FEBS Lett, 1996 Oct 21, 395(2-3), 199 - 203 The aromatic domain 66(YWYWW)70 of subunit VIII of the yeast ubiquinol-cytochrome c oxidoreductase is important for both assembly and activity of the enzyme; Lobo-Hajdu G et al.; The aromatic character of the region 66(YWYWW)70 of the 11-kDa subunit VIII of ubiquinol-cytochrome c oxidoreductase (bc1 complex) of the yeast Saccharomyces cerevisiae has previously been demonstrated to be important for assembly of a functional complex {Hemrika et al . (1994) FEBS Lett . 344, 15-19} . Especially the aromatic nature of residue 66 appeared to be relevant, as the very low level (5%) of bc1 complex in the mutant 66(SASAA)70 was restored to nearly 70% of the wild-type level in a phenotypic revertant with the sequence 66(FASAA)70 . In the present study, three other site-directed mutants (66(SAYAA)70, 66(SASAW)70 and 66(SWYWW)70) were constructed and analysed . The data indicate that the presence of one aromatic residue is enough for a substantial level of assembly and that its position modulates the level of both assembly and electron transfer activity . The results also confirm the relevance of this region of subunit VIII for the formation of the Q(out) reaction domain, as reported by Hemrika et al . {(1993) Eur . J . Biochem . 215, 601-609} . It is further shown that the lowered specific activity of the mutant described by these authors is not due to the introduction of a cysteine in the sequence of subunit VIII. Cell, 1996 Oct 18, 87(2), 277 - 85 A 700 bp cis-acting region controls mating-type dependent recombination along the entire left arm of yeast chromosome III; Wu X et al.; Homothallic switching of the mating-type MATa gene in Saccharomyces cerevisiae results from replacement by gene conversion of MAT-Ya DNA with Y(alpha) sequences copied from one of two unexpressed donors . MATa preferentially recombines with HML(alpha), located near the left end of chromosome III, but can use HMR(alpha), near the right chromosome end . MATa donor preference depends on a 700 bp orientation-independent cis-acting recombination enhancer, located 17 kb proximal to HML . Deletion of this element markedly reduces MATa's use of a donor inserted at any of four different locations along the leftmost 92 kb of chromosome III . This enhancer is sufficient for donor activation, since it stimulates use of the "wrong" donor, when it is inserted 7 kb proximal to HMR. J Biol Chem, 1996 Oct 18, 271(42), 25790 - 6 A yeast Ubc9 mutant protein with temperature-sensitive in vivo function is subject to conditional proteolysis by a ubiquitin- and proteasome-dependent pathway; Betting J et al.; The UBC9 gene of the yeast Saccharomyces cerevisiae is essential for cell viability and encodes a soluble protein of the nucleus that is metabolically stable . Products of mutant alleles selected to confer temperature-sensitive in vivo function were found to be extremely short-lived at the restrictive but long-lived at the permissive condition . An extragenic suppressor mutation was isolated which increased thermoresistance of a ubc9-1 strain . This suppressor turned out to stabilize the mutated gene product, indicating that the physiological activity of ubc9-1 protein is primarily controlled by conditional proteolysis . The labile ubc9-1 protein appears to be a substrate for ubiquitination, and its turnover was substantially reduced by expression of a ubiquitin derivative that interferes with formation of multi-ubiquitin chains . Stabilization resulted also from competitive inhibition of Ubc4-related ubiquitin-conjugating enzymes . Activity of the proteasome complex was crucial to rapid breakdown, whereas vacuolar proteases were dispensable . Thus, the heat-denatured ubc9-1 protein is targeted for proteolysis by the ubiquitin-proteasome pathway and may serve as a useful tool to further define the process by which a misfolded polypeptide is recognized. Gene, 1996 Oct 17, 176(1-2), 111 - 7 The yeast SME1 gene encodes the homologue of the human E core protein; Bordonne R et al.; Removal of introns from pre-messenger RNA (pre-mRNA) requires small nuclear RNAs (snRNAs) packaged into stable small ribonucleoprotein particles (snRNP) . These snRNPs contain specific and common proteins also called Sm proteins . Correct assembly of the snRNAs with the common proteins is an essential step for the biogenesis of snRNP particles . We have identified a new Saccharomyces serevisiae gene, SME1 whose product shows 45% identity with the E core protein of human snRNP . The Sme1p contains the evolutionary conserved residues found in all Sm proteins . Combining genetic and biochemical experiments, we show that SME1 is an essential gene required for pre-mRNA splicing, cap