Biochemical experiments showing that GCD6 and GCD7 copurify and coimmunoprecipitate with GCD1, GCD2, GCN3, and subunits of eIF-2 have confirmed that GCD6 and GCD7 are subunits of the GCD-eIF-2B complex . The fact that all five subunits of yeast eIF-2B were first identified as translational regulators of GCN4 strongly suggests that regulation of guanine nucleotide exchange on eIF-2 is a key control point for translation in yeast cells just as in mammalian cells.

J Cell Biol, 1993 Aug, 122(3), 635 - 44
Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae; Holtzman DA et al.; Abplp is a yeast cortical actin-binding protein that contains an SH3 domain similar to those found in signal transduction proteins that function at the membrane/cytoskeleton interface . Although no detectable phenotypes are associated with a disruption allele of ABP1, mutations that create a requirement for this protein have now been isolated in the previously identified gene SAC6 and in two new genes, SLA1 and SLA2 . The SAC6 gene encodes yeast fimbrin, an actin filament-bundling protein . Null mutations in SLA1 and SLA2 cause temperature-sensitive growth defects . Sla1p contains three SH3 domains and is essential for the proper formation of the cortical actin cytoskeleton . The COOH terminus of Sla2p contains a 200 amino acid region with homology to the COOH terminus of talin, a membrane cytoskeletal protein which is a component of fibroblast focal adhesions . Sla2p is required for cellular morphogenesis and polarization of the cortical cytoskeleton . In addition, synthetic-lethal interactions were observed for double-mutants containing null alleles of SLA2 and SAC6 . In total, the mutant phenotypes, sequences, and genetic interactions indicate that we have identified novel proteins that cooperate to control the dynamic cytoskeletal rearrangements that are required for the development of cell polarity in budding yeast.

J Bacteriol, 1993 Aug, 175(15), 4688 - 98
Regulation of the urea active transporter gene (DUR3) in Saccharomyces cerevisiae; ElBerry HM et al.; The DUR3 gene, which encodes a component required for active transport of urea in Saccharomyces cerevisiae, has been isolated, and its sequence has been determined . The deduced DUR3 protein profile possesses alternating hydrophobic and hydrophilic regions characteristics of integral membrane proteins . Strong negative complementation observed during genetic analysis of the DUR3 locus suggests that the DUR3 product may polymerize to carry out its physiological function . Expression of DUR3 is regulated in a manner similar to that of other genes in the allantoin pathway . High-level expression is inducer dependent, requiring functional DAL81 and DAL82 genes . Maintenance of DUR3 mRNA at uninduced, nonrepressed basal levels requires the negatively acting DAL80 gene product . DUR3 expression is highly sensitive to nitrogen catabolite repression and also has a partial requirement for the GLN3 product.

J Protein Chem, 1993 Aug, 12(4), 413 - 8
Limited proteolysis of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase; Herrera L et al.; Incubation of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with trypsin under native conditions cases a time-dependent loss of activity and the production of protein fragments . Cleavage sites determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and sequence analyses identified protease-sensitive peptide bonds between amino acid residues at positions 9-10 and 76-77 . Additional fragmentation sites were also detected in a region approximately 70-80 amino acids before the carboxyl end of the protein . These results suggest that the enzyme is formed by a central compact domain comprising more than two thirds of the whole protein structure . From proteolysis experiments carried out in the presence of substrates, it could be inferred that CO2 binding specifically protects position 76-77 from trypsin action . Intrinsic fluorescence measurements demonstrated that CO2 binding induces a protein conformational change, and a dissociation constant for the enzyme CO2 complex of 8.2 +/- 0.6 mM was determined.

Biochem Mol Biol Int, 1993 Aug, 30(5), 911 - 9
Transport of mitoribosomal proteins, YmL13 and MRP7, into isolated mitochondria of Saccharomyces cerevisiae; Matsushita Y et al.; Two mitochondrial ribosomal proteins, YmL13 and MRP7, of the yeast Saccharomyces cerevisiae and their derivatives were synthesized in vitro and their transport into isolated yeast mitochondria was examined . Both proteins were transported into yeast mitochondria in an energy-dependent manner . MRP7 protein was transported even when its N-terminal presequence was deleted, and the N-terminal presequence alone was not capable of transporting a fused passenger protein, Chinese hamster DHFR . YmL13 protein, on the other hand, was not transported without its N-terminal presequence, and its presequence was capable of transporting Chinese hamster DHFR into mitochondria . Thus, MRP7 appears to possess a transport signal in its mature part, while YmL13 possesses a signal only in its N-terminal presequence.

Anal Biochem, 1993 Aug 1, 212(2), 537 - 9
Hormone-inducible expression and metal affinity chromatography of recombinant proteins in Saccharomyces cerevisiae; Clausen M et al.; An economical method to express recombinant protein in yeast (Saccharomyces cerevisiae) was developed . We combined two principles: Ni(2+)-agarose-based affinity purification of fusion proteins and expression from a cassette regulated by steroid hormones . After induction by desoxycorticosterone, > 90% pure protein was obtained after a single round of metal affinity chromatography . Gentle lysis conditions allowed the preservation of enzymatic activity . Average yields of 10 mg protein per liter of culture were obtained at a fraction of the cost of other eukaryotic expression systems.

Yeast, 1993 Aug, 9(8), 929 - 31
CLG1, a new cyclin-like gene of Saccharomyces cerevisiae; Matsumoto Y et al.; A region of chromosome VII adjacent to SKI8 has a 453 amino acid open reading frame whose sequence has significant similarity to that of HCS26, a G1 cyclin . A disruption mutation of this open reading frame has no apparent phenotype under the conditions tested . ORFD, an open reading frame adjacent to the CDC48 gene, is even more similar to HCS26.

Yeast, 1993 Aug, 9(8), 915 - 21
Sequence and function analysis of a 4.3 kb fragment of Saccharomyces cerevisiae chromosome II including three open reading frames; Schaaff-Gerstenschlager I et al.; The nucleotide sequence of a fragment of 4337 base pairs of Saccharomyces cerevisiae chromosome II has been determined . The sequence contains three open reading frames, one of them being incomplete . Deletion analysis showed that YBR12.31 is essential for yeast growth, while deletion mutants of YBR12.32 and YBR12.33 are viable . YBR12.33 is identical to SMY2, isolated as a suppressor of a myo2 mutant (Lillie, S.H . and Brown, S.S., unpublished, EMBL M90654).

Yeast, 1993 Aug, 9(8), 889 - 905
Linguistic analysis of chromosome III DNA sequence of Saccharomyces cerevisiae; Kalogeropoulos A; The analysis of the Saccharomyces cerevisiae chromosome III DNA sequence by computer ('in silico') permits the definition of its linguistic characteristics . These characteristics include the designation of non-randomly occurring oligonucleotides, their distribution along the chromosome, and the distribution of some particular homopolymers . All these elements may contribute to the understanding of the organization of information on the chromosome.

J Biotechnol, 1993 Aug, 30(2), 211 - 23
High level expression in Saccharomyces cerevisiae of an artificial gene encoding a repeated tripeptide aspartyl-phenylyalanyl-lysine; Choi SY et al.; A chemically synthesized gene, which encodes a 64 or 128 times-repeated tripeptide, aspartyl-phenylalanyl-lysine, has been cloned onto the yeast expression vector pAM82 containing the PHO5 promoter . The artificial gene (LAP gene) contains the untranslated leader sequence of the E . coli lipoprotein gene (lpp) with its transcription terminator sequence . When yeast AH22 cells transformed by recombinant plasmid containing repeated tripeptide gene were derepressed in low phosphate medium, the artificial polypeptides were synthesized to the amounts of about 30% of the total cell protein . SDS-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the artificial polypeptides synthesized in yeast have molecular weights ranging from about 30,000 and 60,000 and have immunoreactivity with the artificial polypeptides expressed in E . coli . The artificial popypeptides in whole cell extract were insoluble and seem to be synthesized as insoluble aggregates . Electron microscopy showed the presence of inclusion bodies in the cell . These polypeptides can be hydrolyzed to tripeptides with trypsin or chymotrypsin . These properties along with the high expression and easy separation may make the artificial polypeptides a potential raw material for the production of an artificial sweetener, Aspartame.

Infect Immun, 1993 Aug, 61(8), 3313 - 7
Characterization of epitopes recognized by Candida factor 1 and 9 antisera by use of Saccharomyces cerevisiae mnn mutants; Ataoglu H et al.; The use of Saccharomyces cerevisiae mnn mutants has facilitated the study of the epitopes recognized by antisera against several antigenic factors of the genus Candida (Candida Check; Iatron Laboratories, Tokyo, Japan) . We have taken advantage of the very well characterized structure of the mannans of the different mnn mutants to compare their reactivities with the factor antisera used in the identification of different species of the genus Candida . The results of this study provide evidence that one of the antigenic determinants recognized by factor 1 antisera is the O-linked mannose chains of the cell wall mannoproteins, while that recognized by factor 9 antiserum is the alpha 1-6-linked mannose backbone of the outer chain of the N-linked oligosaccharide.

J Biol Chem, 1993 Jul 25, 268(21), 15626 - 32
Cytochrome b-deficient mutants of the ubiquinol-cytochrome c oxidoreductase in Saccharomyces cerevisiae . Consequence for the functional and structural characteristics of the complex; Lemesle-Meunier D et al.; We characterized six novel missense mutations in mitochondrial cytochrome b (C133Y, W142R, S206L, M221K, L282F, and G340E) which impair the respiratory growth of yeast and which have differential effects on the functioning and assembly of the bc1 complex . The mutations have been mapped genetically in exons of the mitochondrial gene coding for apocytochrome b and their nucleotide sequence established . The mutants help to better define the topographical and primary sequence location of the ubiquinol oxidase (center P) and ubiquinone reductase (center N) sites on cytochrome b . Two mutants (C133Y and S206L) resulted in an active assembled complex, with selective disturbances of heme 565 and heme 562, respectively, which is consistent with the assignment of the axial ligands of these hemes; the C133Y mutation induced myxothiazol resistance, whereas the S206L did not modify the antimycin binding site, although perturbing the center N . These two amino acid replacements, along with those described elsewhere (Tron, T., and Lemesle-Meunier, D . (1990) Curr . Genet . 18, 413-419), constitute a novel class of mutants exhibiting appreciable electron transfer activity, despite their impaired ability to grow on respiratory substrates, raising the possibility that these mutants carry alleles which result in "decoupling" of proton translocation from electron transfer . Mutants W142R and M221K had an inactive but well assembled bc1 complex, whereas the G34OE and L282F mutations impaired the assembly of the bc1 complex.

Nucleic Acids Res, 1993 Jul 25, 21(15), 3473 - 7
MGM101, a nuclear gene involved in maintenance of the mitochondrial genome in Saccharomyces cerevisiae; Chen XJ et al.; A nuclear mutation, mgm101, results in temperature sensitive loss of mitochondrial DNA (mtDNA) in the yeast Saccharomyces cerevisiae . The corresponding gene, MGM101, was isolated from a genomic DNA library by complementation . Sequence analysis shows that MGM101 encodes a positively charged protein of 269 amino acids with a calculated molecular weight of 30 kDa . This analysis also reveals that MGM101 is adjacent to the ribosomal protein gene RPS7A on chromosome X and hybridization indicates it occurs in single copy . Creation of a null mutant by targeted disruption showed that the gene has no essential cellular function, aside from its participation in mitochondrial genome maintenance . As no counterpart has been identified in databases it is a novel protein whose role has yet to be determined . Expression of MGM101 is low on glucose medium but on galactose there is a two-fold increase in the level of the transcript.

Gene, 1993 Jul 15, 129(1), 141 - 6
Isolation, overexpression and disruption of a Saccharomyces cerevisiae YNK gene encoding nucleoside diphosphate kinase; Fukuchi T et al.; Nucleoside diphosphate kinase (NDPK) is the major enzyme responsible for the synthesis of all nucleoside triphosphates except ATP . A gene (YNK) encoding NDPK was isolated from the yeast Saccharomyces cerevisiae . The coding region consists of 459 bp encoding 153 amino acid (aa) residues . The M(r) of NDPK, calculated from the deduced aa sequence, is 17,166 . Yeast NDPK was 59% and 58% identical to the rat NDPK alpha and beta, respectively . Overexpression of YNK in yeast showed high NDPK activity . The overproduced NDPK cross-reacted with anti-NDPK antibody raised against rat NDPK by Western blot analysis . Despite the fact that NDPK has features of a housekeeping enzyme, disruption of the YNK locus in a haploid strain was neither lethal nor significantly affected phenotypic behaviors such as growth rate, spore formation, mating ability and morphology . Yeast with a defective YNK still possessed NDPK activity to approximately 10% of the wild-type level . Possible sources of the remaining enzyme activity are discussed.

Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6771 - 5
The Saccharomyces cerevisiae PRP21 gene product is an integral component of the prespliceosome; Arenas JE et al.; In Saccharomyces cerevisiae, the prp21 mutation causes accumulation of unspliced pre-mRNA at the nonpermissive temperature . We have cloned the PRP21 gene by complementation of its temperature-sensitive phenotype and found it to be the same as SPP91, an extragenic suppressor of the prp9 mutation previously studied in vivo by Chapon and Legrain {Chapon, C . & Legrain, P . (1992) EMBO J . 11, 3279-3288} . We have analyzed the effects of the prp21 mutation on splicing in vitro and have found that PRP21 is a splicing factor required for prespliceosome assembly . We also have analyzed the interaction of PRP21 with splicing complexes using anti-PRP21 antibodies and found that the RNA components of the prespliceosome--U1 and U2 small nuclear RNA (snRNA) particles and pre-mRNA--are specifically coimmunoprecipitated under splicing conditions in the presence of 0.2 M KCl . At higher KCl concentrations, U1 snRNP dissociates from splicing complexes; nevertheless, U2 snRNA and pre-mRNA are still efficiently immunoprecipitated . Immunoprecipitation of both U1 and U2 snRNA as well as pre-mRNA is ATP-dependent and requires a pre-mRNA capable of supporting prespliceosome assembly . Analysis of the unbound complexes in native gels confirmed that prespliceosomes are specifically immunoprecipitated by anti-PRP21 antibodies . These results demonstrate that PRP21 is an integral component of the prespliceosome and establishes a stable interaction with U2 snRNP and/or pre-mRNA in that complex.

Proc Natl Acad Sci U S A, 1993 Jul 15, 90(14), 6621 - 5
Transcription factors are required for the meiotic recombination hotspot at the HIS4 locus in Saccharomyces cerevisiae; White MA et al.; The full activity of a recombination initiation site located 5' of HIS4 requires the binding of the transcription factors RAP1, BAS1, and BAS2 . Two RAP1 binding sites can substitute for the wild-type initiation site . A 51-bp region of telomeric DNA inserted upstream of either HIS4 or ARG4 very strongly stimulates recombination . We suggest that the ability of transcription factors to induce recombination is a consequence of an altered chromatin structure that favors the entry of proteins that initiate recombination, rather than an effect of these factors on transcription.

J Biol Chem, 1993 Jul 15, 268(20), 15080 - 7
An RNA-binding protein gene (RBP1) of Saccharomyces cerevisiae encodes a putative glucose-repressible protein containing two RNA recognition motifs; Lee FJ et al.; A gene, termed RNA-binding protein (RBP1), was cloned from Saccharomyces cerevisiae . RBP1 contains an open reading frame of 2016 nucleotides that encodes a 672-amino acid protein with a calculated M(r) of approximately 75,000 . Southern blots of genomic DNA from wild-type and RBP1-disrupted strains were consistent with the presence of homologous genes . RNA blots revealed a major 2.7-kb RNA band and two minor bands of 1.5 and 1.1 kb . The sequence of the putative RBP1 protein contains two copies of an RNA recognition motif, two glutamine stretches, an asparagine-rich region, a methionine-rich region, and two long potential alpha-helixes . In addition, recombinant RBP1 fusion protein can bind to RNA and single-stranded DNA but not double-stranded DNA . RBP1 is a glucose-repressible gene . Disruption of RBP1 increased cell growth rate in the early log phase . Overexpression of RBP1 or reduction in its translation by expression of antisense RNA decreased or increased the cell growth rate, respectively . From these observations, we infer that RBP1 may be involved in growth regulation, possibly through its participation in RNA metabolism.

J Biol Chem, 1993 Jul 15, 268(20), 14750 - 6
Translation initiation factor eIF-5A, the hypusine-containing protein, is phosphorylated on serine in Saccharomyces cerevisiae; Kang HA et al.; Translation initiation factor eIF-5A (formerly called eIF-4D) is a small, highly conserved protein in eukaryotic cells that undergoes a unique modification at one of its lysine residues to form hypusine . eIF-5A stimulates in vitro the synthesis of methionyl-puromycin, a model reaction for formation of the first peptide bond . In Saccharomyces cerevisiae eIF-5A is encoded by two highly homologous genes, TIF51A and TIF51B, and each gene gives rise to two hypusinated isoelectric variants, eIF-5Aa (more acidic) and eIF-5Ab (more basic) . In order to study the structural and functional differences between the two isoforms, both isoelectric forms were purified from a yeast strain overexpressing TIF51A and were shown to stimulate identically the synthesis of methionyl-puromycin in a heterologous mammalian assay system . Pulse-chase labeling of yeast cells with {35S}methionine showed that the basic form, eIF-5Ab, is a precursor form of the acidic form, eIF-5Aa . Immunoprecipitation of 32P-labeled cell lysates with rabbit antibodies specific for yeast eIF-5A, phosphoprotein phosphatase treatment of eIF-5Aa, and phosphoamino acid analysis demonstrated that eIF-5Aa is generated by phosphorylation of eIF-5Ab on serine . Therefore eIF-5A undergoes two post-translational modifications, hypusination and phosphorylation, where the activity of the factor is dependent on the first but is not influenced in vitro by the second.

Biochim Biophys Acta, 1993 Jul 10, 1164(2), 143 - 51
Identification of reactive vicinal cysteines in Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phospho enol pyruvate carboxykinases; Rojas MC et al.; Saccharomyces cerevisiae (ATP) and cytosolic rat liver (GTP) phospho enol pyruvate carboxykinases (EC 4.1.1.49/32) have been labeled with N-(1-pyrenyl)-iodoacetamide . Reagent incorporation was completely prevented by the presence of the respective nucleoside diphosphate plus MnCl2 . Under appropriate conditions, 2 mol of reagent per mol of enzyme subunit were incorporated . The fluorescence spectra of the labeled proteins showed the pyrene excimer emission band . The pyrenyl-derivatized enzymes were digested with trypsin after carboxymethylation, and two labeled peptides were isolated for each carboxykinase upon reverse-phase high-performance liquid chromatography . Automated Edman degradation of the labeled peptides indicated that cysteines 364 and 457 (yeast enzyme), and cysteines 288 and 413 (rat enzyme) were labeled with the fluorescence SH-specific reagent . The relative reactivity of these residues was characterized . Labeling experiments utilizing the 5,5'-dithiobis(2-nitrobenzoate)-oxidized enzymes suggested that the reactive SH-groups occupy a vicinal position in the tertiary structure of the proteins, probably in the nucleotide-binding region.

FEBS Lett, 1993 Jul 5, 325(3), 242 - 6
Selective ubiquitination of calmodulin by UBC4 and a putative ubiquitin protein ligase (E3) from Saccharomyces cerevisiae; Parag HA et al.; A putative ubiquitin protein ligase (E3-CaM) which cooperates with UBC4 in selectively ubiquitinating calmodulin has been partially purified from Saccharomyces cerevisiae . Ca2+ was required for this activity and monoubiquitinated calmodulin was the main product of the reaction . The apparent Km of E3-CaM for calmodulin was approximately 1 microM which is of the same order of magnitude as the concentration of calmodulin in yeast cells . Proteins which are good substrates for other E3s (E3 alpha or E3-R) were not ubiquitinated by E3-CaM . Lower but significant activities of E3-CaM were observed when UBC1 replaced UBC4.

FEBS Lett, 1993 Jul 5, 325(3), 191 - 5
The pde2 gene of Saccharomyces cerevisiae is allelic to rca1 and encodes a phosphodiesterase which protects the cell from extracellular cAMP; Wilson RB et al.; The high affinity cAMP phosphodiesterase, encoded by PDE2, is an important component of the cAMP-dependent protein kinase signaling system in Saccharomyces cerevisiae . An unexpected phenotype of pde2 mutants is sensitivity to external cAMP . This trait has been found independently for rca1 mutants and has been used to monitor the effects of cAMP on several biological processes . We demonstrate here that RCA1 is identical to PDE2 . Further analysis of the phenotype of pde2 deletions reveal that exogenously added cAMP results in an increase in the internal level of cAMP . This increase slows down the rate of cell division by increasing the length of the G1 phase of the cell cycle and leads to increased cell volume . Also, cells with a disrupted PDE2 gene previously arrested by nutrient starvation rapidly lose thermotolerance when incubated with exogenous cAMP . From these observations we propose that a role of the PDE2-encoded phosphodiesterase may be to help insulate the internal cAMP pools from the external environment . This protective role might also be important in other eukaryotic organisms where cAMP is a key second messenger.

Cell Signal, 1993 Jul, 5(4), 435 - 41
Glucose activation of adenylate cyclase in Saccharomyces cerevisiae mutants lacking glucose-phosphorylating enzymes; Pardo LA et al.; The "in vitro" activation by glucose of the RAS-adenylate cyclase system in membranes from a strain of Saccharomyces cerevisiae lacking any functional glucose kinase activity presents similar features to those of the wild type . However, this triple mutant appears to be unable to produce the glucose-induced increase of cAMP levels "in vivo" . The results obtained in vitro indicate that the signal transduction mechanism is active in the mutant cells and suggest that the absence of intracellular acidification in vivo might be responsible for the lack of response to glucose.

Biokhimiia, 1993 Jul, 58(7), 1053 - 61
{Characteristics of polyphosphatase activity of vacuoles in Saccharomyces cerevisiae cells}; Andreeva NA et al.; Vacuoles of the Saccharomyces cerevisiae yeast possess a polyphosphatase activity which differs from other known vacuolar phosphohydrolase activities by pH-optimum, sensitivity towards inhibitors and distribution between the tonoplast and vacuolar sap . The polyphosphatase activity is inhibited by EDTA, molybdate, ortho-vanadate and fluoride . Nearly 77% of this activity is located in the vacuolar sap, while 25%--in the tonoplast fraction . Both the soluble and membrane-bound polyphosphatase activities are maximal at pH 6.8-7.2 . Bivalent metal cations stimulate this activity in the following order: Zn2+ > Mg2+ > Co2+ > Mn2+ . Other ions (Fe2+, Cu2+, Ca2+) inhibit this activity at all concentrations tested . The polyphosphatase activity of both the vacuolar sap and the tonoplast increases 2.4-and 2-fold, respectively, with an increase in the degree of substrate polymerization--from n = 3 to n = 208.

Curr Genet, 1993 Jul-Aug, 24(1-2), 26 - 31
'Phase variation'-type regulation of gene expression and gene replacement mediated by FLP recombinase in the yeast Saccharomyces cerevisiae; Saveliev SV et al.; Expression of a neomycin phosphotransferase II (NPTII) gene has been designed to be regulated by an FLP-mediated switching of the orientation of the NPTII coding region located on the invertible DNA segment in episomal yeast plasmids . Inversion of the segment from inverted to direct orientation with respect to the promoter resulted in a dramatic increase in G418 resistance . FLP also promoted a double reciprocal exchange between the transforming and the resident 2-microns plasmid, leading to insertion of the FLP and REP2 genes into the transforming plasmid . The results demonstrate a possible use of FLP recombinase for 'phase variation'-type regulation of gene expression and gene replacement in eukaryotic cells.

Arch Biochem Biophys, 1993 Jul, 304(1), 197 - 204
Characteristics, substrate analysis, and intracellular location of Saccharomyces cerevisiae UMP kinase; Jong A et al.; The yeast Saccharomyces cerevisiae SOC8 DNA fragment was isolated as a wildtype dominant suppressor of the cdc8 mutation . The SOC8 has previously been proved to be allelic with URA6, encoding the UMP kinase in yeast cells . The protein has been purified to homogeneity . In this report, we describe the characteristics of the UMP kinase from yeast . The yeast enzyme requires a divalent cation and is active over the entire range of pH from 6 to 9.5 . The enzyme can use UMP and dUMP as phosphate acceptors with high activity; to a lesser extent, it can also use IMP, GMP, dGMP, 5-iodo-dUMP, XMP, and dTMP as substrates . ATP and dATP are the best phosphate donors; the enzyme could use GTP, dGTP, dCTP, and dTTP to some degree (30-50%) . CTP and UTP were poor phosphate donors for the UMP kinase reaction (10-14%) . Like other monophosphate kinases, UMP kinase contains a conserved nucleotide-binding site at its N-terminus following a cysteine residue, and its enzymatic activity is inhibited by sulfhydryl inhibitors such as 5,5'-dithio-bis(2-nitrobenzoic acid) and N-ethylmaleimide . Subcellular localization studies indicate that the UMP kinase locates primarily in the cytoplasm (approximately 80%) and also in the nucleus (approximately 20%), but not in the mitochondria . These results suggest that it may exert its function in the nucleus, such as in RNA synthesis, as well as in the cytoplasm, but not in the mitochondria . The presence of UMP kinase in the nucleus might facilitate its suppression of cdc8 mutant cells, which are defective in nuclear DNA synthesis.

Mol Cell Biol, 1993 Jul, 13(7), 4351 - 64
A mutation in PLC1, a candidate phosphoinositide-specific phospholipase C gene from Saccharomyces cerevisiae, causes aberrant mitotic chromosome segregation; Payne WE et al.; We identified a putative Saccharomyces cerevisiae homolog of a phosphoinositide-specific phospholipase C (PI-PLC) gene, PLC1, which encodes a protein most similar to the delta class of PI-PLC enzymes . The PLC1 gene was isolated during a study of yeast strains that exhibit defects in chromosome segregation . plc1-1 cells showed a 10-fold increase in aberrant chromosome segregation compared with the wild type . Molecular analysis revealed that PLC1 encodes a predicted protein of 101 kDa with approximately 50 and 26% identity to the highly conserved X and Y domains of PI-PLC isozymes from humans, bovines, rats, and Drosophila melanogaster . The putative yeast protein also contains a consensus EF-hand domain that is predicted to bind calcium . Interestingly, the temperature-sensitive and chromosome missegregation phenotypes exhibited by plc1-1 cells were partially suppressed by exogenous calcium.

J Gen Microbiol, 1993 Jul, 139 ( Pt 7), 1617 - 26
Distribution of 64Cu in Saccharomyces cerevisiae: kinetic analyses of partitioning; Lin CM et al.; The cell association of copper in the yeast Saccharomyces cerevisiae can involve both binding to the cell wall and the accumulation of copper within the cell . The former process requires the concurrent generation of H2S by the cell via the reduction of sulphate . The contributions of each of these processes to the uptake of 64Cu by wild type and met3-containing (ATP sulphurylase-deficient) strains have been kinetically dissected . The Michaelis constant for uptake (4 microM) is independent of the type of cell association which is occurring, suggesting, although not requiring, that both processes are associated with a common kinetic intermediate . The time dependence of the cell-association of 64Cu also suggests the presence of this intermediate pool of bound copper . The Vmax for uptake includes a constant contribution from accumulation of 64Cu within the plasmalemma {0.1 nmol min-1 (mg protein)-1} plus that fraction of the 64Cu within the intermediate pool which diffuses away and is trapped on the cell wall as a metal sulphide . This latter contribution to Vmax can be two- to three-times greater than the intracellular uptake depending on the amount and type of sulphur supplementation provided in the 64Cu2+ uptake buffer . Both processes are energy-dependent although the sulphide-dependent periplasmic accumulation is somewhat more sensitive to metabolic inhibition . This can be attributed to the ATP required for the activation of sulphate prior to its reduction to the level of sulphite and then sulphide . Periplasmic 64Cu accumulation is strongly inhibited by Zn2+ and Ni2+ . This inhibition is due to competition for cell-generated sulphide; in the presence of 65Zn2+, the decrease in 64Cu bound is quantitatively related to the amount of 65Zn which becomes cell-associated . In contrast, intracellular 64Cu uptake is not inhibited by these two metals (at 50 microM) showing that the copper translocation pathway is metal-specific . These observations suggest a model for the way newly arrived copper is handled at the cell membrane and is partitioned for intracellular uptake.

J Gen Microbiol, 1993 Jul, 139 ( Pt 7), 1409 - 15
Clonal size-variation of rDNA cluster region on chromosome XII of Saccharomyces cerevisiae; Chindamporn A et al.; Using pulsed-field gel electrophoresis (PFGE), we have demonstrated clonal variation in the size of chromosome XII in a diploid strain of Saccharomyces cerevisiae X2180-2D . The sizes of the two chromosome XII homologues were very different: 2600 (L-type) and 1450 kb (S-type) . The frequency with which we detected clonal size variation in the diploid, compared to that of the parental clones, was about 15-50% of the progeny clones and the range of the size variation of the homologues was 2580-2680 kb (L-type) and 1340-1500 kb (S-type), respectively . The homologue of the L-type appeared to be more frequently variable than that of the S-type . The size variation was shown to be derived from size changes in the rDNA cluster region, which is present in chromosome XII, by digesting the chromosome with XhoI, whose cutting site is not present in a rDNA repeat unit, and hybridizing to rDNA probes . The clonal size variation was also investigated in haploids from spores after meiosis . The L-type and S-type chromosomes segregated 2:2 in an ascus and the sizes of all the S-type chromosomes were shifted up, compared to the original diploid, though the L-type ones were stable . The S-type sizes of 1340, 1450 and 1780 kb in the original diploids changed into the ranges of 1475-1610 kb, 1520-1680 kb and 1820-2010 kb, respectively, in the segregants . Furthermore, we observed that the size of S-type chromosomes in haploid cells was gradually increasing in mitosis during successive subcultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Yeast, 1993 Jul, 9(7), 783 - 5
Vector YFRp1 allows transformant selection in Saccharomyces cerevisiae via resistance to formaldehyde; Wehner EP et al.; Formaldehyde (FA), a chemical with low toxic potential, is used as sole selective agent for transformation in the yeast Saccharomyces cerevisiae . Neither stable auxotrophic markers in recipient cells nor defined synthetic media are needed when multicopy vector YFRp1, containing the yeast SFA gene, is employed for yeast transformation . The SFA gene gives stability to the vector and its yeast (and other) passenger genes when transformants are propagated in complex media supplemented with 3-5 mM-FA . Use of inexpensive FA and non-synthetic, undefined media will lower the cost of yeast transformant propagation considerably and thus make feasible large-volume industrial application of transformants containing YFRp1 derivatives.

Yeast, 1993 Jul, 9(7), 771 - 82
Cloning and sequencing of the Saccharomyces cerevisiae gene LYP1 coding for a lysine-specific permease; Sychrova H et al.; The LYP1 gene of Saccharomyces cerevisiae was cloned by complementation in lysine-permease-deficient recipient yeast cells, and its nucleotide sequence was determined . An open reading frame of 1833 nucleotides was found encoding a polypeptide of 611 amino acids, with a calculated molecular weight of 68 118 . Analysis of the deduced primary structure of the protein revealed ten membrane-spanning regions and three potential N-glycosylation sites . Analysis of the deduced sequence of protein LYP1 indicates homology with other yeast amino-acid permeases, in particular with CAN1, and also the lysine-specific permease of Escherichia coli . The strain transformed by a multi-copy plasmid harbouring the LYP1 gene, showed a 20-fold increase in the maximum velocity of lysine uptake over that in the wild type, with no changes in the affinity of the permease for its substrate.

Yeast, 1993 Jul, 9(7), 703 - 13
Molecular characterization of the SEC1 gene of Saccharomyces cerevisiae: subcellular distribution of a protein required for yeast protein secretion; Egerton M et al.; Strains of Saccharomyces cerevisiae harbouring temperature-sensitive mutations in the SEC1 and SEC5 genes exhibit an accumulation of post-Golgi secretory vesicles at 37 degrees C . We have cloned a fragment of yeast DNA which carries two distinct genes, one of which complements a sec1 mutation, and the other a sec5 mutation . Genetic test confirm that the sec1-complementing gene is indeed SEC1, and is essential for cell growth . Nucleotide sequence analysis reveals that the cloned SEC1 gene is the same as a previously sequenced sec1-complementing gene . The SEC1 sequence encodes a protein of 724 amino acids with a predicted molecular mass of 83 kDa . Antibodies purified from a polyclonal antiserum raised against the protein product of the cloned gene recognize a yeast protein of apparent molecular mass 78 kDa which is found in a detergent-resistant association with a rapidly sedimenting yeast subcellular fraction, behaviour which is suggestive of an interaction with a component of the yeast cytoskeleton.

Curr Genet, 1993 Jul-Aug, 24(1-2), 21 - 5
The glycerol kinase (GUT1) gene of Saccharomyces cerevisiae: cloning and characterization; Pavlik P et al.; The GUT1 gene of Saccharomyces cerevisiae, encoding glycerol kinase, was cloned and sequenced . The cloned genomic DNA fragment contains an open reading frame potentially coding for a protein of 709 amino acids with homology to bacterial glycerol kinases (40.8% identity over 502 amino acids, and 42.1% identity over 496 amino acids, in comparison to the smaller E . coli and B . subtilis enzymes) . Disruption of GUT1 showed that the gene is required for growth on glycerol, but not on glucose or ethanol media . No glycerol kinase activity was detected in the disruption mutant . According to enzyme activity and transcript analysis, synthesis of glycerol kinase is repressed by glucose, and derepression is ADR1-dependent.

Curr Genet, 1993 Jul-Aug, 24(1-2), 136 - 40
Molecular cloning and analysis of the nuclear gene MRP-L6 coding for a putative mitochondrial ribosomal protein from Saccharomyces cerevisiae; Harrer R et al.; The Saccharomyces cerevisiae nuclear gene MRP-L6 was cloned by complementation of the respiratory-deficient mutant pet-ts 2523 with a library of wild-type yeast genomic DNA . The isolated gene was part of a 3.8-kb sequenced DNA fragment containing, in addition to MRP-L6, two unassigned reading frames, ORF1 and ORF2 . MRP-L6 codes for a basic protein of 205 amino acids and a molecular mass of 22.8 kDa . The protein exhibits significant sequence similarity to the ribosomal protein L6 of bacteria and chloroplasts . Unlike the corresponding bacterial proteins, however, the MRP-L6 protein (MRP-L6p) contains at its N-terminus a 16 amino-acid leader sequence exhibiting the known characteristics of mitochondrial import signals . Disruption of MRP-L6 leads to the phenotype of a mitochondrial translation-defective, rho-negative yeast mutant . The results are consistent with MRP-L6p representing an essential component of yeast mitochondrial ribosomes . Expression of MRP-L6 was examined, under conditions of glucose repression and derepression, in wild-type cells and in a series of catabolite repression-defective yeast mutants . In most cases, a distinct though small influence of the carbon source on the expression of an MRP-L6/lacZ reported construct was observed.

Curr Genet, 1993 Jul-Aug, 24(1-2), 126 - 35
Characterization of a second nuclear gene, AEP1, required for expression of the mitochondrial OLI1 gene in Saccharomyces cerevisiae; Payne MJ et al.; Due to mutation in a single nuclear locus, AEP1, the temperature-conditional pet mutant ts1860 of Saccharomyces cerevisiae fails to synthesize mitochondrial ATP synthase subunit 9 at the restrictive temperature of 36 degrees C . The presence at this temperature of near-normal levels of the cognate oli1 mRNA in mutant ts1860 indicates that, as previously shown, the product of the AEP1 gene is required for translation of the mitochondrial oli1 transcript . In this study the AEP1 gene has been cloned from a wild-type yeast genomic library by genetic complementation of a temperature-conditional aep1 strain at the restrictive temperature . A 2,330-bp genomic fragment which restores subunit 9 synthesis in aep1 mutant strains was characterized . This fragment encoded five open reading frames: the longest of these, at 1,554 nucleotides, was identified as the AEP1 gene, since disruption of this reading frame generated a non-conditional pet strain unable to synthesize subunit 9 . The predicted product of AEP1 is a basic, hydrophilic protein of 59,571 Da which possesses a putative mitochondrial address sequence . Hybridization studies with AEP1-specific probes indicate that the gene is located on chromosome XIII and produces several poly(A)+ transcripts ranging in size from 0.9 to 2.7 kb . None of the identified reading frames share significant homologies with entries of several data bases.