modification and U1, U2, U4 and U5 snRNA stability . We show also that the human E core protein complements a yeast SME1 disruption demonstrating the functional equivalence of Sme1p and the human E core protein. Mol Gen Genet, 1996 Oct 16, 252(5), 608 - 21 Genetic interactions indicate a role for Mdg1p and the SH3 domain protein Bem1p in linking the G-protein mediated yeast pheromone signalling pathway to regulators of cell polarity; Leberer E et al.; The pheromone signal in the yeast Saccharomyces cerevisiae is transmitted by the beta and gamma subunits of the mating response G-protein . The STE20 gene, encoding a protein kinase required for pheromone signal transduction, has recently been identified in a genetic screen for high-gene-dosage suppressors of a partly defective G beta mutation . The same genetic screen identified BEM1, which encodes an SH3 domain protein required for polarized morphogenesis in response to pheromone, and a novel gene, designated MDG1 (multicopy suppressor of defective G-protein) . The MDG1 gene was independently isolated in a search for multicopy suppressors of a bem1 mutation . The MDG1 gene encodes a predicted hydrophilic protein of 364 amino acids with a molecular weight of 41 kDa that has no homology with known proteins . A fusion of Mdg1p with the green fluorescent protein from Aequorea victoria localizes to the plasma membrane, suggesting that Mdg1p is an extrinsically bound membrane protein . Deletion of MDG1 causes sterility in cells in which the wild-type G beta has been replaced by partly defective G beta derivatives but does not cause any other obvious phenotypes . The mating defect of cells deleted for STE20 is partially suppressed by multiple copies of BEM1 and CDC42, which encodes a small GTP-binding protein that binds to Ste20p and is necessary for the development of cell polarity . Elevated levels of STE20 and BEM1 are capable of suppressing a temperature-sensitive mutation in CDC42 . This complex network of genetic interactions points to a role for Bem1p and Mdg1p in G-protein mediated signal transduction and indicates a functional linkage between components of the pheromone signalling pathway and regulators of cell polarity during yeast mating. Genomics, 1996 Oct 15, 37(2), 183 - 6 Associations of UBE2I with RAD52, UBL1, p53, and RAD51 proteins in a yeast two-hybrid system; Shen Z et al.; The yeast RAD52-dependent pathway is involved in DNA recombination and double-strand break repair . Yeast ubiquitin-conjugating enzyme UBC9 participates in S- and M-phase cyclin degradation and mitotic control . Using the human RAD52 protein as the "bait" in a yeast two-hybrid system, we have identified a human homolog of yeast UBC9, designated UBE2I, that interacts with RAD52, RAD51, p53, and a ubiquitin-like protein UBL1 . These interactions are UBE2I-specific, since another DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), does not interact with these proteins . The interaction of UBE2I with RAD52 is mediated by RAD52's self-association region . These results suggest that the RAD52-dependent processes, cell cycle control, p53-mediated pathway(s), and ubiquitination interact through human UBE2I. Anal Biochem, 1996 Oct 15, 241(2), 220 - 7 Electrophoretic behavior of L- and D-alanine-scanning analogs of a yeast tridecapeptide pheromone in a fused-silica capillary; Zhang YL et al.; Electrophoretic behavior of synthetic tridecapeptide diastereomers has been systematically investigated using a series of L-Ala- and D-Ala-scanning analogs of {Nle12} alpha-factor {WHWLQLKPGQP(Nle)Y}, a tridecapeptide mating pheromone of Saccharomyces cerevisiae . The effects of buffer pH, buffer concentration, voltage, and temperature on diastereomer separation were tested . Among 13 pairs of diastereomers, those with L-Ala/D-Ala replacement in the middle of the peptide chain exhibited much higher diastereomeric resolution than those with identical replacement near the peptide termini . The fact that D-Ala9 and D-Ala12 homologs exhibited abnormal mobility differences compared to their L-diastereomers is probably related to the conformational restriction imposed by a Pro-D-Ala sequence . The results on the alpha-factor analogs represent the first observations of the influence of peptide secondary structure on mobility during capillary electrophoresis. EMBO J, 1996 Oct 15, 15(20), 5701 - 14 RRP5 is required for formation of both 18S and 5.8S rRNA in yeast; Venema J et al.; Three of the four eukaryotic ribosomal RNA molecules (18S, 5.8S and 25-28S) are synthesized as a single precursor which is subsequently processed into the mature rRNAs by a complex series of cleavage and modification reactions . In the yeast Saccharomyces cerevisiae, the early pre-rRNA cleavages at sites A0, A1 and A2, required for the synthesis of 18S rRNA, are inhibited in strains lacking RNA or protein components of the U3, U14, snR10 and snR30 small nucleolar ribonucleoproteins (snoRNPs) . The subsequent cleavage at site A3, required for formation of the major, short form of 5.8S rRNA, is carried out by another ribonucleoprotein, RNase MRP . A screen for mutations showing synthetic lethality with deletion of the non-essential snoRNA, snR10, identified a novel gene, RRP5, which is essential for viability and encodes a 193 kDa nucleolar protein . Genetic depletion of Rrp5p inhibits the synthesis of 18S rRNA and, unexpectedly, also of the major short form of 5.8S rRNA . Pre-rRNA processing is concomitantly impaired at sites A0, A1, A2 and A3 . This distinctive phenotype makes Rrp5p the first cellular component simultaneously required for the snoRNP-dependent cleavage at sites A0, A1 and A2 and the RNase MRP-dependent cleavage at A3 and provides evidence for a close interconnection between these processing events . Putative RRP5 homologues from Caenorhabditis elegans and humans were also identified, suggesting that the critical function of Rrp5p is evolutionarily conserved. Science, 1996 Oct 11, 274(5285), 249 - 52 Cdc13p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance; Nugent CI et al.; The CDC13 gene has previously been implicated in the maintenance of telomere integrity in Saccharomyces cerevisiae . With the use of two classes of mutations, here it is shown that CDC13 has two discrete roles at the telomere . The cdc13-2est mutation perturbs a function required in vivo for telomerase regulation but not in vitro for enzyme activity, whereas cdc13-1ts defines a separate essential role at the telomere . In vitro, purified Cdc13p binds to single-strand yeast telomeric DNA . Therefore, Cdc13p is a telomere-binding protein required to protect the telomere and mediate access of telomerase to the chromosomal terminus. J Biol Chem, 1996 Oct 11, 271(41), 25590 - 7 Determination of the transmembrane topology of yeast Sec61p, an essential component of the endoplasmic reticulum translocation complex; Wilkinson BM et al.; Sec61p is a highly conserved integral membrane protein that plays a role in the formation of a protein-conducting channel required for the translocation of polypeptides into, and across, the membrane of the endoplasmic reticulum . As a major step toward elucidating the structure of the endoplasmic reticulum translocation apparatus, we have determined the transmembrane topology of Sec61p using a combination of C-terminal reporter-domain fusions and the in situ digestion of specifically inserted factor Xa protease cleavage sites . Our data indicate the presence of 10 transmembrane domains, including several with surprisingly limited hydrophobicity . Furthermore, we provide evidence for complex intramolecular interactions in which these weakly hydrophobic domains require C-terminal sequences for their correct topogenesis . The incorporation of sequences with limited hydrophobicity into the bilayer may play a vital role in the formation of an aqueous membrane channel required for the translocation of hydrophilic polypeptide chains. J Biol Chem, 1996 Oct 11, 271(41), 25208 - 12 Covalent attachment of FAD derivatives to a fusion protein consisting of 6-hydroxy-D-nicotine oxidase and a mitochondrial presequence . Folding, enzyme activity, and import of the modified protein into yeast mitochondria; Stoltz M et al.; Autoflavinylation of 6-hydroxy-D-nicotine oxidase (6-HDNO) was successfully employed to modify the protein covalently with FAD derivatives . The model compounds N6-(2-aminoethyl)-FAD and N6-(6-carboxyhexyl)-FAD were spontaneously bound to a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa F0-ATPase subunit 9 (Su9) attached to 6-HDNO . When translated in the rabbit reticulocyte lysate, Su9-6-HDNO was in the trypsin-sensitive apoenzyme form; when translated in the presence of flavins it adopted a trypsin-resistant conformation characteristic of the 6-HDNO holoenzyme . With flavin derivatives, Su9-6-HDNO exhibited approximately 50% of the 6-HDNO activity observed with FAD . The covalently modified Su9-6-HDNO was imported into Saccharomyces cerevisiae mitochondria with an efficiency equal to that of the apoenzyme . Apparently the increase in size and charge of the FAD moiety did not hamper translocation across the mitochondrial membranes . Yeast mutant ssc1-2 mitochondria deficient in mtHsp70 unfoldase activity imported the flavinylated Su9-6-HDNO protein . In mutant ssc1-3 mitochondria deficient in both mtHsp70 unfoldase and translocase activity Su9-6-HDNO was trapped as translocation intermediate; the