Curr Genet, 1993 Jul-Aug, 24(1-2), 122 - 5
Symmetrical transcription in the tRNA region of the mitochondrial genome of Saccharomyces cerevisiae; Grisanti P et al.; The occurrence of discrete transcripts originating from the non-coding strand of the yeast mitochondrial genome is described . The region under investigation is localized in the large tRNA gene cluster between the LSU ribosomal RNA and OXI 1 genes . The transcripts originating from the non-coding strand were detected in a wild-type strain and in a rho- mutant . Their size range includes transcripts of about 2000 nucleotides able to accommodate more than one "anti-tRNA" . In some cases their extremities can be mapped near highly-conserved nonanucleotides that could function as origins of transcription . The involvement of the tRNA-processing machinery in the cleavage of these transcripts is also hypothesized.

Curr Genet, 1993 Jul-Aug, 24(1-2), 1 - 6
Asd-homothallism of Saccharomyces cerevisiae: identification of asd1-1 as an allele of sir4 and detection of alpha-specific suppressors of it; Ono B et al.; Asd-homothallism of Saccharomyces cerevisiae involves a life cycle characterized by a non-mating phenotype and endomitotic diploidization . The former trait is determined by a single mutation, asd1-1 . This mutation was mapped between hom2 and lys4 on the right arm of chromosome IV and was complemented by the cloned SIR4 gene . Therefore, we conclude that asd1-1 is an allele of sir4-11 and renamed it sir4-11 . Endomitotic diploidization of asd-homothallism is caused by the collaboration of three to four mutations including sir4-11 . In the course of this study, we detected alpha-specific suppressors of sir4-11.

Genetics, 1993 Jul, 134(3), 701 - 16
TSF1 to TSF6, required for silencing the Saccharomyces cerevisiae GAL genes, are global regulatory genes; Chen S et al.; The Saccharomyces cerevisiae GAL1 and GAL10 genes are controlled in response to the availability of galactose and glucose by multiple activating and repressing proteins bound at adjacent or overlapping sites in UASG . Negative control elements in UASG, designated GAL operators GALO1 to GALO6, are required to silence basal level transcription of GAL1 and GAL10 when galactose is absent . We isolated and characterized recessive mutations in six nuclear genes, TSF1 to TSF6, that impair silencing of GAL1 and GAL10 gene expression . Surprisingly, the results of several experiments suggest that the TSF genes encode global regulatory factors . tsf1 to tsf6 mutations derepressed expression from yeast CYC-GAL hybrid promoters (fused to lacZ) that harbor a variety of operator sequences, and caused pleiotropic defects in cell growth, mating, and sporulation . S1 mapping and Northern blot results for tsf3 suggest that the molecular defect is at the transcriptional level . Mutant phenotypes were additive in certain combinations of tsf double mutants, implying that more than one silencing pathway is involved in TSF1 to TSF6 function . Most significantly, mutations in all six TSF1 to TSF6 genes activated expression from GAL1 and CYC1 promoters (fused to lacZ) lacking upstream activating sequences . Combined, the simplest interpretation of these results is that TSF1 to TSF6 encode factors that control the function of the basic RNA polymerase II transcriptional machinery.

EMBO J, 1993 Jul, 12(7), 2831 - 40
The TIP1 gene of Saccharomyces cerevisiae encodes an 80 kDa cytoplasmic protein that interacts with the cytoplasmic domain of Sec20p; Sweet DJ et al.; The SEC20 gene of Saccharomyces cerevisiae encodes a 50 kDa type II integral membrane glycoprotein that is required for endoplasmic reticulum (ER) to Golgi transport . Here, we have used a genetic screen, based on the lethal effect of overexpressing the cytoplasmic domain of Sec20p, to identify a novel cytosolic factor that interacts with SEC20 . This factor is an 80 kDa cytoplasmic protein encoded by the TIP1 (SEC twenty interacting protein) gene . Coimmunoprecipitation and immunofluorescence using Tip1p and Sec20p or its cytoplasmic domain showed that the two proteins physically interact to form a stable complex . Like SEC20, TIP1 is required for ER to Golgi transport and depletion of Tip1p results in accumulation of an extensive network of ER plus small transport vesicles . We therefore propose that Sec20p and Tip1p act together as a functional unit in the ER to Golgi transport step.

Mol Cell Biol, 1993 Jul, 13(7), 4445 - 57
A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5; Kitada K et al.; We have isolated a multicopy suppressor of the temperature-sensitive growth phenotype of organisms carrying mutations of DBF4, a gene that is required for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae and that interacts with the CDC7 protein kinase . Nucleotide sequence analysis of the suppressor gene, provisionally named MSD2, revealed an open reading frame encoding a protein with a calculated M(r) of 81,024, with amino acid sequence similarity to the catalytic domains of protein kinases . Both genetic linkage and complementation analyses indicated that MSD2 is identical to the cell division cycle gene CDC5 . An activity that phosphorylated exogenously added casein was immunoprecipitated by antiserum against a TrpE-Cdc5 fusion protein from lysates of wild-type cells containing CDC5 on a multicopy plasmid but not of cells bearing a small deletion in the predicted protein kinase domain of CDC5 on the plasmid . Deletion of CDC5 was lethal and resulted in a dumbbell-shaped terminal morphology, with the nuclei almost divided but still connected . Consistent with the function at the G2/M boundary, the CDC5 transcript accumulated periodically during the cell cycle, peaking at the G2/M boundary . CDC5 on a multicopy plasmid also suppresses temperature-sensitive cdc15, cdc20, and dbf2 mutations which affect mitosis during the cell cycle.

Mol Cell Biol, 1993 Jul, 13(7), 4331 - 41
Evidence that the SKI antiviral system of Saccharomyces cerevisiae acts by blocking expression of viral mRNA; Widner WR et al.; The SKI2 gene is part of a host system that represses the copy number of the L-A double-stranded RNA (dsRNA) virus and its satellites M and X dsRNA, of the L-BC dsRNA virus, and of the single-stranded replicon 20S RNA . We show that SKI2 encodes a 145-kDa protein with motifs characteristic of helicases and nucleolar proteins and is essential only in cells carrying M dsRNA . Unexpectedly, Ski2p does not repress M1 dsRNA copy number when M1 is supported by aN L-A cDNA clone; nonetheless, it did lower the levels of M1 dsRNA-encoded toxin produced . Since toxin secretion from cDNA clones of M1 is unaffected by Ski2p, these data suggest that Ski2p acts by specifically blocking translation of viral mRNAs, perhaps recognizing the absence of cap or poly(A) . In support of this idea, we find that Ski2p represses production of beta-galactosidase from RNA polymerase I {no cap and no poly(A)} transcripts but not from RNA polymerase II (capped) transcripts.

Mol Cell Biol, 1993 Jul, 13(7), 4260 - 75
Genetic evidence for in vivo cross-specificity of the CaaX-box protein prenyltransferases farnesyltransferase and geranylgeranyltransferase-I in Saccharomyces cerevisiae; Trueblood CE et al.; Two protein prenyltransferase enzymes, farnesyltransferase (FTase) and geranylgeranyltransferase-I (GGTase-I), catalyze the covalent attachment of a farnesyl or geranylgeranyl lipid group to the cysteine of a CaaX sequence (cysteine {C}, two aliphatic amino acids {aa}, and any amino acid {X} . In vitro studies reported here confirm previous reports that CaaX proteins with a C-terminal serine are farnesylated by FTase and those with a C-terminal leucine are geranylgeranylated by GGTase-I . In addition, we found that FTase can farnesylate CaaX proteins with a C-terminal leucine and can transfer a geranylgeranyl group to some CaaX proteins . Genetic data indicate that FTase and GGTase-I have the same substrate preferences in vivo as in vitro and also show that each enzyme can prenylate some of the preferred substrates of the other enzyme in vivo . Specifically, the viability of yeast cells lacking FTase is due to prenylation of Ras proteins by GGTase-I . Although this GGTase-I dependent prenylation of Ras is sufficient for growth, it is not sufficient for mutationally activated Ras proteins to exert deleterious effects on growth . The dependence of the activated Ras phenotype on FTase can be bypassed by replacing the C-terminal serine with leucine . This altered form of Ras appears to be prenylated by both GGTase-I and FTase, since it produces an activated phenotype in a strain lacking either FTase or GGTase-I . Yeast cells can grow in the absence of GGTase-I as long as two essential substrates are overexpressed, but their growth is slow . Such strains are dependent on FTase for viability and are able to grow faster when FTase is overproduced, suggesting that FTase can prenylate the essential substrates of GGTase-I when they are overproduced.

Mol Cell Biol, 1993 Jul, 13(7), 3937 - 50
Substrate length requirements for efficient mitotic recombination in Saccharomyces cerevisiae; Jinks-Robertson S et al.; An ectopic recombination system using ura3 heteroalleles varying in size from 80 to 960 bp has been used to examine the effect of substrate length on spontaneous mitotic recombination . The ura3 heteroalleles were positioned either on nonhomologous chromosomes (heterochromosomal repeats) or as direct or inverted repeats on the same chromosome (intrachromosomal repeats) . While the intrachromosomal events occur at rates at least 2 orders of magnitude greater than the corresponding heterochromosomal events, the recombination rate for each type of repeat considered separately exhibits a linear dependence on substrate length . The linear relationships allow estimation of the corresponding minimal efficient processing segments, which are approximately 250 bp regardless of the relative positions of the repeats in the yeast genome . An examination of the distribution of recombination events into simple gene conversion versus crossover events indicates that reciprocal exchange is more sensitive to substrate size than is gene conversion.

Mol Cell Biol, 1993 Jul, 13(7), 3919 - 28
Epigenetic switching of transcriptional states: cis- and trans-acting factors affecting establishment of silencing at the HMR locus in Saccharomyces cerevisiae; Sussel L et al.; In this study, we used the ADE2 gene in a colony color assay to monitor transcription from the normally silent HMR mating-type locus in Saccharomyces cerevisiae . This sensitive assay reveals that some previously identified cis- and trans-acting mutations destabilize silencing, causing genetically identical cells to switch between repressed and derepressed transcriptional states . Deletion of the autonomously replicating sequence (ARS) consensus element at the HMR-E silencer or mutation of the silencer binding protein RAP1 (rap1s) results in the presence of large sectors within individual colonies of both repressed (Ade-, pink) and derepressed (Ade+, white) cells . These results suggest that both the ARS consensus element and the RAP1 protein play a role in the establishment of repression at HMR . In diploid cells, the two copies of HMR appear to behave identically, suggesting that the switching event, though apparently stochastic, reflects some property of the cell rather than a specific event at each HMR locus . In the ADE2 assay system, silencing depends completely upon the function of the SIR genes, known trans-acting regulators of the silent loci, and is sensitive to the gene dosage of two SIR genes, SIR1 and SIR4 . Using the ADE2 colony color assay in a genetic screen for suppressors of rap1s, silencer ARS element deletion double mutants, we have identified a large number of genes that may affect the establishment of repression at the HMR silent mating-type locus.

Mol Cell Biol, 1993 Jul, 13(7), 3872 - 81
Two homologous zinc finger genes identified by multicopy suppression in a SNF1 protein kinase mutant of Saccharomyces cerevisiae; Estruch F et al.; The MSN2 gene was selected as a multicopy suppressor in a temperature-sensitive SNF1 protein kinase mutant of Saccharomyces cerevisiae . MSN2 encodes a Cys2His2 zinc finger protein related to the yeast MIG1 repressor and to mammalian early growth response and Wilms' tumor zinc finger proteins . Deletion of MSN2 caused no phenotype . A second similar zinc finger gene, MSN4, was isolated, and deletion of both genes caused phenotypic defects related to carbon utilization . Overexpression of the zinc finger regions was deleterious to growth . LexA-MSN2 and LexA-MSN4 fusion proteins functioned as strong transcriptional activators when bound to DNA . Functional roles of this zinc finger protein family are discussed.

J Bacteriol, 1993 Jul, 175(13), 4235 - 8
The INO1 promoter of Saccharomyces cerevisiae includes an upstream repressor sequence (URS1) common to a diverse set of yeast genes; Lopes JM et al.; The INO1 promoter of Saccharomyces cerevisiae includes a copy of an upstream repression sequence (URS1; 5'AGCCGCCGA 3') observed in the promoters of several unrelated yeast genes . Expression of INO1-lacZ and CYC1-lacI'Z, activated by the INO1 UASINO, is significantly decreased by the INO1 URS1.

Genes Dev, 1993 Jul, 7(7A), 1160 - 75
CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae; Schwob E et al.; The functions of the Cdc28 protein kinase in DNA replication and mitosis in Saccharomyces cerevisiae are thought to be determined by the type of cyclin subunit with which it is associated . G1-specific cyclins encoded by CLN1, CLN2, and CLN3 are required for entry into the cell cycle (Start) and thereby for S phase, whereas G2-specific B-type cyclins encoded by CLB1, CLB2, CLB3, and CLB4 are required for mitosis . We describe a new family of B-type cyclin genes, CLB5 and CLB6, whose transcripts appear in late G1 along with those of CLN1, CLN2, and many genes required for DNA replication . Deletion of CLB6 has little or no effect, but deletion of CLB5 greatly extends S phase, and deleting both genes prevents the timely initiation of DNA replication . Transcription of CLB5 and CLB6 is normally dependent on Cln activity, but ectopic CLB5 expression allows cells to proliferate in the absence of Cln cyclins . Thus, the kinase activity associated with Clb5/6 and not with Cln cyclins may be responsible for S-phase entry . Clb5 also has a function, along with Clb3 and Clb4, in the formation of mitotic spindles . Our observation that CLB5 is involved in the initiation of both S phase and mitosis suggests that a single primordial B-type cyclin might have been sufficient for regulating the cell cycle of the common ancestor of many, if not all, eukaryotes.

Genes Dev, 1993 Jul, 7(7A), 1146 - 59
RAP1 and telomere structure regulate telomere position effects in Saccharomyces cerevisiae; Kyrion G et al.; To investigate the role of the yeast telomere-, silencing-, and UAS-binding protein RAP1 in telomere position effects, we have characterized two sets of mutant cells: (1) a set of rap1 alleles (termed the rap1t alleles) that produce truncated RAP1 proteins missing the carboxy-terminal 144-165 amino acids; and (2) null mutants of the RIF1 gene, encoding a protein capable of interaction with the carboxyl terminus of RAP1 . The data presented here indicate that loss of the carboxyl terminus of RAP1 abolishes position effects at yeast telomeres and diminishes silencing at the HML locus . Elimination of position effects in these cells is associated with increased accessibility to the Escherichia coli dam methylase in vivo . Thus, the carboxy-terminal domain of RAP1 is required for telomere position effects . In contrast, rif1 deletion alleles increase the frequency of repressed cells . Using the rap1t alleles to generate wild-type cells differing only in telomere tract lengths, we also show that telomere position effects are highly sensitive to changes in the size (or structure) of the telomeric tract . Longer poly(G1-3T) tracts can increase the frequency of transcriptional repression at the telomere, suggesting that telomeric poly(G1-3T) tracts play an active role in the formation or stability of subtelomeric transcriptional states.

Plant Physiol, 1993 Jul, 102(3), 783 - 8
Apple ripening-related cDNA clone pAP4 confers ethylene-forming ability in transformed Saccharomyces cerevisiae; Wilson ID et al.; The apple ripening-related cDNA insert of clone pAP4 (G.S . Ross, M.L . Knighton, M . Lay-Yee {1992} Plant Mol Biol 19: 231-238) has previously been shown to have considerable nucleic acid and predicted amino acid sequence similarity to the insert of a tomato ripening-related cDNA clone (pTOM13) that is known to encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase (A.J . Hamilton, G.W . Lycett, D . Grierson {1990} Nature 346: 284-287; A.J . Hamilton, M . Bouzayen, D . Grierson {1991} Proc Natl Acad Sci USA 88: 7434-7437) . The cDNA insert from the clone pAP4 was fused between the galactose-inducible promoter and the terminator of the yeast expression vector pYES2 . Transformation of Saccharomyces cerevisiae strain F808- with this DNA construct and incubation of the yeast in the presence of D{+}-galactose allowed these cells to convert ACC to ethylene . The transformed yeast converted 1-amino-2-ethylcyclopropane-1-carboxylate isomers to 1-butene with the same 1R,2S-stereoselectivity as achieved by the native ACC oxidase from applies . Both ascorbate and Fe2+ ions stimulated the rate of the production of ethylene from ACC by the transformed yeast, whereas Cu2+ and Co2+ were strongly inhibitory; these are features of ACC oxidase . Northern analysis of the total RNA from nontransformed and transformed yeast showed that the ability to convert the ACC to ethylene was correlated with the synthesis and accumulation of a novel 1.2-kb mRNA that hybridized to the cDNA clone pAP4 . We conclude that the cDNA sequence of the clone pAP4 encodes ACC oxidase.

J Environ Pathol Toxicol Oncol, 1993 Jul-Sep, 12(3), 143 - 7
Study of spermine and spermidine effects on Saccharomyces cerevisiae . Polyamine production in different growth conditions and in the presence of interleukin-2; Del Carratore R et al.; The role of polyamines during cell growth is still uncertain . Yeast cells possess an amine pathway similar to that described for animal cells . We studied the relationship between growth and polyamine production in yeast cells of Saccharomyces cerevisiae grown under various conditions . Polyamines were determined in homogenates of yeast cells by a rapid enzymatic assay, confirmed by thin-layer chromatography . Polyamine production is dependent on the growth conditions . The effects of spermine, spermidine, and interleukin-2 on yeast cells were investigated . Exogenous polyamines affected the endogenous pathway, and interleukin-2 treatment increase yeast growth and polyamine production.