Su9 presequence was passed to the matrix where it was proteolytically cleaved by the mitochondrial processing peptidase; (MPP); the translocation-arrested 6-HDNO moiety adopted a trypsin-sensitive conformation . Our results indicate that unfolding of the FAD-stabilized flavin-binding domain of 6-HDNO in passage through the mitochondrial general insertion pore does not require the activity of mtHsp70. Cell, 1996 Oct 4, 87(1), 85 - 94 The major cytoplasmic histone acetyltransferase in yeast: links to chromatin replication and histone metabolism; Parthun MR et al.; We have isolated the predominant cytoplasmic histone acetyltransferase activity from Saccharomyces cerevisiae . This enzyme acetylates the lysine at residue 12 of free histone H4 but does not modify histone H4 when packaged in chromatin . The activity contains two proteins, Hat1p and Hat2p . Hat1p is the catalytic subunit of the histone acetyltransferase and has an intrinsic substrate specificity that modifies lysine in the recognition sequence GXGKXG . The specificity of the enzyme in the yeast cytoplasm is restricted relative to recombinant Hat1p suggesting that it is negatively regulated in vivo . Hat2p, which is required for high affinity binding of the acetyltransferase to histone H4, is highly related to Rbap48, which is a subunit of the chromatin assembly factor, CAF-1, and copurifies with the human histone deacetylase HD1 . We propose that the Hat2p/Rbap48 family serve as escorts of histone metabolism enzymes to facilitate their interaction with histone H4. Science, 1996 Oct 4, 274(5284), 103 - 6 Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon; Rep M et al.; Afg3p and Rca1p are adenosine triphosphate (ATP)-dependent metalloproteases in yeast mitochondria . Cells lacking both proteins exhibit defects in respiration-dependent growth, degradation of mitochondrially synthesized proteins, and assembly of inner-membrane complexes . Defects in growth and protein assembly, but not in degradation, were suppressed by overproduction of yeast mitochondrial Lon, an ATP-dependent serine protease . Suppression by Lon was enhanced by inactivation of the proteolytic site and was prevented by mutation of the ATP-binding site . It is suggested that the mitochondrial proteases Lon, Afg3p, and Rca1p can also serve a chaperone-like function in the assembly of mitochondrial protein complexes. J Biol Chem, 1996 Oct 4, 271(40), 24761 - 8 Elements of neural adhesion molecules and a yeast vacuolar protein sorting receptor are present in a novel mammalian low density lipoprotein receptor family member; Yamazaki H et al.; Normal cell development depends to a large part on multifunctional proteins that have evolved by recombination of proven modular elements . We now have discovered and characterized in rabbit such a multi-domain protein, and classify it as novel member of the low density lipoprotein (LDL) receptor gene family . The extracellular portion of the approximately 250-kDa membrane protein, termed LR11, contains a cluster of 11 LDL receptor ligand binding repeats, a group of 5 LDL receptor "YWTD" repeats, a large hexarepeat domain of structural elements found in neural cell adhesion molecules, and a domain with similarity to a yeast receptor for vacuolar protein sorting, VPS10 . The cytoplasmic domain exhibits features typical of endocytosis-competent coated-pit receptors . The mosaic, and presumably multifunctional, receptor is expressed abundantly in brain, in particular the hippocampus, dentate gyrus, and cerebral cortex, and is present at significant levels in liver, adrenal glands, and testis . Western blotting of tissues and ligand blotting of LR11-transfected cells demonstrated that the novel protein binds apolipoprotein E-containing lipoproteins . In contrast to the LDL receptor, hepatic expression of LR11 is unaffected by hyperlipidemia . The identification of this highly conserved and superbly complex protein offers the opportunity to gain new insights into the emergence of multifunctional mosaic proteins akin to the ever expanding LDL receptor gene family. J Biol Chem, 1996 Oct 4, 271(40), 24498 - 504 Purification and characterization of an RNA polymerase II phosphatase from yeast; Chambers RS et al.; RNA polymerase (RNAP) II is subject to extensive phosphorylation on the heptapeptide repeats of the C-terminal domain (CTD) of the largest subunit . An activity that is required for the dephosphorylation of yeast RNAP II in vitro has been purified from a yeast whole cell extract by >30,000-fold . The yeast CTD phosphatase activity copurified with two bands with apparent molecular masses of 100 and 103 kDa . The properties of the yeast CTD phosphatase are similar to those of a previously characterized CTD phosphatase from HeLa cells . These properties include stimulation by the general transcription factor IIF (TFIIF), competitive inhibition by RNAP II, magnesium dependence, and resistance to okadaic acid . Both the HeLa and yeast CTD phosphatases are highly specific for their cognate polymerases . Neither phosphatase functions upon the polymerase molecule from the other species, even though the heptapeptide repeats of the CTDs in yeast RNAP II and mammalian RNAP II are essentially identical . The activity of the highly purified CTD phosphatase is stimulated >300-fold by a partially purified fraction of TFIIF . Recombinant TFIIF did not substitute for the TFIIF fraction, indicating that an additional factor present in the TFIIF fraction is required for CTD phosphatase activity . These results show that yeast contains a CTD phosphatase activity similar to that of mammalian cells that is likely composed of at least two components, one of which is 100 and/or 103 kDa. J Biol Chem, 1996 Oct 4, 271(40), 24458 - 64 Mitochondrial and cytosolic branched-chain amino acid transaminases from yeast, homologs of the myc oncogene-regulated Eca39 protein; Kispal G et al.; We have isolated a high copy suppressor of a temperature-sensitive mutation in ATM1, which codes for an ABC transporter of Saccharomyces cerevisiae mitochondria . The suppressor, termed BAT1, encodes a protein of 393 amino acid residues with an NH2-terminal extension that directs Bat1p to the mitochondrial matrix . A highly homologous protein, Bat2p, of 376 amino acid residues was found in the cytosol . Both Bat proteins show striking similarity to the mammalian protein Eca39, which is one of the few known targets of the myc oncogene . Deletion of a single BAT gene did not impair growth of yeast cells . In contrast, deletion of both genes resulted in an auxotrophy for branched-chain amino acids (Ile, Leu, and Val) and in a severe growth reduction on glucose-containing media, even after supply of these amino acids . Mitochondria and cytosol isolated from bat1 and bat2 deletion mutants, respectively, contained largely reduced activities for the conversion of branched-chain 2-ketoacids to their corresponding amino acids . Thus, the Bat proteins represent the first known isoforms of yeast branched-chain amino acid transaminases . The severe growth defect of the double deletion mutant observed even in the presence of branched-chain amino acids suggests that the Bat proteins, in addition to the supply of these amino acids, perform another important function in the cell. J Biol Chem, 1996 Oct 4, 271(40), 24442 - 8 Synchronized heat flux oscillations in yeast cell populations; Teusink B et al.; Microcalorimetry was adapted to the study of glycolytic oscillations in suspensions of intact yeast cells . A correction procedure was developed for the distortion of the amplitude and phase of the heat signal, caused by the slow response of the calorimeter . This made it possible to observe oscillations in the heat production rate with a period of less than 1 min, and a relative amplitude of 5-10% . By simultaneously measuring the heat flux and concentrations of glycolytic metabolites, and by comparing acetaldehyde-induced phase shifts of the heat flux oscillations with those of NADH oscillations, the heat flux was found to be 100 degrees out of phase with glucose 6-phosphate, 80 degrees out of phase with fructose 1, 6-bisphosphate, and in phase with NADH . The flux measurement made possible by microcalorimetry allowed the recognition of (i) changes in metabolic capacity that may affect glycolytic dynamics, (ii) implications of glucose carrier kinetics for glycolytic dynamics and (iii) the continued requirement for an acetaldehyde trapping agent for the oscillations. J Biol Chem, 1996 Oct 4, 271(40), 24425 - 32 Characterization of an upstream activation sequence and two Rox1p-responsive sites controlling the induction of the yeast HEM13 gene by oxygen and heme deficiency; Amillet JM et al.; The Saccharomyces cerevisiae HEM13 gene codes for coproporphyrinogen oxidase, an oxygen-requiring enzyme catalyzing the sixth step of heme biosynthesis . Its transcription has been shown to be induced 40-50-fold in response to oxygen or heme deficiency, in part through relief of repression exerted by Rox1p and in part by activation mediated by an upstream activation sequence (UAS) . This report describes an analysis of HEM13 UAS and of the Rox1p-responsive sites by electrophoretic mobility shift assays, DNase I footprinting, and mutational mapping . HEM13 UAS is composed of two subelements: a 16-base pair sequence binding a constitutive factor acting as a transcriptional activator, and a 5'-flanking 