Enzyme Microb Technol, 1993 Jul, 15(7), 593 - 600
Enhanced recovery and purification of Aspergillus glucoamylase from Saccharomyces cerevisiae by the addition of poly(aspartic acid) tails; Suominen I et al.; Poly(aspartic acid) tails of different lengths were fused to the glucoamylase (GA) of Aspergillus awamori by genetic engineering techniques . Tails consisting of 5, 7, and 10 aspartate residues were fused to the N-terminus of the full-length mature GA (aa 1-616) downstream from the intact leader peptide to produce fusion proteins designated GAND5, GAND7, and GAND10, respectively . Three fusion proteins with C-terminal tails were also constructed, designated GACD0, GACD5, and GACD10 (0, 5, and 10 aspartate residues, respectively) . For the C-terminal fusion proteins, the tails were fused to a catalytically active but truncated form of GA (aa 1-484) . All of the charged tails had the general sequence Met-Ala-Aspn-Tyr, where n = 0, 5, 7, or 10 . The modified genes were expressed in the yeast Saccharomyces cerevisiae and the proteins secreted into the culture medium . The enzymes were subsequently purified by affinity chromatography . The specific activity of each purified enzyme was found to be comparable to the wild-type enzyme . The C-terminal tails did not interfere with expression, whereas decreased extracellular glucoamylase activities corresponding to increased tail length were found for the N-terminal fusion proteins . Amino-terminal amino acid sequence analysis of the purified GAND proteins confirmed the authenticity of the amino termini of the modified proteins and showed that both the leader peptidase and KEX2 protease cleavages had occurred faithfully . The increased net negative charge of the GAND and GACD proteins was indicated by both nondenaturing PAGE and isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1993 Jun 30, 193(3), 1297 - 303
Effects of unsaturated fatty acid supplementation on phospholipid and triacylglycerol biosynthesis in Saccharomyces cerevisiae; Casey WM et al.; A fatty acid desaturase mutant was used to study the regulatory effects of unsaturated fatty acids on glycerolipid biosynthesis in yeast . Cells grown on palmitoleic acid (16:1) contain approximately twice the amount of phospholipids and triacylglycerols (per mg . dry weight) compared to those grown on oleic acid (18:1) . The in vitro specific activity of glycerol-3-phosphate acyl transferase was two fold higher when palmitoyl-CoA was used as a substrate relative to oleoyl-CoA . In vivo methylation studies revealed that cells grown on palmitoleic acid produce 2.6 fold more phosphatidylcholine via the CDP-DAG (methylation) pathway than cells grown on oleic acid, although oleic acid facilitated the direct phosphorylation of exogenously supplied choline . These data indicate that unsaturated fatty acids may act as key regulatory molecules which influence the glycerolipid biosynthetic matrix in yeast.

J Biol Chem, 1993 Jun 25, 268(18), 13344 - 51
Characterization of human tissue factor pathway inhibitor variants expressed in Saccharomyces cerevisiae; Petersen JG et al.; Human tissue factor pathway inhibitor (TFPI) and three derivatives with deletions of: 1) the complete COOH-terminal third of the polypeptide including the third Kunitz domain, 2) the third Kunitz domain alone, or 3) the penultimate basic COOH-terminal region alone were expressed in yeast as secreted products . High expression yield was obtained only with the derivative that lacked both the third Kunitz domain and the penultimate COOH tail (TFPI1-161) . The purified short form was heterogeneously glycosylated with a high mannose glycan . The specific activities of the different mutant polypeptides toward FXa.tissue factor.FVIIa in a chromogenic assay were similar to that of TFPI expressed in baby hamster kidney cells, suggesting that correct folding takes place in yeast and that neither the third Kunitz domain nor the COOH-terminal region is required for this activity . However, in a clotting assay the anticoagulant activities of yeast-produced TFPI and the shortened derivative TFPI1-161 were about 5- and 50-fold lower, respectively, than for full-length TFPI from mammalian cells . Clotting assays with purified short form TFPI showed that it acted mainly via inhibition of FVIIa.tissue factor rather than FXa . The anticoagulant activity of short form TFPI was comparable with that of high affinity antibodies toward tissue factor.

J Biol Chem, 1993 Jun 15, 268(17), 12895 - 900
A putative homologue of the human autoantigen Ku from Saccharomyces cerevisiae; Feldmann H et al.; We have identified and purified a new DNA binding protein, designated HDF (high affinity DNA-binding factor) from Saccharomyces cerevisiae . HDF binds in a sequence-independent manner to the ends of double-stranded DNA . The protein appears as a stable heterodimer of two polypeptides with molecular masses of 70 and 85 kDa . We have cloned and sequenced the 70-kDa subunit of the HDF protein . The amino acid sequence shows a weak but significant homology with the p70 subunit of the human Ku autoantigen, a protein that also binds to the ends of double-stranded DNA . Hdf- strains generated by one-step gene disruption show a temperature-sensitive phenotype for growth at 37 degrees C . Cells arrest growth at 37 degrees C and after several hours appear as enlarged single-budded cells with abnormally high DNA content indicating a defect in the regulation of DNA replication coupled with or causing a cell cycle arrest in G2 or mitosis.

J Biol Chem, 1993 Jun 15, 268(17), 12749 - 57
The Saccharomyces cerevisiae VMA6 gene encodes the 36-kDa subunit of the vacuolar H(+)-ATPase membrane sector; Bauerle C et al.; The yeast vacuolar membrane proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral catalytic, and integral membrane domains . At least eight proteins cofractionate with purified preparations of the enzyme including 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa polypeptides (Kane, P.M., Yamashiro, C.T., and Stevens, T.H . (1989a) J . Biol . Chem . 264, 19236-19244) . We took a reverse genetic approach to clone the structural gene for the 36-kDa subunit of the V-ATPase, VMA6, vma6 null mutants displayed growth characteristics typical of other vma mutants including sensitivity to media buffered at neutral pH or media containing 100 mM Ca2+ . Vacuolar acidification was defective in vma6 cells and isolated vacuolar membrane preparations contained no detectable V-ATPase activity . The VMA6 gene encodes a hydrophilic polypeptide of 345 amino acids (predicted molecular mass 39.8-kDa) . We present evidence that the VMA6 gene product (Vma6p) is a non-integral membrane component of the membrane pore domain and is required for V-ATPase complex assembly . Vma6p was removed from wild type vacuolar membranes by strong chaotropic agents such as alkaline Na2CO3 or 5M urea, which did not remove integral membrane polypeptides . In yeast cells lacking the integral membrane portion of the V-ATPase complex, Vma6p was unable to stably associate with vacuolar membranes . Conversely, in mutants specifically lacking Vma6p, components of the V-ATPase integral membrane domain were destabilized, and peripheral subunits failed to assemble onto vacuolar membranes . These results are discussed in the context of a developing model for V-ATPase assembly in yeast.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5399 - 403
Model system for DNA replication of a plasmid DNA containing the autonomously replicating sequence from Saccharomyces cerevisiae; Ishimi Y et al.; A negatively supercoiled plasmid DNA containing autonomously replicating sequence (ARS) 1 from Saccharomyces cerevisiae was replicated with the proteins required for simian virus 40 DNA replication . The proteins included simian virus 40 large tumor antigen as a DNA helicase, DNA polymerase alpha.primase, and the multisubunit human single-stranded DNA-binding protein from HeLa cells; DNA gyrase from Escherichia coli, which relaxes positive but not negative supercoils, was included as a "swivelase." DNA replication started from the ARS region, proceeded bidirectionally with the synthesis of leading and lagging strands, and resulted in the synthesis of up to 10% of the input DNA in 1 h . The addition of HeLa DNA topoisomerase I, which relaxes both positive and negative supercoils, to this system inhibited DNA replication, suggesting that negative supercoiling of the template DNA is required for initiation . These results suggest that DNA replication starts from the ARS region where the DNA duplex is unwound by torsional stress; this unwound region can be recognized by a DNA helicase with the assistance of the multisubunit human single-stranded DNA-binding protein.

J Biol Chem, 1993 Jun 15, 268(17), 12925 - 32
Isolation and characterization of a previously undetected human cAMP phosphodiesterase by complementation of cAMP phosphodiesterase-deficient Saccharomyces cerevisiae; Michaeli T et al.; We have established a highly sensitive functional screen for the isolation of cDNAs encoding cAMP phosphodiesterases (PDEs) by complementation of defects in a Saccharomyces cerevisiae strain lacking both endogenous cAMP PDE genes, PDE1 and PDE2 . Three groups of cDNAs corresponding to three distinct human genes encoding cAMP-specific PDEs were isolated from a human glioblastoma cDNA library using this functional screen . Two of these genes are closely related to the Drosophila dunce cAMP-specific PDE . The third gene, which we named HCP1, encoded a novel cAMP-specific PDE . HCP1 has an amino acid sequence related to the sequences of the catalytic domains of all cyclic nucleotide PDEs . HCP1 is a high affinity cAMP-specific PDE (Km = 0.2 microM) that does not share other properties of the cAMP-specific PDE family, i.e . extensive sequence homology to the Drosophila dunce cAMP PDE and sensitivity to rolipram and R020-1724 . The PDE activity of HCP1 is not sensitive to cGMP or other inhibitors of the cGMP-inhibitable PDEs, such as milrinone . The biochemical and pharmacological properties of HCP1 suggest that it is a member of a previously undiscovered cyclic nucleotide PDE family . Northern blot analysis indicates that high levels of HCP1 mRNA are present in human skeletal muscle.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5450 - 4
The FKB2 gene of Saccharomyces cerevisiae, encoding the immunosuppressant-binding protein FKBP-13, is regulated in response to accumulation of unfolded proteins in the endoplasmic reticulum; Partaledis JA et al.; The FKB2 gene of Saccharomyces cerevisiae encodes a homolog of mammalian FKBP-13, an FK506/rapamycin-binding protein that localizes to the lumen of the endoplasmic reticulum (ER) . We have found that FKB2 mRNA levels increase in response to the accumulation of unfolded precursor proteins in the ER . FKB2 mRNA levels are elevated in cells blocked in N-glycosylation--i.e., in wild-type cells treated with tunicamycin and in the sec53-6 mutant grown at the nonpermissive temperature . Mutations that block other steps in secretion have no effect on FKB2 mRNA levels, indicating that increases in FKB2 mRNA are not the consequence of a general block in secretion . The increase in FKB2 mRNA in response to unfolded proteins in the ER is mediated through a 21-bp unfolded-protein response (UPR) element located in the 5' noncoding region of FKB2 . UPR elements present in other ER chaperone genes, such as yeast KAR2 (BiP), mammalian GRP78 (BiP), and GRP94, function in an analogous manner to that in FKB2 . As with KAR2, FKB2 mRNA levels are also elevated by heat shock . The similarities in the regulation of FKB2 and other ER chaperone genes suggest that FKBP-13 may play a role in protein trafficking in the ER.

J Mol Biol, 1993 Jun 5, 231(3), 658 - 67
Structural and functional requirements for the chromatin transition at the PHO5 promoter in Saccharomyces cerevisiae upon PHO5 activation; Fascher KD et al.; The PHO5 promoter from Saccharomyces cerevisiae can exist in two chromatin configurations depending on its state of activity . In the repressed promoter a short hypersensitive site containing a binding site for the transcription factor PHO4 is flanked by specifically positioned nucleosomes . After induction two nucleosomes upstream and two downstream of the hypersensitive site are disrupted, and the entire promoter becomes accessible . We have investigated mechanisms responsible for setting up the structure of the repressed state and for the transition . Episomal centromeric plasmids bearing the PHO5 promoter show the same chromatin structure as the endogenous chromosomal copy arguing that the chromosomal context is not essential and that the nucleosomal organization is not set up from a distance . Deleting most of the hypersensitive region including the PHO4 binding site also leaves the positioning of the adjacent nucleosomes in the repressed promoter unchanged indicating that histone-DNA interactions play an important role in setting up nucleosome positions . However, when half of the DNA of a nucleosome is deleted a new nucleosome forms at the same location with respect to the neighboring nucleosome indicating that boundary effects also contribute to nucleosome positioning in the native promoter . Disruption of the nucleosomes under activating conditions is shown to require interaction of PHO4 with its binding site located within the hypersensitive region . This disruption takes place also in two independent constructs in which the TATA box had been deleted and as a result the gene was not transcribed . This result shows for the first time that the generation of active chromatin at a regulated promoter is not the result of gene expression but occurs prior to transcription.

J Biol Chem, 1993 Jun 5, 268(16), 12104 - 15
Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant . II . Structure of novel Man6-10GlcNAc2 processing intermediates on secreted invertase; Verostek MF et al.; Alg3 yeast mutants synthesize endoglycosidase H-resistant oligosaccharides whose precursor for elongation is Man1 alpha-->2Man1 alpha-->2Man1 alpha-->3(Man1 alpha-->6)Man1 beta-->4GlcNAc2 (Verostek, M.F., Atkinson, P.H., and Trimble, R . B . (1991) J . Biol . Chem . 266, 5547-5551) . To characterize alg3 glycan elongation in vivo, oligosaccharides on alg3,sec18 invertase synthesized and secreted at 26 degrees C were released with peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase and purified by Bio-Gel P-4 chromatography . Large (Man > 30GlcNAc2) and intermediate (Man5-10GlcNAc2) sized oligosaccharides were pooled separately, and the smaller ones were exchanged with 2H2O for one- and two-dimensional DQF-COSY 1H NMR analyses at 500 MHz . Although there was no detectable substitution of the terminal alpha 1,6-core-linked mannose, addition of alpha 1,6-, alpha 1,2-, and alpha 1,3-mannoses to the alpha 1,3-linked core branch of a majority of the Man5 precursor was analogous to core-filling reactions seen on wild type invertase glycans (Trimble, R.B., and Atkinson, P.H . (1986) J . Biol . Chem . 261, 9815-9824) . Two additional types of oligosaccharide structures were found; those which retained glucose and those consistent with mannan elongation . Glucose retention appeared to be due to inefficient trimming from minor glucosylated intermediates, while mannan elongation was by extension of a new alpha 1,6-linked branch from the alpha 1,3-core-linked residue as seen in wild-type core oligosaccharides (Hernandez, L.M., Ballou, L., Alvarado, E., Gillece-Castro, B.L., Burlingame, A.L., and Ballou, C . E . (1989) J . Biol . Chem . 264, 11849-11856) or mnn1,mnn2,mnn10 processing intermediates (Ballou, L., Alvarado, E., Tsai, P-k., Dell, A., and Ballou, C.E . (1989) J . Biol . Chem . 264, 11857-11864) . Thus, the alpha 1,6-linked branch additions which form Man9GlcNAc2-PP-dolichol from Man5GlcNAc2-PP-dolichol appear to provide important structural information enabling efficient recognition by the endoplasmic reticulum-glucosyltransferases forming oligosaccharide-lipid as well as the glucosidases involved in early trimming reactions, but the alg3 mutant documents that they are unnecessary for normal yeast mannan elongation.

J Biol Chem, 1993 Jun 5, 268(16), 12095 - 103
Glycoprotein biosynthesis in the alg3 Saccharomyces cerevisiae mutant . I . Role of glucose in the initial glycosylation of invertase in the endoplasmic reticulum; Verostek MF et al.; Oligosaccharides on invertase restricted to the endoplasmic reticulum (ER) in alg3,sec18 yeast at 37 degrees C were found to be 20% wild type Man8GlcNAc and 80% Man1 alpha-->2Man1 alpha-->2Man1 alpha-->3(Man1 alpha-->6)Man1 beta-->4GlcNAc2 (Verostek, M.F., Atkinson, P.H., and Trimble, R . B . (1991) J . Biol . Chem . 266, 5547-5551) . These results suggested that alg3 was slightly leaky, but did not address whether the oligosaccharide-lipid Man9GlcNAc2 and Man5GlcNAc2 precursors were glucosylated in alg3 yeast . Therefore, an alg3,sec18,gls1 strain was constructed to delete the GLS1-encoded glucosidase I responsible for trimming the terminal alpha 1,2-linked glucose from newly transferred Glc3ManxGlcNAc2 oligosaccharides . Invertase activity was overexpressed 5-10-fold on transforming this strain with a multicopy plasmid (pRB58) carrying the SUC2 gene, and preparative amounts of the ER form of external invertase, derepressed and accumulated at 37 degrees C, were purified . The N-linked glycans were released by sequential treatment with endo-beta-N-acetylglucosaminidase H (endo H) and peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase . Oligosaccharide pools were sized separately on Bio-Gel P-4, which showed that endo H released about 17% of the carbohydrate as Glc3Man8GlcNAc, while peptide-N4-N-acetyl-beta-glucosaminyl asparagine amidase released the remainder as Hex8GlcNAc2 and Man5GlcNAc2 in a 1:4 ratio . Glycan structures were assigned by 500-MHz two-dimensional DQF-COSY 1H NMR spectroscopy, which revealed that the endo H-resistant Hex8GlcNAc2 pool contained Glc3Man5GlcNAc2 and Man8GlcNAc2 in a 6:4 ratio, the latter a different isomer from that formed by the ER alpha 1,2-mannosidase (Byrd, J . C., Tarentino, A . L., Maley, F., Atkinson, P . H., and Trimble, R . B . (1982) J . Biol . Chem . 257, 14657-14666) . Recovery of Glc3Man8GlcNAc and not the ER form of Man8GlcNAc provided an internal control indicating the absence of glucosidase I, which was confirmed by incubation of {3H}Glc3{14C}Man9GlcNAc with solubilized membranes from either alg3,sec18,gls1 or alg3,sec18,GLS1 strains . Chromatographic analysis of the products showed that {3H}Glc was removed only in the presence of the GLS1 gene product . Thus, the vast majority of the N-linked glycosylation in the ER of alg3 yeast (> 75%) occurs by transfer of Man5GlcNAc2 without prior addition of the 3 glucoses normally found on the lipid-linked precursor.

Biochim Biophys Acta, 1993 Jun 5, 1148(2), 303 - 7
The interaction of Saccharomyces cerevisiae trehalase with membranes; de Araujo PS et al.; Plasma membranes isolated from cells of Saccharomyces cerevisiae previously submitted to a heat-shock showed a 10-fold increase in membrane-bound trehalase activity . Trehalase was purified to a high specific activity and was shown to be inhibited by glucose 6-phosphate and by the addition of a neutral phospholipid-like surfactant . Purified trehalase binds spontaneously to egg phosphatidylcholine small unilamellar vesicles, when in its active, phosphorylated form . When the enzyme was treated with alkaline phosphatase no binding was observed . The significance of this reversible binding for the control of trehalose metabolism in yeast cells is still unknown.