20-base pair GC-rich region . Both subelements were required additively for transcription, but each element alone was sufficient for almost normal control by oxygen/heme deficiency . Mutations in both elements decreased the induction ratio 3-4-fold . HEM13 UAS conferred a 2-4-fold oxygen/heme control on a heterologous reporter gene . Two Rox1p-responsive sites, R1 and R3, were identified, which accounted for the 6-7-fold repression by Rox1p . A factor bound to a sequence close to site R3 . This DNA-binding activity was only detected in protein extracts of aerobic heme-sufficient ROX1 TUP1 cells, suggesting a possible role in site R3 function. J Biol Chem, 1996 Oct 4, 271(40), 24337 - 40 The human homologue of the yeast CHL1 gene is a novel keratinocyte growth factor-regulated gene; Frank S et al.; Keratinocyte growth factor (KGF) is a potent and specific mitogen for different types of epithelial cells, including keratinocytes of the skin . To gain insight into the mechanisms of KGF action in this tissue, we attempted to identify genes that are regulated by KGF in keratinocytes . Using the differential display reverse transcription polymerase chain reaction technology, a gene was identified which was strongly induced in these cells by treatment with KGF but not with serum growth factors or pro-inflammatory cytokines . This gene seems to be part of a multigene family as assessed by Southern blot analysis . Molecular cloning and sequencing of the full-length cDNA revealed a strong homology with the yeast CHL1 gene . The latter encodes a putative helicase, which is involved in correct chromosome transmission and cell cycle progression . Furthermore, the CHL1 gene product and the protein encoded by the novel KGF-regulated gene were identical in size, indicating that we had cloned the human CHL1 homologue . This finding suggests a novel and specific role of KGF in correct chromosome segregation and/or cell cycle progression. Gene, 1996 Oct 3, 174(2), 289 - 92 A gene encoding a yeast equivalent of mammalian NADPH-adrenodoxin oxidoreductases; Lacour T et al.; Adrenodoxin oxidoreductase (ADR) and adrenodoxin (ADX) are the two proteins involved in electron transport to mammalian mitochondrial P-450s capable of steroid modifications . The cloning and sequencing of a S . cervisiae ADR homologue (YADR) is presented here . The YADR protein sequence shares 36 and 37% of identical amino acids with human and bovine ADR respectively . The physiological role of this ADR homologue in yeast is unknown . We intend to study the interaction of this YADR with bovine ADX in vitro and in vivo. Gene, 1996 Oct 3, 174(2), 225 - 33 Cloning and characterization of two yeast genes encoding members of the CCCH class of zinc finger proteins: zinc finger-mediated impairment of cell growth; Thompson MJ et al.; Members of the CCCH zinc finger (Zf) protein family have in common two or more repeats of a novel Zf motif consisting of Cys and His residues in the form Cx8Cx5Cx3H {where x is a variable amino acid (aa)} . We used a degenerate polymerase chain reaction (PCR) strategy to clone members of this gene family from Saccharomyces cerevisiae . The deduced aa sequences encoded by these genes, designated CTH1 and CTH2, share 46% overall identity and 59% similarity, largely due to the two highly conserved Zf domains . We found readily detectable expression of a 1.4-kb mRNA encoding Cth1p . The 1.1-kb mRNA encoding Cth2p was barely detectable under normal growth conditions; however, disruption of CTH1 resulted in at least a threefold increase in CTH2 mRNA accumulation . No change in phenotype was detected following disruption of CTH1 and CTH2, either singly or together . In contrast, overexpression of the CTH genes or one of the related mammalian genes, tris-tetraprolin (TTP), caused delayed entry of cell cultures into exponential growth, and a decrease in final cell density . Removal of the Zf domain of Cth1p by truncation or deletion completely reversed this slow growth phenotype, indicating that it was mediated through this highly conserved structural motif. Biochem Biophys Res Commun, 1996 Oct 3, 227(1), 140 - 6 The inhibition of estrogen receptor-mediated responses by chloro-S-triazine-derived compounds is dependent on estradiol concentration in yeast; Tran DQ et al.; The chloro-S-triazine derived compounds atrazine, atrazine desisopropyl, cyanazine, and simazine are commonly used herbicides . These compounds do not have estrogenic activity in yeast expressing human estrogen receptor (hER) and an estrogen-sensitive reporter . In the presence of a concentration of estradiol (20 nM) that induced maximal reporter activity in yeast, the triazines did not inhibit reporter activity . However, the