J Biol Chem, 1993 Jun 5, 268(16), 11737 - 41
COX3 mRNA-specific translational activator proteins are associated with the inner mitochondrial membrane in Saccharomyces cerevisiae; McMullin TW et al.; Three yeast proteins coded by nuclear genes, PET54, PET122, and PET494, are specifically required to activate translation of the mitochondrially coded COX3 mRNA, and one of them, PET122, has been shown to interact functionally with both the mitochondrial ribosomal small subunit and the COX3 mRNA leader . To determine the location of these proteins within mitochondria, we have used antisera directed against them to assay for their presence in submitochondrial fractions . Approximately half of the PET54 protein present in mitochondria from wild-type cells pelleted with membranes, while half was released from disrupted mitochondria in soluble form . The membrane-bound PET54 floated with the inner membrane during buoyant density gradient centrifugation, but was efficiently solubilized by extraction with alkaline carbonate . The PET122 and PET494 proteins could only be detected in cells overproducing these proteins . PET122 was completely membrane-bound and could not be extracted with alkaline carbonate . Most of the PET494 protein pelleted with membranes and very little could be solubilized by alkaline carbonate . Both PET122 and PET494 floated with membranes during buoyant density gradient centrifugation . These data suggest the possibility that synthesis of the highly hydrophobic subunit III of cytochrome c oxidase is activated by PET54, PET122, and PET494 at the inner mitochondrial membrane . Amino-terminal sequencing of proteins isolated from mitochondria revealed that PET54 is not processed during import to mitochondria while the PET122 precursor is cleaved between the 8th and 9th residues.

J Biol Chem, 1993 Jun 5, 268(16), 11727 - 36
Subunit 9 of the Saccharomyces cerevisiae cytochrome bc1 complex is required for insertion of EPR-detectable iron-sulfur cluster into the Rieske iron-sulfur protein; Phillips JD et al.; Deletion of QCR9, the nuclear gene encoding the 7.3-kDa subunit 9 of the cytochrome bc1 complex, impairs respiration of Saccharomyces cerevisiae, coincident with loss of ubiquinol-cytochrome c oxidoreductase activity . Optical spectra of mitochondrial membranes from yeast in which the gene for subunit 9 is deleted show a diminution of cytochrome b absorption similar to the spectra of membranes from yeast in which the gene for the Rieske iron-sulfur protein is deleted, suggesting an interaction between subunit 9, iron-sulfur protein, and cytochrome b . Synthesis of cytochrome b by mitochondria from the deletion strain is unimpaired, indicating that the diminished b absorption is due to a post-assembly effect on the heme environment resulting from the absence of subunit 9 . Iron-sulfur protein is present in normal amounts and processed to its mature form in the absence of subunit 9, although the protein is more labile to endogenous proteases during the isolation of membranes . EPR spectroscopy of membranes from the subunit 9 deletion strain indicates that the g = 1.90 signal characteristic of the Rieske iron-sulfur cluster is absent, even though mature sized apoprotein is present . Pre-steady state reduction of cytochrome c1 is markedly slowed, but not eliminated, in the subunit 9 deletion strain, which suggests that an EPR-silent, sluggishly reactive derivative of the iron-sulfur cluster is present . These results suggest that in the absence of subunit 9 the conformation of iron-sulfur protein is altered such that the protein is more labile, the iron-sulfur cluster is not properly inserted, and iron-sulfur protein interaction with cytochrome b is modified in a manner which distorts the heme environment . This is the first instance in which deletion of one of the supernumerary subunits of the cytochrome bc1 complex results in the loss of function of a redox center within the complex, without a concomitant loss of other subunits.

Protein Expr Purif, 1993 Jun, 4(3), 247 - 55
Expression, purification and characterization of multigram amounts of a recombinant hybrid HV1-HV2 hirudin variant expressed in Saccharomyces cerevisiae; Lehman ED et al.; Hirudin (HIR), derived from leeches, and tick anticoagulant peptide (TAP) are polypeptide protease inhibitors of thrombin and coagulation factor Xa (fXa), respectively, and they have both shown utility in vitro and in vivo as potent antithrombotic agents . A thorough side-by-side comparison of the in vivo efficacy of factor Xa inhibition compared to thrombin inhibition by TAP and HIR, respectively, required purification and characterization of multigram amounts of hirudin . Therefore, a recombinant Saccharomyces cerevisiae strain was developed using a plasmid containing the gene encoding the MF alpha 1 preproleader, a synthetic hybrid HV1-HV2 HIR gene, and a galactose-inducible promoter which directed the secretion of 44 mg/liter of recombinant HIR (rHIR) after induction . rHIR was purified by a process that consisted of two chromatographic steps and decolorization . Total yield for the purification process was 3.6 g, or 41% . This process gave 59-fold purification of rHIR that was judged to be > 96% pure with regard to polypeptide content by capillary zonal electrophoresis and reversed-phase high-performance liquid chromatography . Single, unique N- and C-termini were obtained by sequencing and were identical to those predicted from the deduced sequence of the cDNA . Determination of the dissociation constant, by thrombin:hirudin inhibition reaction, and anticoagulant activity, by the activated partial thromboplastin time, demonstrated that the hybrid rHIR HV1-HV2 protein discussed in this report was essentially equipotent with rHIR preparations HV1 and HV2 reported by others.

Eur J Biochem, 1993 Jun 1, 214(2), 577 - 85
Mitochondrial transport of mitoribosomal proteins, YmL8 and YmL20, in Saccharomyces cerevisiae; Matsushita Y et al.; Two mitochondrial ribosomal (mitoribosomal) proteins, YmL8 and YmL20, of the yeast Saccharomyces cerevisiae and their derivatives were synthesized in vitro and their transport into isolated yeast mitochondria was examined . Of the two proteins, YmL20 possesses an N-terminal presequence of 18 amino acid residues, while YmL8 has no such presequence . Both proteins were found to be transported into isolated mitochondria in an energy-dependent manner . Furthermore, YmL20 protein without its N-terminal presequence was also transported, despite the fact that the presequence alone was capable of transporting a fused passenger protein, Chinese hamster dihydrofolate reductase (DHFR) . Therefore, YmL20 protein appears to possess redundant transport signals in its structure . Similarly, YmL8 derivatives lacking either 40 or 86 amino acid residues from the N-terminus and/or 52 amino acid residues from the C-terminus were transported . In addition, the N-terminal segment of this protein was capable of transporting Chinese hamster DHFR into mitochondria, while its C-terminal segment was not . Thus, YmL8 protein also appears to possess two or more transport signals in its structure . Perhaps the presence of many basic amino acid residues in these proteins might, at least partly, contribute to their mitochondrial transport.

J Bacteriol, 1993 Jun, 175(12), 3823 - 37
SSG1, a gene encoding a sporulation-specific 1,3-beta-glucanase in Saccharomyces cerevisiae; San Segundo P et al.; In Saccharomyces cerevisiae, the meiotic process is accompanied by a large increase in 1,3-beta-glucan-degradative activity . The molecular cloning of the gene (SSG1) encoding a sporulation-specific exo-1,3-beta-glucanase was achieved by screening a genomic library with a DNA probe obtained by polymerase chain reaction amplification using synthetic oligonucleotides designed according to the nucleotide sequence predicted from the amino-terminal region of the purified protein . DNA sequencing indicates that the SSG1 gene specifies a 445-amino-acid polypeptide (calculated molecular mass, 51.8 kDa) showing extensive similarity to the extracellular exo-1,3-beta-glucanases encoded by the EXG1 gene (C . R . Vazquez de Aldana, J . Correa, P . San Segundo, A . Bueno, A . R . Nebreda, E . Mendez, and F . del Rey, Gene 97:173-182, 1991) . The N-terminal domain of the putative precursor is a very hydrophobic segment with structural features resembling those of signal peptides of secreted proteins . Northern (RNA) analysis reveals a unique SSG1-specific transcript, 1.7 kb long, which can be detected only in sporulating diploids (MATa/MAT alpha) but does not appear in vegetatively growing cells or in nonsporulating diploids (MAT alpha/MAT alpha) when incubated under nitrogen starvation conditions . The meiotic time course of SSG1 induction indicates that the gene is transcribed only in the late stages of the process, beginning at the time of meiosis I and reaching a maximum during spore formation . Homozygous ssg1/ssg1 mutant diploids are able to complete sporulation, although with a significant delay in the appearance of mature asci.

EMBO J, 1993 Jun, 12(6), 2431 - 7
GCR1, a transcriptional activator in Saccharomyces cerevisiae, complexes with RAP1 and can function without its DNA binding domain; Tornow J et al.; In Saccharomyces cerevisiae, efficient expression of glycolytic and translational component genes requires two DNA binding proteins, RAP1 (which binds to UASRPG) and GCR1 (which binds to the CT box) . We generated deletions in GCR1 to test the validity of several different models for GCR1 function . We report here that the C-terminal half of GCR1, which includes the domain required for DNA binding to the CT box in vitro, can be removed without affecting GCR1-dependent transcription of either the glycolytic gene ADH1 or the translational component genes TEF1 and TEF2 . We have also identified an activation domain within a segment of the GCR1 protein (the N-terminal third) that is essential for in vivo function . RAP1 and GCR1 can be co-immunoprecipitated from whole cell extracts, suggesting that they form a complex in vivo . The data are most consistent with a model in which GCR1 is attracted to DNA through contact with RAP1.

Biochem J, 1993 Jun 1, 292 ( Pt 2), 333 - 41
The molecular cloning of the squid (Loligo forbesi) visual Gq-alpha subunit and its expression in Saccharomyces cerevisiae; Ryba NJ et al.; The sequence of the alpha-subunit of the major G-protein from the squid (Loligo forbesi) retina was predicted from its cDNA to be a member of the Gq subclass . The abundance of the squid Gq-alpha in the squid photoreceptor membranes suggests that the protein functions in phototransduction; the sequence of this G-protein is consistent with it mediating the light-dependent activation of a phospholipase C . The squid G-alpha was expressed in the yeast Saccharomyces cerevisiae, where it was unable to replace the function of GPA1, the yeast G-alpha homologue that regulates the mating response, suggesting that Gq-alpha was unable to interact with the endogenous G-beta gamma (STE4-STE18).

Mol Cell Biol, 1993 Jun, 13(6), 3541 - 56
GCN1, a translational activator of GCN4 in Saccharomyces cerevisiae, is required for phosphorylation of eukaryotic translation initiation factor 2 by protein kinase GCN2; Marton MJ et al.; Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) by the protein kinase GCN2 mediates increased translation of the transcriptional activator GCN4 in amino acid-starved yeast cells . We show that this key phosphorylation event and the attendant translational induction of GCN4 are dependent on the product of a previously uncharacterized gene, GCN1 . Inactivation of GCN1 did not affect the level of eIF-2 alpha phosphorylation when mammalian eIF-2 alpha kinases were expressed in yeast cells in place of GCN2, arguing against an involvement of GCN1 in dephosphorylation of eIF-2 alpha . In addition, while GCN1 is required in vivo for phosphorylation of eIF-2 alpha by GCN2, cell extracts from gcn1 delta strains contained wild-type levels of GCN2 eIF-2 alpha-kinase activity . On the basis of these results, we propose that GCN1 is not needed for GCN2 kinase activity per se but is required for in vivo activation of GCN2 in response to the starvation signal, uncharged tRNA . GCN1 encodes a protein of 297 kDa with an 88-kDa region that is highly similar in sequence to translation elongation factor 3 identified in several fungal species . This sequence similarity raises the possibility that GCN1 interacts with ribosomes or tRNA molecules and functions in conjunction with GCN2 in monitoring uncharged tRNA levels during the process of translation elongation.

J Bioenerg Biomembr, 1993 Jun, 25(3), 245 - 57
Mutational analysis of assembly and function of the iron-sulfur protein of the cytochrome bc1 complex in Saccharomyces cerevisiae; Graham LA et al.; The iron-sulfur protein of the cytochrome bc1 complex oxidizes ubiquinol at center P in the protonmotive Q cycle mechanism, transferring one electron to cytochrome c1 and generating a low-potential ubisemiquinone anion which reduces the low-potential cytochrome b-566 heme group . In order to catalyze this divergent transfer of two reducing equivalents from ubiquinol, the iron-sulfur protein must be structurally integrated into the cytochrome bc1 complex in a manner which facilitates electron transfer from the iron-sulfur cluster to cytochrome c1 and generates a strongly reducing ubisemiquinone anion radical which is proximal to the b-566 heme group . This radical must also be sequestered from spurious reactivities with oxygen and other high-potential oxidants . Experimental approaches are described which are aimed at understanding how the iron-sulfur protein is inserted into center P, and how the iron-sulfur cluster is inserted into the apoprotein.

J Bioenerg Biomembr, 1993 Jun, 25(3), 211 - 20
Random mutant generation and its utility in uncovering structural and functional features of cytochrome b in Saccharomyces cerevisiae; Colson AM; The generation of random mutations in the mitochondrial cytochrome b gene of Saccharomyces cerevisiae has been used as a most fruitful means of identifying subregions that play a key role in the bc1 complex mechanism, best explained by the protonmotive Q cycle originally proposed by Peter Mitchell . Selection for center i and center o inhibitor resistance mutants, in particular, has yielded much information . The combined approaches of genetics and structural predictions have led to a two-dimensional folding model for cytochrome b that is most compatible with current knowledge of the protonmotive Q cycle . A three-dimensional model is emerging from studies of distant reversions of deficient mutants . Finally, interactions between cytochrome b and the other subunits of the bc1 complex, such as the iron-sulfur protein, can be affected by a single amino acid change.

Mol Cell Biol, 1993 Jun, 13(6), 3125 - 34
A 5'-3' exonuclease from Saccharomyces cerevisiae is required for in vitro recombination between linear DNA molecules with overlapping homology; Huang KN et al.; When two linear DNA molecules with overlapping, homologous ends were incubated with a yeast nuclear extract, they recombined at the region of homology to produce a joint molecule . We have identified a 5'-3' exonuclease in the extract that is likely to be responsible for the formation of the observed product . We propose that the exonuclease degrades each substrate to reveal regions of complementary sequence which anneal to form a recombinant product . Consistent with this model, we have partially purified the activity that promotes joint molecule formation and found it to cofractionate with a 5'-3' exonuclease activity through three consecutive chromatography steps . We have further characterized the reaction to determine the optimal length of homology . Substrates with homologous terminal overlaps of 29 to 958 bp were capable of product formation, whereas substrates with longer overlaps were not . Extracts prepared from a number of recombination-defective or nuclease-deficient strains revealed no defect in exonuclease activity, indicating that the reaction is likely to be dependent upon the product of an as yet unidentified gene.

Yeast, 1993 Jun, 9(6), 645 - 59
RIM2, MSI1 and PGI1 are located within an 8 kb segment of Saccharomyces cerevisiae chromosome II, which also contains the putative ribosomal gene L21 and a new putative essential gene with a leucine zipper motif; Demolis N et al.; We report the DNA sequence of an 8 kb segment localized on the right arm of chromosome II from Saccharomyces cerevisiae . The sequence reveals the presence of eight open reading frames (ORFs) . Three of them, YBR1402, YBR1405 and YBR1406 are previously sequenced genes, respectively the RIM2 (replication in mitochondria), MSI1 (multicopy suppressor of IRA1 gene) and PGI1 (phosphoglucoisomerase) genes . The predicted product of the ORF YBR1401 could be the putative yeast ribosomal protein L21 . A new essential gene, YBR1403, has been identified by disruption; it possesses a leucine zipper motif.

Yeast, 1993 Jun, 9(6), 607 - 11
Assay of trehalose with acid trehalase purified from Saccharomyces cerevisiae; Kienle I et al.; An enzymatic end-point assay of trehalose using acid trehalase from yeast is described . After quantitative hydrolysis of trehalose by acid trehalase, the resulting glucose is assayed with the commercially available glucose oxidase/peroxidase dye system . Pre-existing glucose is determined in a control reaction from which acid trehalase is omitted . When intact cells are analysed for trehalose, pre-existing glucose can be washed out with ice-cold water without reducing the trehalose content of the cells . A convenient method for extraction of trehalose from intact yeast cells is heating for 20 min at 95 degrees C followed by centrifugation . The specificity of the assay is determined by the specificity of the acid trehalase preparation used . As described previously (Mittenbuhler, K . and Holzer, H., 1988, J . Biol . Chem . 263, 8537-8543; Mittenbuhler, K., 1988, Thesis, University of Freiburg), the following sugars and sugar derivatives do not form glucose when incubated with purified acid trehalase: sucrose, cellobiose, mellobiose, raffinose, maltose, lactose, glucose-6-phosphate, glucose-1-phosphate, galactose . The application of the new trehalose assay to yeast cells grown to different growth stages and at various temperatures is presented.

Yeast, 1993 Jun, 9(6), 575 - 82
Alteration of cell population structure due to cell lysis in Saccharomyces cerevisiae cells overexpressing the GAL4 gene; Martegani E et al.; Transformed Saccharomyces cerevisiae cells overexpressing the Escherichia coli LacZ gene and the transcriptional activator GAL4, release in the external medium a fraction (from 2 to 10%) of the total beta-galactosidase activity (Porro et al., 1992b) . It is known that this abnormal release of a cytoplasmic protein is related to a partial cell lysis of the yeast population, which is likely to be caused by the overexpression of the transcriptional activator GAL4 . In the present paper we have characterized the GAL4-induced cell lysis phenomenon . The expression of the GAL4 gene causes morphological modifications and alteration of the cell size distribution . The cell lysis is independent of the expression of the heterologous LacZ gene and occurs in a specific subpopulation of cells (the parent cells) independently of the genealogical age, growth phase conditions and cell cycle progression . Lysis is preceded by a loss of the plasma membrane integrity as indicated by the uptake of ethidium bromide in unfixed cells . Computer analysis of simulated protein distributions indicates that cell lysis takes place in a sizeable aliquot (about 50%) of the parent cells, therefore profoundly altering the age structure of the population.

Comp Biochem Physiol C, 1993 Jun, 105(2), 175 - 8
Interaction among heavy metals and methanol affecting superoxide dismutase activity in Saccharomyces cerevisiae; Manzano M et al.; 1 . Three strains of Saccharomyces cerevisiae have been exposed to methanol both in the presence and absence of heavy metal ions . The growth curves and the superoxide dismutase activity were determined . 2 . The presence of alcohol, copper or cadmium alone did not give strong cytotoxic effects, while methanol plus cadmium yielded a growth inhibition in two strains . 3 . SOD levels were stimulated by copper, while methanol did not affect SOD in this non-methylotrophic yeast, indicating the need of alcohol assimilation to stimulate SOD . Cadmium had no inducing effects on SOD levels.

Oncogene, 1993 Jun, 8(6), 1465 - 75
Functional domains of the v-erbA protein necessary for oncogenesis are required for transcriptional activation in Saccharomyces cerevisiae; Smit-McBride Z et al.; The v-erbA oncogene is a retrovirus-transduced and altered copy of a cellular gene (c-erbA-alpha) for a thyroid hormone receptor . In this paper we show that the v-erbA domains required for transcriptional activation in yeast and for oncogenic function in animal cells are closely congruent . We conclude that the behavior of the v-erbA protein as a transcriptional activator in yeast appears to closely reflect the same biochemical requirements that are necessary for v-erbA function in the neoplastic vertebrate cell . Intriguingly, parallel analyses of c-erbA-alpha and -beta demonstrated unexpected differences in the activities of the two thyroid hormone receptor isoforms in the yeast, perhaps reflecting different functions of these genes in vertebrates . Furthermore, results obtained by analysis of chimeric v-/c-erbA genes suggest that the basal and the hormone-induced transcriptional activity of the nuclear hormone receptors can be modulated independently by distinct structural features within the protein molecule.

J Cell Sci, 1993 Jun, 105 ( Pt 2), 519 - 28
The lethality of p60v-src in Saccharomyces cerevisiae and the activation of p34CDC28 kinase are dependent on the integrity of the SH2 domain; Boschelli F et al.; The lethal effects of the expression of the oncogenic protein tyrosine kinase p60v-src in Saccharomyces cerevisiae are associated with a loss of cell cycle control at the G1/S and G2/M checkpoints . Results described here indicate that the ability of v-Src to kill yeast is dependent on the integrity of the SH2 domain, a region of the Src protein involved in recognition of proteins phosphorylated on tyrosine . Catalytically active v-Src proteins with deletions in the SH2 domain have little effect on yeast growth, unlike wild-type v-Src protein, which causes accumulation of large-budded cells, perturbation of spindle microtubules and increased DNA content when expressed . The proteins phosphorylated on tyrosine in cells expressing v-Src differ from those in cells expressing a Src protein with a deletion in the SH2 domain . Also, unlike the wild-type v-Src protein, which drastically increases histone H1-associated Cdc28 kinase activity, c-Src and an altered v-Src protein have no effect on Cdc28 kinase activity . These results indicate that the SH2 domain is functionally important in the disruption of the yeast cell cycle by v-Src.

Antimicrob Agents Chemother, 1993 Jun, 37(6), 1264 - 9
Bleomycin affects cell wall anchorage of mannoproteins in Saccharomyces cerevisiae; Beaudouin R et al.; Bleomycin induces strand breakage in DNA through disruption of glycosidic linkages . We investigated the ability of bleomycin to damage yeast cell walls, which are composed primarily of carbohydrate . Bleomycin treatment of intact yeast cells facilitated enzymatic conversion of yeasts to spheroplasts . Bleomycin treatment also altered anchorage of mannoproteins to the cell wall matrix in intact cells or isolated cell walls . Cell surface mannoproteins were labelled with 125I, and their solubilization was monitored . Seventeen hour treatments with bleomycin released some of the label directly into treatment supernatants and facilitated extraction of mannoproteins by dithiothreitol and lytic enzymes . Bleomycin treatments as short as 10 min caused changes in extraction of mannoproteins from intact cells . Specifically, cell wall anchorage of several mannoproteins was affected by the drug . There were drug-induced changes in extractability of mannoproteins with apparent molecular weights of 96,000, 80,000, 61,000, 41,000, 31,500, and 21,000 (determined after deglycosylation with endo-N-acetylglucosaminidase H) . The similarity of results obtained in the presence and absence of cycloheximide, the appearance of cell wall effects after only 10 min of treatment, and the similarity of effects in intact cells and isolated cell walls are consistent with direct drug-induced damage and inconsistent with a mechanism dependent on expression of bleomycin-damaged genes or other intracellular mediators . The results are consistent with bleomycin-mediated increases in cell wall permeability through disruption of glycosidic cross-linking structures in the cell wall.

Nucleic Acids Res, 1993 May 25, 21(10), 2331 - 8
Chromatin structures and transcription of rDNA in yeast Saccharomyces cerevisiae; Dammann R et al.; The chromatin structure of yeast ribosomal DNA was analyzed in vivo by crosslinking intact cells with psoralen . We found that in exponentially growing cultures the regions coding for the 35S rRNA precursor fall into two distinct classes . One class was highly accessible to psoralen and associated with nascent RNAs, characteristic for transcriptionally active rRNA genes devoid of nucleosomes, whereas the other class showed a crosslinking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rRNA gene copies . By crosslinking the same strain growing in complex or minimal medium, we have shown that yeast cells can modulate the proportion of active (non-nucleosomal) and inactive (nucleosomal) rRNA gene copies in response to variations in environmental conditions which suggests that yeast can regulate rRNA synthesis by varying the number of active gene copies, in contrast to the vertebrate cells studied so far . Whereas intergenic spacers flanking inactive rRNA gene copies are packaged in a regular nucleosomal array, spacers flanking active genes show an unusual crosslinking pattern suggesting a complex interaction of regulatory factors and histones with DNA.

J Biol Chem, 1993 May 25, 268(15), 11113 - 8
Saccharomyces cerevisiae CDC25 (1028-1589) is a guanine nucleotide releasing factor for mammalian ras proteins and is oncogenic in NIH3T3 cells; Chevallier-Multon MC et al.; The best characterized yeast guanine nucleotide releasing factor is CDC25, which acts on RAS and thereby stimulates cAMP production in Saccharomyces cerevisiae . In order to determine if CDC25 could be a specific GDP-GTP releasing factor for the mammalian proteins Ha-ras, Ki-ras, and N-ras, its functions were studied both in vitro and in NIH3T3 cells . The 561 amino acid composing the C-terminal domain of CDC25 (CDC25 C-domain) released guanine nucleotides (both GDP and GTP) from Ha-, Ki-, and N-ras but not from Rap1A, Rab5, and Rab11 . CDC25 acted on oncogenically activated Ha-ras even if the last 23 amino acids (167-189) of the Ras proteins were not present . CDC25 transformed NIH3T3 cells; its transforming capacity was enhanced by overexpression of wild-type Ha-ras . CDC25 C-domain probably exerts its effects through the activation of cellular Ras proteins . These data suggest that the CDC25 C-domain can function as an upstream activator of Ras proteins in a heterologous system and therefore could be a useful tool to study the regulation of Ras activation by growth factor receptors.

FEBS Lett, 1993 May 24, 323(1-2), 113 - 8
Inositol 1,4,5-trisphosphate releases Ca2+ from vacuolar membrane vesicles of Saccharomyces cerevisiae; Belde PJ et al.; Inositol 1,4,5-trisphosphate (IP3) induces a release of Ca2+ from vacuolar membrane vesicles of Saccharomyces cerevisiae . The amount released is dependent on IP3 concentration (concentration for half maximal effect, Km, apparent = 0.4 microM) . Myo-inositol, and inositol 1,4-bisphosphate up to 50 microM have no effect on Ca2+ levels in the vesicles . The IP3-induced Ca2+ release is blocked by dantrolene and 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate-HCl (TMB-8), which are known to block Ca2+ release from Ca2+ stores in animal cells . IP3-induced release of Ca2+ also occurs when Ca2+ is accumulated by means of an artificial pH gradient, indicating that the effect of IP3 is not due to an effect on the vacuolar H(+)-ATPase . The IP3-induced Ca2+ release is not accompanied by a change in the pH gradient, which indicates that it is not due to a reversal of the Ca2+/nH+ antiport or to a decrease in delta pH by IP3 . The present results suggest that IP3 may act as a second messenger in the mobilization of Ca2+ in yeast cells . As in plant cells, the vacuolar membrane of yeast seems to contain a Ca2+ channel, which can be opened by IP3 . In this respect the vacuole could function as an IP3-regulated intracellular Ca2+ store, equivalent to the endoplasmic- and sarcoplasmic reticulum in animal cells, and play a role in Ca(2+)-dependent signal transduction in yeast cells.

FEBS Lett, 1993 May 24, 323(1-2), 77 - 82
Saccharomyces cerevisiae multifunctional protein RAP1 binds to a conserved sequence in the Polyoma virus enhancer and is responsible for its transcriptional activity in yeast cells; Pollice A et al.; The Polyoma virus enhancer (A + B domain) activates transcription in Saccharomyces cerevisiae when joined to appropriate yeast promoter elements . We demonstrate by DNase I footprints and inhibition of binding by specific antibody, that the yeast protein RAP1 binds to the B-domain of the Polyoma enhancer and, at least in some promoter contexts, is responsible for transcriptional activity of the enhancer B-domain in yeast . Close matches to a consensus RAP1-binding site are also present in other viral enhancers.

Eur J Biochem, 1993 May 15, 214(1), 163 - 72
Interactions between glucose metabolism and oxidative phosphorylations on respiratory-competent Saccharomyces cerevisiae cells; Beauvoit B et al.; The purpose of this work was to analyze the interactions between oxidative phosphorylations and glucose metabolism on yeast cells aerobically grown on lactate as carbon source and incubated in a resting cell medium . On such respiratory-competent yeast cells, four different metabolic steady states have particularly been studied: (a) glucose feeding under anaerobiosis, (b) ethanol supply under aerobiosis, (c) glucose supply under aerobiosis and (d) glucose plus ethanol under aerobiosis . For each condition, we measured: (a) the cellular ATP/ADP ratio and NADH content sustained under these conditions, (b) the glucose consumption rate (glucose conditions) and the respiratory rate (aerobic conditions) . Under aerobic conditions, when ethanol is used as substrate, the ATP/ADP ratio and NADH level are very high as compared with glucose feeding . However, the rate of oxygen consumption is similar under both conditions . The main observation is a large increase in the respiratory rate when both glucose and ethanol are added . This increase corresponds to an ATP/ADP ratio and a NADH level lower than those observed with ethanol but higher than those with glucose . Therefore the response of the respiratory rate to the ATP/ADP ratio depends on the redox potential . We studied the way in which the ATP-consuming activity was increased under glucose+ethanol conditions . By NMR experiments, it appears that neither the futile cycle at the level of the phosphofructo-1-kinase/fructo-1,6-bisphosphatase couple nor the synthesis of carbohydrate stores could account for the increase in oxidative phosphorylation . However, it is shown that, in the presence of glucose+ethanol, ATP consumption is strongly stimulated . It is hypothesized that this consumption is essentially due to the combination of the well-known plasma membrane proton-ATPase activation by glucose and the high phosphate potential due to oxidative ethanol metabolism . While it is well documented that oxidative phosphorylations inhibit the glycolytic flux, i.e . the Pasteur effect, we clearly show in this work that the glycolytic pathway limits the ability of mitochondria to maintain a cellular phosphate potential.

Eur J Biochem, 1993 May 15, 214(1), 121 - 7
The glucose-6-phosphate-isomerase reaction is essential for normal glucose repression in Saccharomyces cerevisiae; Sierkstra LN et al.; Wild-type Saccharomyces cerevisiae and a strain carrying a deletion in the glucose-6-phosphate-isomerase gene (pgi1) were grown in carbon-limited continuous cultures on a mixture of fructose and galactose . Pulses of glucose, fructose and galactose were given to these cultures to investigate whether the pgi1 strain was capable of normal glucose repression . Glucose and galactose pulses inhibited fructose consumption and thus glycolysis in the pgi1 strain by a combination of competition between glucose and fructose at the uptake and/or phosphorylation level and inhibition of fructose uptake and/or phosphorylation by glucose 6-phosphate . Fructose pulses administered to the pgi1 strain transiently decreased the glycolytic flux downstream of fructose-1,6-bisphosphate . Transcriptional induction of the PDC1 gene (encoding pyruvate decarboxylase) was observed after glucose or galactose pulses were applied to the pgi1 strain, demonstrating that metabolism of these sugars beyond glucose 6-phosphate is dispensable for PDC1 induction . Fructose also induced PDC1 transcription, indicating that intracellular sugars could act as trigger for PDC1 induction or, alternatively, that two inductors are present . In contrast to the wild-type transcriptional inhibition of the glucose-repressible genes, HXK1 and GAL10 (encoding hexokinase isoenzyme 1 and uridine diphosphoglucose-4-epimerase, respectively) did not occur upon addition of glucose or fructose to the pgi1 mutant . Transcriptional repression was observed after application of the fructose pulse when the yeast had resumed metabolism of fructose . These results demonstrate that the initial signal for catabolite repression is not generated by high sugar concentrations or high concentrations of intermediates; moreover a simple role for the hexokinases can also be excluded . The absence of an increased glycolytic flux in the pgi1 mutant after administration of the sugar pulses while the concentrations of sugar and glycolytic intermediates were high, suggests that the initial signal for glucose repression could be linked to an increased glycolytic flux . The occurrence of PDC1 induction in the pgi1 strain while GAL10/HXKI repression is absent, demonstrates that the initial signals for catabolite induction and catabolite repression are different.

Proc Natl Acad Sci U S A, 1993 May 15, 90(10), 4693 - 7
Oxygen toxicity in a polyamine-depleted spe2 delta mutant of Saccharomyces cerevisiae; Balasundaram D et al.; When a mutant of Saccharomyces cerevisiae (spe2 delta) that cannot make spermidine or spermine was incubated in a polyamine-deficient medium in oxygen, there was a rapid cessation of cell growth and associated cell death . In contrast, when the mutant cells were incubated in the polyamine-deficient medium in air or anaerobically, the culture stopped growing more gradually, and there was no significant loss of cell viability . We also found that the polyamine-deficient cells grown in air, but not those grown anaerobically, showed a permanent loss of functional mitochondria ("respiratory competency"), as evidenced by their inability to grow on glycerol as the sole carbon source . These data support the postulation that polyamines act, in part, by protecting cell components from damage resulting from oxidation . However, since the mutant cells still required spermidine or spermine for growth when incubated under strictly anaerobic conditions, polyamines must also have other essential functions.

J Biol Chem, 1993 May 15, 268(14), 9964 - 71
A comparative analysis of the kinetic mechanism and peptide substrate specificity of human and Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase; Rocque WJ et al.; Human myristoyl-CoA:protein N-myristoyltransferase (hNmt) catalyzes the transfer of myristate from CoA to the amino-terminal Gly residue of a number of cellular proteins involved in signal transduction pathways, to structural and nonstructural proteins encoded by retroviruses, hepadnaviruses, picornaviruses, and reoviruses, as well as to several transforming tyrosine kinases . hNmt has been purified 230-fold from an erythroleukemia cell line . The monomeric enzyme has no associated methionyl aminopeptidase activity . To determine the enzyme's kinetic mechanism, we examined the effect of covariation of subsaturating concentrations of myristoyl-CoA and peptide substrate on initial velocity . Double-reciprocal plots excluded a double displacement (ping-pong) mechanism . Product inhibition studies indicated that CoA was a noncompetitive inhibitor against myristoyl-CoA and a mixed-type inhibitor against peptide substrates . Together these results are consistent with a sequential ordered mechanism where, in a typical catalytic cycle, myristoyl-CoA binds to apoenzyme before peptide followed by release of the CoA and then myristoylpeptide products . This kinetic mechanism is identical to that described for Saccharomyces cerevisiae N-myristoyl-transferase (Nmt1p) and emphasizes the impact that regulation of myristoyl-CoA pool size and accessibility may have in modulating protein N-myristoylation in these two species . Comparative studies of the peptide substrate specificities of hNmt and Nmt1p using a panel of 12 octapeptides revealed distinct differences in their tolerance for amino acid substitutions at positions 3, 4, 7, and 8 of parental peptides derived from the amino-terminal sequences of known N-myristoyl-proteins . This finding contrasts with our recent observation that the acyl-CoA substrate specificities of hNmt and Nmt1p are highly conserved and suggests that these differences in peptide recognition provide an opportunity to develop species-specific enzyme inhibitors.

J Biol Chem, 1993 May 15, 268(14), 10558 - 63
Analysis of the sequence requirements for glycosylphosphatidylinositol anchoring of Saccharomyces cerevisiae Gas1 protein; Nuoffer C et al.; The Saccharomyces cerevisiae Gas1 protein is synthesized as a precursor with a hydrophobic extension at the carboxyl terminus which is removed and replaced with an inositol containing glycolipid that anchors the protein to the plasma membrane . We performed saturation mutagenesis on the anchor attachment site (Asn506) and showed that only a subset of amino acids with small side chains could act as substrates for peptide cleavage and glycolipid addition . After Asn, which is the most efficient anchor attachment site, Ser, Gly, Ala, Asp, and Cys function with decreasing effectiveness . Mutational analysis also revealed that the 2 adjacent amino acids to the carboxyl side of the anchor attachment site are important for efficient anchoring . These two amino acids should have relatively short side chains with the second position being more critical . Analysis of the region between the anchor attachment site and the carboxyl-terminal hydrophobic region indicated that this region may not simply perform a spacer function.

J Biol Chem, 1993 May 15, 268(14), 10524 - 33
Eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae . Cloning, characterization, and expression of the gene encoding the 45,346-Da protein; Chakravarti D et al.; Eukaryotic translation initiation factor 5 (eIF-5) catalyzes hydrolysis of GTP bound to a 40 S ribosomal initiation complex with the subsequent joining of a 60 S ribosomal subunit to form an 80 S initiation complex . The yeast gene that encodes eIF-5, designated TIF5, has been isolated and expressed in Escherichia coli to yield a catalytically active eIF-5 protein . TIF5 is a single-copy gene that maps on yeast chromosome XVI and is essential for cell viability . The gene contains an intron-free open reading frame that encodes a protein of calculated M(r) 45,346 in close agreement with the apparent molecular weight of eIF-5 isolated from yeast cells . Sequence analysis of the gene reveals several interesting features . First, the presence of two in-frame translational start sites located 51 base pairs apart suggests the possibility that two proteins, differing by an amino-terminal extension of 17 amino acids, could be generated from the TIF5 gene via differential translational starts . This would explain the presence, in yeast cell lysates, of two forms of eIF-5 differing in molecular weight by about 2,000 . Second, the predicted amino acid sequence of eIF-5 contains sequence motifs characteristic of proteins of the GTPase superfamily.