triazines decreased reporter activity in a dose dependent manner in the presence of a submaximal concentration of estradiol (0.5 nM) . The estradiol-dependent activity of a mutant hER lacking the amino terminus was not inhibited by the triazines in yeast . Competition binding assays demonstrated that the triazines displaced radiolabeled estradiol from recombinant hER . These results suggest that the ability of the triazines to inhibit estrogen receptor-mediated responses in yeast occur through their interaction with hER and is dependent on the concentration of estradiol. Zhonghua Yi Xue Za Zhi, 1996 Oct, 76(10), 747 - 9 {Yeast artificial chromosome cloning and physical mapping of retinitis pigmentosa 3 (RP3) locus}; Miao W et al.; OBJECTIVE: To clone retinitis pigmentosa region by yeast artificial chromosome (YAC) and establish the restriction enzyme physical map . METHODS: The ornithine transcarbamoylase (OTC) cDNA probe, which is closely linked to the RP3 locus, was chosen to screen the X chromosome YAC library by colony in situ hybridization . Size determination, sequence taged site (STS) analysis and long range physical mapping were performed with positive YACs . The results obtained were used to map these YACs . RESULTS: We obtained a 1.6 Mb YAC contig containing information on RP3 range, restriction enzyme sites, CpG islands location and YAC position . CONCLUSION: The work provides a good basis for identification and cloning of the RP3 gene. J Enzyme Inhib, 1996 Oct, 11(2), 141 - 9 Inactivation of yeast glutathione reductase by O-phthalaldehyde; Pandey A et al.; Yeast glutathione reductase was inactivated by the bifunctional reagent, o-phthalaldehyde . The initial rate of inactivation followed pseudo-first order kinetics . Fluorescence spectral properties of modified enzyme indicated the formation of an isoindole derivative from cysteine and lyaine residues present in close proximity as shown by typical fluorescence emission and excitation maximum at 410 nm and 337 nm, respectively . The fluorescence spectral studies with o-phthalaldehyde in the presence and absence of N-ethylmaleimide indicated that both the inhibitors react with the same cysteine residue, which is non-essential for enzyme activity . The coenzyme NADPH did not protect the enzyme against the o-phthalaldehyde reaction while oxidised glutathione prevented o-phthalaldehyde inactivation . This could be due to reaction of the amino group of glutathione with o-phthalaldehyde . Stoichiometry of the reaction showed that the formation of approximately 2 isoindole derivatives per subunit of glutathione reductase is accompanied by 75% loss of activity . The results suggest that o-phthalaldehyde binds to non-essential cysteine and lysine residues present in close proximity which results in conformational changes leading to enzyme inactivation. Cell Struct Funct, 1996 Oct, 21(5), 395 - 402 Signaling toward yeast 1,3-beta-glucan synthesis; Inoue SB et al.; 1,3-beta-glucan synthase catalyzes the synthesis of a 1,3-beta-linked glucan polymer which produces the main rigidity of the yeast cell wall . Recent success in purification of this enzyme by product entrapment (21) has provided new insights into the dynamic aspects of the cell wall . This relatively simple procedure made it possible to identify the genes encoding the catalytic subunits of glucan synthase . In addition, the involvement of a rho type GTPase in the regulation of glucan synthase was demonstrated with the purified enzyme . Based on intracellular localization of the glucan synthase subunits, we have proposed a model in which assembly of the subunits is important for the activation of glucan synthase at sites of polarized growth . In this article, we will focus on biochemistry of 1,3-beta-glucan synthase and signaling through rho type GTPase. Appl Biochem Biotechnol, 1996 Oct-Nov, 61(1-2), 67 - 74 Thermostability of yeast hexokinase and yeast glucose-6-phosphate dehydrogenase; Zaitzeva EA et al.; Kinetic study of the mechanism of the temperature-induced loss of the catalytic activity by yeast hexokinase (HK) and yeast glucose-6-phosphate dehydrogenase (G-6-PDG) has shown the dissociative nature of the processes . In the temperature range 40-47 degrees C, they are satisfactorily described in terms of consecutive reactions in which steps of irreversible denaturation of the monomeric units follow the reversible dissociation of inactive oligomeric forms into the active units, resulting in an increase in catalytic activity . The experimental data have been analyzed in the framework of the dissociative mechanism, and a semiquantitative method has been developed for calculating the individual rate constants. Indian J Physiol Pharmacol, 