Gene, 1993 May 15, 127(1), 139 - 44
A random mutagenesis procedure: application to the POL3 gene of Saccharomyces cerevisiae; Simon M et al.; To obtain a broad spectrum of mutations in the POL3 gene, we have developed an efficient random mutagenesis procedure . Partially extended, primed, single-stranded templates were used for forced misincorporation of non-complementary nucleotides, extended to completion and ligated . Linear fragments of the resulting amplified mutagenized library were then used to transform a Saccharomyces cerevisiae strain by the marker replacement technique . This procedure has proven to be very efficient when applied to the C-terminal moiety of POL3, yielding 24 temperature-sensitive mutants and six extragenic revertants.

J Biol Chem, 1993 May 15, 268(14), 10386 - 92
The DNA helicase activities of Rad3 protein of Saccharomyces cerevisiae and helicase II of Escherichia coli are differentially inhibited by covalent and noncovalent DNA modifications; Naegeli H et al.; Rad3 protein of Saccharomyces cerevisiae is a DNA-dependent ATPase that acts as a DNA helicase on partially duplex substrates . Rad3 protein is required for damage-specific incision of DNA during the nucleotide excision repair (NER) pathway in yeast . Helicase II of Escherichia coli is also a DNA helicase, but it is involved in postincision events in NER . Previous investigations have demonstrated that the DNA helicase activities of Rad3 protein and helicase II are both inhibited by DNA damage . In the present study we have compared the response of yeast Rad3 protein and E . coli helicase II to a broad spectrum of DNA modifications . The Rad3 helicase activity is considerably more sensitive to ultraviolet radiation damage and cisplatin adducts in DNA than to drugs that interact noncovalently with duplex DNA . Conversely, E . coli helicase II is highly sensitive to noncovalent DNA modifications but less sensitive than Rad3 protein to ultraviolet radiation damage or cisplatin adducts . We also show that Rad3 protein and helicase II differ in their ability to form stable protein-DNA complexes at sites of DNA damage . Hence, DNA helicases that catalyze distinct steps in NER respond differently to chemical and conformational states of the DNA substrate . The observation that Rad3 protein is particularly sensitive to covalent but not noncovalent alterations in DNA structure is consistent with the hypothesis that this enzyme may have adopted a highly specialized role in damage-specific recognition during NER.

Biochim Biophys Acta, 1993 May 8, 1177(1), 25 - 30
Identification and characterization of nuclear calmodulin-binding proteins of Saccharomyces cerevisiae; Hiraga K et al.; Nuclear calmodulin-binding proteins of the yeast Saccharomyces cerevisiae were investigated . The soluble fractions after serial treatments of the isolated nuclei with buffers containing the nonionic detergent NP-40 (F1), 0.5 M KCl (F2) and 2.0 M KCl (F3) in this order, and the residual proteins (F4) were obtained . The calmodulin-binding proteins of the nucleus and nuclear subfractions were identified using the gel overlay method using 125I-calmodulin . Each subnuclear fraction contained a large number of components that bound calmodulin in a Ca(2+)-dependent or -independent manners . The calmodulin-binding proteins were isolated from F1 and F2 subnuclear fractions by affinity chromatography . The affinity-purified proteins bound calmodulin in a Ca(2+)-dependent manner when analyzed using the gel overlay method . The major calmodulin-binding components of F1 were 44, 42, 36, 32 and 29 kDa proteins, and those of F2 were 200, 100, 40, 42, 36, 34 and 32 kDa proteins . The isolated proteins also contained several Coomassie-blue stained proteins that did not bind calmodulin and, therefore, may represent the proteins associated with the calmodulin-binding proteins . Antisera raised against the affinity-purified preparation of F1 and F2 recognized almost all of the calmodulin-binding proteins present in the fraction and several other proteins of the nucleus . The presence of Ca(2+)-dependent protein phosphatase (type 2B) in the nucleus was demonstrated by Western blotting . The enzyme was localized predominantly in F1 and F4.

Appl Environ Microbiol, 1993 May, 59(5), 1487 - 94
Role of D-ribose as a cometabolite in D-xylose metabolism by Saccharomyces cerevisiae; van Zyl C et al.; The influence of D-ribose as a cosubstrate on the uptake and metabolism of the non-growth substrate D-xylose by Saccharomyces cerevisiae ATCC 26602 was investigated . Xylose was taken up by means of low- and high-affinity glucose transport systems . In cells exposed for 2 days to a mixture of xylose and ribose, only the high-affinity system could be detected . Glucose strongly inhibited the transport of xylose by both systems . Starvation or exposure to either xylose or ribose resulted in inactivation of xylose transport, which did not occur in the presence of a mixture of ribose and xylose . A constitutive non-glucose-repressible NADPH2-dependent xylose reductase with a specific activity of ca . 5 mU/mg of protein that converted xylose to xylitol was present in a glucose-grown culture . No activity converting xylitol to xylulose or vice versa was found in crude extracts . Both xylose and ribose were converted to their corresponding polyols, xylitol and ribitol, as indicated by 13C nuclear magnetic resonance spectroscopy . Furthermore, ethanol was detected, and this implied that pathways for the complete catabolism of xylose and ribose exist . However, the NADPH2 required for the conversion of xylose to xylitol is apparently not supplied by the pentose phosphate pathway since the ethanol produced from D-{1-13C}xylose was labelled only in the C-2 position . Acetic acid was produced from ribose and may assist in the conversion of xylose to xylitol by cycling NADPH2.

Genetics, 1993 May, 134(1), 81 - 150
Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs; Riles L et al.; Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae are presented . In order of increasing size, they are chromosomes I, VI, III, IX, V and VIII, comprising 2.49 megabase pairs of DNA . The maps are based on the analysis of an overlapping set of lambda and cosmid clones . Overlaps between adjacent clones were recognized by shared restriction fragments produced by the combined action of EcoRI and HindIII . The average spacing between mapped cleavage sites is 2.6 kb . Five of the six chromosomes were mapped from end to end without discontinuities; a single internal gap remains in the map of chromosome IX . The reported maps span an estimated 97% of the DNA on the six chromosomes; nearly all the missing segments are telomeric . The maps are fully cross-correlated with the previously published SfiI/NotI map of the yeast genome by A . J . Link and M . V . Olson . They have also been cross-correlated with the yeast genetic map at 51 loci.

Genetics, 1993 May, 134(1), 5 - 19
Genetic evidence that the meiotic recombination hotspot at the HIS4 locus of Saccharomyces cerevisiae does not represent a site for a symmetrically processed double-strand break; Porter SE et al.; In the yeast Saccharomyces cerevisiae, the binding of the Rap1 protein to a site located between the 5' end of the HIS4 gene and the 3' end of BIK1 stimulates meiotic recombination at both flanking loci . By using strains that contain mutations located in HIS4 and BIK1, we found that most recombination events stimulated by the binding of Rap1 involve HIS4 or BIK1, rather than bidirectional events including both loci . The patterns of aberrant segregation indicate that most of the Rap1-stimulated recombination events do not represent the symmetric processing of a double-strand DNA break located at the Rap1-binding site.

Genetics, 1993 May, 134(1), 43 - 56
Mutations in POL1 increase the mitotic instability of tandem inverted repeats in Saccharomyces cerevisiae; Ruskin B et al.; Tandem inverted repeats (TIRs or hairpins) of 30 and 80 base-pair unit lengths are unstable mitotically in yeast (Saccharomyces cerevisiae) . TIR instability results from deletions that remove part or all of the presumed hairpin structure from the chromosome . At least one deletion endpoint is always at or near the base of the hairpin, and almost all of the repaired junctions occur within short direct sequence repeats of 4 to 9 base pairs . The frequency of this event, which we call "hairpin excision," is influenced by chromosomal position, length of the inverted repeats, and the distance separating the repeat units; increasing the distance between the inverted repeats as little as 25 base pairs increases their chromosomal stability . The frequency of excision is not affected by representative rad mutations, but is influenced by mutations in certain genes affecting DNA synthesis . In particular, mutations in POL1/CDC17, the gene that encodes the large subunit of DNA polymerase I, increase the frequency of hairpin deletions significantly, implicating this protein in the normal maintainance of genomic TIRs.

Genetics, 1993 May, 134(1), 21 - 8
Nuclear mutations in Saccharomyces cerevisiae that affect the escape of DNA from mitochondria to the nucleus; Thorsness PE et al.; We have inserted a yeast nuclear DNA fragment bearing the TRP1 gene and its associated origin of DNA replication, ARS1, into the functional mitochondrial chromosome of a strain carrying a chromosomal trp1 deletion . TRP1 was not phenotypically expressed within the organelle . However, this Trp- strain readily gave rise to respiratory competent Trp+ clones that contained the TRP1/ARS1 fragment, associated with portions of mitochondrial DNA (mtDNA), replicating in their nuclei . Thus the Trp+ clones arose as a result of DNA escaping from mitochondria and migrating to the nucleus . We have isolated 21 nuclear mutants in which the rate of mtDNA escape is increased by screening for increased rates of papillation to Trp+ . All 21 mutations were recessive and fell into six complementation groups, termed YME1-YME6 . In addition to increasing the rate of mtDNA escape, yme1 mutations also caused a heat-sensitive respiratory deficient phenotype at 37 degrees and a cold-sensitive growth defect on complete glucose medium at 14 degrees . While the other yme mutations had no detectable growth phenotypes, synergistic interactions were observed in two double mutant combinations: a yme1, yme2 double mutant failed to respire at 30 degrees and a yme4, yme6 double mutant failed to respire at all temperatures tested . None of the respiratory defects were caused by loss of functional mtDNA . These findings suggest that yme1, yme2, yme4 and yme6 mutations alter mitochondrial functions and thereby lead to an increased rate of DNA escape from the organelle.

Genetics, 1993 May, 134(1), 159 - 73
Extragenic suppressors of mutations in the cytoplasmic C terminus of SEC63 define five genes in Saccharomyces cerevisiae; Nelson MK et al.; Mutations in the SEC63 gene of Saccharomyces cerevisiae affect both nuclear protein localization and translocation of proteins into the endoplasmic reticulum . We now report the isolation of suppressors of sec63-101 (formerly npl1-1), a temperature-sensitive allele of SEC63 . Five complementation groups of extragenic mutations, son1-son5 (suppressor of npl1-1), were identified among the recessive suppressors . The son mutations are specific to SEC63, are not bypass suppressors, and are not new alleles of previously identified secretory (SEC61, SEC62, KAR2) or nuclear protein localization genes (NPL3, NPL4, NPL6) . son1 mutations show regional specificity of suppression of sec63 alleles . At low temperatures, son1 mutants grow slowly and show partial mislocalization of nuclear antigens . The SON1 gene maps to chromosome IV and encodes a nuclear protein of 531 amino acids that contains two acidic stretches and a putative nuclear localization sequence . We show that son1 mutations suppress sec63-101 by elimination of Son1p function.

Genetics, 1993 May, 134(1), 151 - 7
Direct cloning of yeast genes from an ordered set of lambda clones in Saccharomyces cerevisiae by recombination in vivo; Erickson JR et al.; We describe a technique that facilitates the isolation of yeast genes that are difficult to clone . This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids . The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L . Riles et al . in 1993 . The Escherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert . When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura+ transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences . Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype . This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E . coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score . We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning.

Mol Gen Genet, 1993 May, 239(1-2), 28 - 32
Inducible removal of UV-induced pyrimidine dimers from transcriptionally active and inactive genes of Saccharomyces cerevisiae; Waters R et al.; The prior UV irradiation of alpha haploid Saccharomyces cerevisiae with a UV dose of 25 J/m2 substantially increases the repairability of damage subsequently induced by a UV dose of 70 J/m2 given 1 h after the first irradiation . This enhancement of repair is seen at both the MAT alpha and HML alpha loci, which are, respectively, transcriptionally active and inactive in alpha haploid cells . The presence in the medium of the protein synthesis inhibitor, cycloheximide in the period between the two irradiations eliminated this effect . Enhanced repair still occurred if cycloheximide was present only after the final UV irradiation . This indicated that the first result is not due to cycloheximide merely blocking the synthesis of repair enzymes associated with a hypothetical rapid turnover of such molecules . The enhanced repairability is not the result of changes in chromatin accessibility without protein synthesis, merely caused by the repair of the damage induced by the prior irradiation . The data clearly show that a UV-inducible removal of pyrimidine dimers has occurred which involves the synthesis of new proteins . The genes known to possess inducible promoters, and which are involved in excision are RAD2, RAD7, RAD16 and RAD23 . Studies with the rad7 and rad16 mutants which are defective in the ability to repair HML alpha and proficient in the repair of MAT alpha showed that in rad7, preirradiation enhanced the repair at MAT alpha, whereas in rad16 this increased repair of MAT alpha was absent . The preirradiation did not modify the inability to repair HML alpha in either strain . Thus RAD16 has a role in this inducible repair.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA Cell Biol, 1993 May, 12(4), 363 - 70
Mutation analysis of Saccharomyces cerevisiae CDC6 promoter: defining its UAS domain and cell cycle regulating element; Zhou C et al.; Using beta-galactosidase as the reporter gene, we carried out mutagenesis experiments to investigate the 5' promoter region of the CDC6 gene . Our results showed that the DNA element, between -262 and -170, is important for the upstream activating sequence (UAS) activities . On the basis of the DNA sequence, there is a Mlu I (-204) and a Mlu I-like (-216) element located within the middle of the UAS region . Insertion and deletion mutagenesis analysis of the Mlu I sequence has indicated that the internal CGCG sequence of the Mlu I site (ACGCGT) is important for gene expression . Furthermore, when DNA elements containing the Mlu I sites were subcloned into the tester plasmid, periodic expression of a reporter gene throughout the cell cycle was observed, as evidenced by the beta-galactosidase activities and lacZ mRNA . Because the possible transcriptional initiation sites of the CDC6 transcript have been previously defined (Zhou and Jong, 1990, J . Biol . Chem . 264, 9022-9029), we propose a model regarding the construct of the CDC6 promoter region . This 5' promoter construct contains a UAS region and a Mlu I element (MCB box) typical of a family of cell cycle-regulated genes involved in DNA metabolism . Previous genetic studies have not completely defined the CDC6 execution point in the functional yeast cell cycle map . Our results favor the possibility that the CDC6 gene is required, and directly involved, in the initiation of DNA replication.

J Bacteriol, 1993 May, 175(10), 3174 - 81
Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry; Woldringh CL et al.; The pattern of volume growth of Saccharomyces cerevisiae a/alpha was determined by image cytometry for daughter cells and consecutive cycles of parent cells . An image analysis program was specially developed to measure separately the volume of bud and mother cell parts and to quantify the number of bud scars on each parent cell . All volumetric data and cell attributes (budding state, number of scars) were stored in such a way that separate volume distributions of cells or cell parts with any combination of properties--for instance, buds present on mothers with two scars or cells without scars (i.e., daughter cells) and without buds--could be obtained . By a new method called intersection analysis, the average volumes of daughter and parent cells at birth and at division could be determined for a steady-state population . These volumes compared well with those directly measured from cells synchronized by centrifugal elutriation . During synchronous growth of daughter cells, the pattern of volume increase appeared to be largely exponential . However, after bud emergence, larger volumes than those predicted by a continuous exponential increase were obtained, which confirms the reported decrease in buoyant density . The cycle times calculated from the steady-state population by applying the age distribution equation deviated from those directly obtained from the synchronized culture, probably because of inadequate scoring of bud scars . Therefore, for the construction of a volume-time diagram, we used volume measurements obtained from the steady-state population and cycle times obtained from the synchronized population . The diagram shows that after bud emergence, mother cell parts continue to grow at a smaller rate, increasing about 10% in volume during the budding period . Second-generation daughter cells, ie., cells born from parents left with two scars, were significantly smaller than first-generation daughter cells . Second- and third-generation parent cells showed a decreased volume growth rate and a shorter budding period than that of daughter cells.

Genes Dev, 1993 May, 7(5), 822 - 32
The Cdk-associated protein Cks1 functions both in G1 and G2 in Saccharomyces cerevisiae; Tang Y et al.; The CKS1 gene of Saccharomyces cerevisiae encodes a small essential protein shown to interact genetically and physically with the Cdc28 protein kinase . To investigate the specific functions of the CKS1 gene product, conditional temperature-sensitive mutant alleles were generated . The mutations were found to impair the ability of cells to undergo both the G1/S-phase and G2/M-phase transitions of the cell cycle, as well as the ability to bud . Mutants were not defective, however, in their ability to activate Cdc28 kinase as assayed in vitro on the substrate histone H1 . It is likely, therefore, that Cks1 mediates a more specialized function of the Cdc28 kinase such as its ability to form specific multimeric complexes or to localize properly in cellular compartments.

J Cell Biol, 1993 May, 121(3), 503 - 12
NDC10: a gene involved in chromosome segregation in Saccharomyces cerevisiae; Goh PY et al.; A mutant, ndc10-1, was isolated by anti-tubulin staining of temperature-sensitive mutant banks of budding yeast . ndc10-1 has a defect chromosome segregation since chromosomes remains at one pole of the anaphase spindle . This produces one polyploid cell and one aploid cell, each containing a spindle pole body (SPD . NDC10 was cloned and sequenced and is identical to CBF2 (Jiang, W., J . Lechnermn and J . Carbon . 1993 . J . Cell Biol . 121:513) which is the 110-kD component of a centromere DNA binding complex (Lechner, J., and J . Carbon . 1991 . Cell . 61:717-725) . NDC10 is an essential gene . Antibodies to Ndc10p labeled the SPB region in nearly all the cells examined including nonmitotic cells . In some cells with short spindles which may be in metaphase, staining was also observed along the spindle . The staining pattern and the phenotype of ndc10-1 are consistent with Cbf2p/Ndc10p being a kinetochore protein, and provide in vivo evidence for its role in the attachment of chromosomes to the spindle.

Mol Cell Biol, 1993 May, 13(5), 2959 - 70
A U5 small nuclear ribonucleoprotein particle protein involved only in the second step of pre-mRNA splicing in Saccharomyces cerevisiae; Horowitz DS et al.; The PRP18 gene, which had been identified in a screen for pre-mRNA splicing mutants in Saccharomyces cerevisiae, has been cloned and sequenced . Yeast strains bearing only a disrupted copy of PRP18 are temperature sensitive for growth; even at a low temperature, they grow extremely slowly and do not splice pre-mRNA efficiently . This unusual temperature sensitivity can be reproduced in vitro; extracts immunodepleted of PRP18 are temperature sensitive for the second step of splicing . The PRP18 protein has been overexpressed in active form in Escherichia coli and has been purified to near homogeneity . Antibodies directed against PRP18 precipitate the U4/U5/U6 small nuclear ribonucleoprotein particle (snRNP) from yeast extracts . From extracts depleted of the U6 small nuclear RNA (snRNA), the U4 and U5 snRNAs can be immunoprecipitated, while no snRNAs can be precipitated from extracts depleted of the U5 snRNA . PRP18 therefore appears to be primarily associated with the U5 snRNP . The antibodies against PRP18 inhibit the second step of pre-mRNA splicing in vitro . Together, these results imply that the U5 snRNP plays a role in the second step of splicing and suggest a model for the action of PRP18.

Mol Cell Biol, 1993 May, 13(5), 2815 - 21
In Xenopus laevis, the product of a developmentally regulated mRNA is structurally and functionally homologous to a Saccharomyces cerevisiae protein involved in translation fidelity; Tassan JP et al.; We have performed a differential screen of a Xenopus egg cDNA library and selected two clones (Cl1 and Cl2) corresponding to mRNA which are specifically adenylated and recruited into polysomes after fertilization . Sequence analysis of Cl1 reveals that the corresponding protein is 67.5% identical (83% similar) to the product of the Saccharomyces cerevisiae SUP45 (also called SUP1 or SAL4) gene . This gene, when mutated, is an omnipotent suppressor of nonsense codons . When expressed in a sup45 mutant, the Xenopus Cl1 cDNA was able to suppress sup45-related phenotypes, showing that the structural homology reflects a functional homology . Our discovery of a structural and functional homolog in Xenopus cells implies that the function of SUP45 is not restricted to lower eukaryotes and that the SUP45 protein may perform a crucial cellular function in higher eukaryotes.

Curr Genet, 1993 May-Jun, 23(5-6), 382 - 7
Lack of correlation between trehalase activation and trehalose-6 phosphate synthase deactivation in cAMP-altered mutants of Saccharomyces cerevisiae; Arguelles JC et al.; The rise in cAMP level that follows the addition of glucose or 2,4-dinitrophenol (DNP) to stationary-phase cells of Saccharomyces cerevisiae was accompanied by a marked activation of trehalase (3-fold increase) and a concomitant deactivation of trehalose-6 phosphate synthase (50% of the basal levels) . In glucose-grown exponential cells, which are deficient in glucose-induced cAMP signalling, the addition of glucose also prompted a decrease in trehalose-6 phosphate synthase, but had no effect on trehalase activity . Mutants defective in the RAS-adenylate cyclase pathway (ras1 ras2 bcy1 strain), as well as mutants containing greatly reduced protein kinase activity either cAMP-dependent (tpkw1 BCY1 strains) or cAMP-independent (tpk1w1 bcy1 strains), were unable to show glucose- or DNP-induced trehalase activation but still displayed a clear decrease in trehalose-6 phosphate synthase activity upon addition of these compounds . These data suggest that the activity of trehalose-6 phosphate synthase, as opposed to that of trehalase, is not controlled by the cAMP signalling pathway "in vivo" . Trehalose-6 phosphate synthase was competitively inhibited by glucose (Ki = 15 mM) and resulted unaffected by ATP in assays performed "in vitro".

EMBO J, 1993 May, 12(5), 1997 - 2003
A Saccharomyces cerevisiae UAS element controlled by protein kinase A activates transcription in response to a variety of stress conditions; Marchler G et al.; Transcription of the Saccharomyces cerevisiae CTT1 gene encoding the cytosolic catalase T is activated by a variety of stress conditions: it is derepressed by nitrogen starvation and induced by heat shock . Furthermore, it is activated by osmotic and oxidative stress . This study shows that a CTT1 upstream region previously found to be involved in nitrogen, cAMP and heat control (base pairs -382 to -325) contains a UAS element (STRE, -368 to -356), which is sufficient for the activation of a reporter gene by all types of stress acting on CTT1 . Gel retardation experiments demonstrated the existence of a factor specifically binding to STRE, but to a lesser extent to mutated elements having partly or entirely lost the ability to mediate stress control . Heat activation of STRE, but not of a canonical heat shock element, is enhanced by a ras2 defect mutation, which enhances thermotolerance, and is dramatically reduced by a bcy1 disruption mutation, which decreases thermotolerance . It can be hypothesized, therefore, that the novel stress control element is important for the establishment of induced stress tolerance.

EMBO J, 1993 May, 12(5), 1955 - 68
Comparison of the Saccharomyces cerevisiae G1 cyclins: Cln3 may be an upstream activator of Cln1, Cln2 and other cyclins; Tyers M et al.; In the budding yeast Saccharomyces cerevisiae, the G1 cyclins Cln1, Cln2 and Cln3 regulate entry into the cell cycle (Start) by activating the Cdc28 protein kinase . We find that Cln3 is a much rarer protein than Cln1 or Cln2 and has a much weaker associated histone H1 kinase activity . Unlike Cln1 and Cln2, Cln3 is not significantly cell cycle regulated, nor is it down-regulated by mating pheromone-induced G1 arrest . An artificial burst of CLN3 expression early in G1 phase accelerates Start and rapidly induces at least five other cyclin genes (CLN1, CLN2, HCS26, ORFD and CLB5) and the cell cycle-specific transcription factor SWI4 . In similar experiments, CLN1 is less efficient than CLN3 at activating Start . Strikingly, expression of HCS26, ORFD and CLB5 is dependent on CLN3 in a cln1 cln2 strain, possibly explaining why CLN3 is essential in the absence of CLN1 and CLN2 . To explain the potent ability of Cln3 to activate Start, despite its apparently weak biochemical activity, we propose that Cln3 may be an upstream activator of the G1 cyclins which directly catalyze Start . Given the large number of known cyclins, such cyclin cascades may be a common theme in cell cycle control.

Mol Cell Biol, 1993 May, 13(5), 2697 - 705
Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences; Schiestl RH et al.; When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained . In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment . In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I . The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.

Yeast, 1993 May, 9(5), 551 - 6
Identification of a gene encoding a novel zinc finger protein in Saccharomyces cerevisiae; Breitwieser W et al.; In the course of a comprehensive genomic screen for cell cycle regulated genes in the yeast Saccharomyces cerevisiae (Price et al., 1991) we identified and characterized a transcriptional unit encoding a putative zinc finger protein named FZF1 (EMBL accession number: X67787) . This gene encodes a protein containing five zinc fingers of the Cys2His2 class, three of which are positioned in tandem at the N-terminus . The fourth and fifth finger follow after an interruption of 61 and 66 amino acids, respectively . While FZF1 is constitutively expressed at a very low level, its deletion is not essential for growth . Its similarity with known transcription factors, however, suggests that the FZF1 gene product may serve as a transcription factor in yeast.

Yeast, 1993 May, 9(5), 523 - 6
Increased transformation levels in intact cells of Saccharomyces cerevisiae aculeacin A-resistant mutants; Gallego C et al.; Several Saccharomyces cerevisiae mutants resistant to the cell wall synthesis inhibitor aculeacin A exhibit higher transformation levels than the parental strain . Mutant acr2 has been studied in more detail . It is transformed up to ten-fold more efficiently than the wild-type strain with episomal, centromeric and integrative plasmids, and dimethyl sulfoxide has an additive effect improving transformation efficiency . Transformation with linear DNA molecules is not as much affected in acr2 cells . The observed effects may be caused by the altered plasma membrane composition of the mutants.

Yeast, 1993 May, 9(5), 519 - 21
Endomitotic diploidization of Saccharomyces cerevisiae by heat treatment during spore germination; Tani Y et al.; Diploid cells with ability to mate, hereafter referred to as diploid mater cells, were obtained at significant frequencies by the heat treatment of haploid spores at the early germination stage in Saccharomyces cerevisiae heterothallic strain CG5M (a/alpha diploid cells heterozygous for five auxotrophic markers) . The highest frequency (ca . 11%) of diploidization was obtained from viable cells after heat treatment at 55 degrees C for 10 min when spores were precultivated for 30 min in liquid medium to initiate the germination . The diploid mater cells obtained were homozygous for mating type and for the auxotrophic markers . The diploidization of a spore is thus concluded to be due to endomitotic events in germinating heat-treated spores.

Curr Genet, 1993 May-Jun, 23(5-6), 423 - 9
Time-dependent mitotic recombination in Saccharomyces cerevisiae; Steele DF et al.; The time-dependent appearance of prototrophic recombinants between heterologously located artificial repeats has been studied in Saccharomyces cerevisiae . While initial prototrophic colony numbers from independent cultures were highly variable, additional recombinants were found to arise daily at roughly constant rates irrespective of culture . These late-appearing recombinants could be accounted for neither by detectable growth on the selective media nor by delayed appearance of recombinants present at the time of selective plating . Significantly, at no time did the distributions of recombinants fully match those expected according to the Luria-Delbruck model and, in fact, after the first day, the distributions much more closely approximated a Poisson distribution . Prototrophic recombinants accumulated not only on the relevant selective medium, but also on media unrelated to the acquired prototrophy.

Curr Genet, 1993 May-Jun, 23(5-6), 402 - 7
The use of a double-marker shuttle vector to study DNA double-strand break repair in wild-type and radiation-sensitive mutants of the yeast Saccharomyces cerevisiae; Jha B et al.; An episomal DNA vector (YpJA18), encoding two selectable recombinant yeast genes (TRP1, URA3), was constructed to assess the fidelity of DNA repair in haploid repair-competent (RAD) wild-type yeast and several radiation-sensitive mutants . Either a DNA double-strand break (DSB) or a double-strand gap of 169 bp (DSG) was introduced by restriction enzymes in-vitro within the coding sequence of the URA3 gene of this vector . To eliminate transfer artefacts, selection was first applied for the undamaged TRP1 gene followed by counter selection for URA3 gene activity, which indicated correct repair of the DSB and DSG . Correct repair of the damaged URA3 gene was found to be about 90% in RAD cells (normalized for the expression of undamaged URA3 in TRP+ transformants) . Plasmids isolated from the transformants (URA+TRP+) carry both unique sites (ApaI and NcoI) within the URA3 gene indicating the precise restitution of the 169-bp gap . An excision-repair-defective rad4-4 mutant repaired these lesions as correctly as RAD cells, whereas the mutants rad50-1, rad51-1 and rad54-1, proven to be defective in DSB repair and mitotic recombination, showed less than 5% correct repair of such lesions . In contrast, a representative of the RAD6 epistasis group of genes, the rev2-1 mutant which is sensitive towards UV and ionizing radiation, had a significantly reduced ability (about 20%) for the correct repair of both DSBs and DSGs.

Curr Genet, 1993 May-Jun, 23(5-6), 392 - 6
Phenotypic identification of amplifications of the ADH4 and CUP1 genes of Saccharomyces cerevisiae; Dorsey MJ et al.; Primary gene amplification, i.e., mutation from one gene copy to multiple gene copies per genome, is important in genomic evolution, as a means of producing anti-cancer drug resistance, and is associated with the progression of tumor malignancy . Primary amplification has not been studied in normal eukaryotic cells because amplifications are extremely rare in these cells . A system has been developed to phenotypically identify co-amplifications of the ADH4 and CUP1 genes of Saccharomyces cerevisiae and 21 independent spontaneous amplifications have been isolated.

Curr Genet, 1993 May-Jun, 23(5-6), 388 - 91
Reversible pseudohyphal growth in haploid Saccharomyces cerevisiae is an aerobic process; Wright RM et al.; Pseudohyphal growth in Saccharomyces cerevisiae has been postulated to be an adaptation to foraging for nitrogen during nitrogen starvation . This process was described as a strictly diploid phenomenon which did not occur in haploid yeast cells and was under the genetic control of both the mating-type locus and a group of five genes, the BUD genes, regulating bud formation . We have also observed a dimorphic growth pattern in yeast growing on various nitrogen-limiting synthetic media . However, and in contrast to a previous report, we find that pseudohyphal growth is not precluded in haploid cells . We demonstrate that haploid pseudohyphal growth is strictly oxygen-dependent and is rapidly reversible, defining pseudohyphal growth as a reversible developmental pathway in yeast.

Curr Genet, 1993 May-Jun, 23(5-6), 375 - 81
Structure and regulation of the isocitrate lyase gene ICL1 from the yeast Saccharomyces cerevisiae; Scholer A et al.; The ICL1 gene encoding the isocitrate lyase from Saccharomyces cerevisiae was cloned and sequenced . A reading frame of 557 amino acids showing significant similarity to isocitrate lyases from seven other species could be identified . Construction of icl1 null mutants led to growth defects on C2 carbon sources while utilization of sugars or C3 substrates remained unaffected . Using an ICL1-lacZ fusion integrated at the ICL1 locus, a more than 200-fold induction of beta-galactosidase activity was observed after growth on ethanol when compared with glucose-repressed conditions . A preliminary analysis of the ICL1 upstream region identified a 364-bp fragment necessary and sufficient for this regulatory phenotype . Sequence motifs also present in the upstream regions of co-regulated genes were found within this region.

Lett Appl Microbiol, 1993 May, 16(5), 251 - 3
Involvement of potassium ions in the action of various antineoplastic drugs on the growth of Saccharomyces cerevisiae; Tiligada E et al.; The involvement of potassium ions in the action of some antineoplastic drugs on the growth of Saccharomyces cerevisiae was studied by incubating yeast cells in the presence of drugs at various concentrations and KCl at concentrations of 50 mmol l-1 and 100 mmol l-1 . The presence of 6.25-50 micrograms ml-1 amsacrine or melphalan alone in the culture medium had no significant effect on yeast growth . Addition of KCl significantly increased the sensitivity to these drugs . On the contrary, incubation of yeast cells with KCl had no effect on the cytotoxic action of doxorubicin, methotrexate or 5-fluorouracil.

Biochemistry, 1993 Apr 27, 32(16), 4231 - 6
The acidic phosphoproteins from Saccharomyces cerevisiae ribosomes . NH2-terminal acetylation is a conserved difference between P1 and P2 proteins; Santos C et al.; Isoelectrofocusing gels of acidic ribosomal proteins from most yeast strains reveal the presence of up to 10 bands which are the product of only 4 genes . The proteins have been characterized by NH2-terminal amino acid sequencing, specific antibodies, HPLC, and by taking advantage of acidic protein-defective yeast strains obtained by gene disruption methods . The four most basic proteins coincide with the phosphorylated and dephosphorylated forms of the YP2 proteins, YP2 alpha and YP2 beta, formerly named L44 and L45 . Amino-terminal sequencing has shown that these two polypeptides have free amino-terminal ends starting at the first methionine residue . The bands defined earlier as L44' correspond to the phosphorylated and dephosphorylated processed forms of protein YB1 beta lacking the first eight amino acids . The formation of this truncated YP1 beta form seems to be stimulated by salt during protein extraction and is also favored by some modifications at the amino termini of the protein . On the other hand, the previously uncharacterized band, called Ax, corresponds to an NH2-terminal acetylated form of YP1 beta which starts at the serine in the second position of the nucleotide-derived sequence . Finally, the most acidic band is the phosphorylated product of the fourth acidic protein gene . This protein, called YP1 alpha, which is very poorly stained by silver and Coomassie blue, has not been characterized in detail previously . It is also monophosphorylated in the ribosome and, like YP1 beta, is present as an NH2-terminal acetylated form starting at the second serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1993 Apr 26, 321(2-3), 159 - 62
Insight into topological and functional relationships of cytochrome c oxidase subunit I of Saccharomyces cerevisiae by means of intragenic complementation; Meunier B et al.; In yeast, revertants were selected from four respiratory deficient mutants carrying mutations in the cytochrome c oxidase subunit I gene . Intragenic second site mutations revealed amino acids which are functionally complementary to the original mutated position and may be in topological interaction with it . The results provide additional data in favour of the model proposed for the structure of the binuclear centre in proton-motive oxidases.

Nucleic Acids Res, 1993 Apr 25, 21(8), 1975 - 82
Molecular analysis of the PHO81 gene of Saccharomyces cerevisiae; Creasy CL et al.; The PHO81 gene product is a positive regulatory factor required for the synthesis of the phosphate repressible acid phosphatase (encoded by the PHO5 gene) in Saccharomyces cerevisiae . Genetic analysis has suggested that PHO81 may be the signal acceptor molecule; however, the biochemical function of the PHO81 gene product is not known . We have cloned the PHO81 gene and sequenced the promoter . A PHO81-LacZ fusion was shown to be a valid reporter since its expression is regulated by the level of inorganic phosphate and is controlled by the same regulatory factors that regulate PHO5 expression . To elucidate the mechanism by which PHO81 functions, we have isolated and cloned dominant mutations in the PHO81 gene which confer constitutive synthesis of acid phosphatase . We have demonstrated that overexpression of the negative regulatory factor, PHO80, but not the negative regulatory factor PHO85, partially blocks the constitutive acid phosphatase synthesis in a strain containing a dominant constitutive allele of PHO81 . This suggests that PHO81 may function by interacting with PHO80 or that these molecules compete for the same target.

J Biol Chem, 1993 Apr 25, 268(12), 8990 - 8
Phenotypic analysis of proteinase A mutants . Implications for autoactivation and the maturation pathway of the vacuolar hydrolases of Saccharomyces cerevisiae; Woolford CA et al.; We have isolated a number of mutants deficient in activity of the vacuolar hydrolase proteinase A (PrA) . The mutations were sequenced and although they all map in the PEP4 gene, which encodes the precursor to PrA, three distinguishable phenotypes have surfaced . The properties of the pep4-7 missense mutant suggested that the activation of the precursor to proteinase A is due to an autocatalytic cleavage . PrA active site mutations were constructed and resulted in accumulation of PrA antigen in the inactive precursor form . Although protease B (PrB), another vacuolar hydrolase, is not required for the production of active PrA, the active form of PrA that accumulates in a strain lacking PrB is larger than that found in a strain containing active PrB . We have purified this larger form of PrA and determined that it bears 7 additional amino acids at its NH2 terminus . It has become apparent from all the studies performed on the maturation pathway of the vacuolar hydrolases that there is a great deal of redundancy built into the system.

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