1996 Oct, 40(4), 367 - 71 Central modulation of Brewer's yeast-induced peripheral inflammation by neuropeptides bradykinin and substance P; Hore SK et al.; Possible modulation of the Brewer's yeast-induced peripheral inflammation by two central neuropeptides, bradykinin and substance P (SP), was investigated in rats . Centrally administered bradykinin significantly increased pedal oedema and pain threshold whereas, SP produced significant augmentation of oedema volume and nociception . The results of the present study indicate that central bradykinin exerts pro-inflammatory and analgesic effects whereas, central SP exerts pro-inflammatory and pro-nociceptive effects on Brewer's yeast-induced peripheral inflammation. Mol Biotechnol, 1996 Oct, 6(2), 99 - 104 A simple assay for optimizing yeast-mammalian cell fusion conditions; Markie D; Polyethylene glycol (PEG)-induced cell fusion can be a useful method for the transfer of yeast artificial chromosomes (YACs) from yeast spheroplasts to mammalian cells in culture, although success varies between recipient cell types . Experiments aimed at determining optimum fusion conditions can also be very time-consuming . To minimize this difficulty, a reporter plasmid has been constructed that allows yeast-mammalian cell fusion rates to be determined within 3 d . The speed and sensitivity of the assay should allow a more systematic evaluation of cell lines for their capacity to fuse with yeast, and for rapid optimization of fusion parameters. J Biochem (Tokyo), 1996 Oct, 120(4), 759 - 65 Characterization of rat monoamine oxidase A with noncovalently-bound FAD expressed in yeast cells; Hiro I et al.; The FAD-binding cysteine of rat liver monoamine oxidase A (MAO A), Cys406, was converted to an alanine by site-directed mutagenesis of the cDNA . The wild-type and mutated enzymes were expressed in yeast cells and catalytic activities were assayed, using as substrates serotonin, tyramine, and kynuramine . Specific activities of the Ala-mutant for these substrates, calculated as the activities per pargyline-sensitive molecule, were about half of those of the wild-type enzyme . The Km values of the mutant enzyme for the substrates were similar to those of the wild-type enzyme . An adduct between FAD and pargyline, a mechanism-based inhibitor, was attached to the apoprotein in the wild-type enzyme, while in the Ala-mutant it was detached from the apoprotein, thereby indicating the presence of noncovalently bound FAD in the mutant enzyme . The Ala-mutant rapidly lost activity during incubation, whereas the wild-type enzyme retained the initial activity . Partial protection from inactivation occurred in the presence of FAD, but not of FMN . Recovery of the enzyme activity was nil when FAD was added after the inactivation . Thus, while the covalent attachment of FAD in MAO A is not required for the catalytic activity, it may function as a structural core for the active conformation in the membrane. Yeast, 1996 Oct, 12(13), 1351 - 7 Intergenic Flip Flop, a method for systematic gene disruption and cloning in yeast; Mallet L et al.; We have developed a strategy named Intergenic Flip Flop which, for each gene, allows us to produce in one experiment both a disrupting cassette and a plasmid for gap repair . The same method can also be used to insert a reporter gene downstream from the promoter . This approach extends the polymerase chain reaction (PCR)-based strategy proposed by Maftahi et al . 1996 . Our method consists of PCR amplification of the two flanking intergenic regions of the open reading frame (ORF) of interest, using two sets of oligonucleotides . Each PCR product is flanked by two short defined nucleotidic sequences with a unique restriction site, allowing subsequent hybridization between them . The association of the two amplimers by the complementary sequences either in the same orientation as in genomic DNA or in the opposite orientation, allows the generation, after PCR, of two distinct cassettes which can be cloned into suitable vectors . When the amplimer in the head-to-tail orientation is cloned in a vector containing a selective marker for yeast such as G418 resistance, it provides a disrupting cassette after cleavage at the unique restriction site introduced by the PCR between the two intergenic amplimers . The amplimer with a direct orientation cloned into a yeast vector, after cleavage at the unique restriction site between the intergenic regions, permits cloning by gap repair of the gene of interest in yeast . Finally, a reporter gene can be inserted in the same plasmid . We report here the successful application of this strategy to an ORF of chromosome XIV of Saccharomyces cerevisiae: N1216. Yeast, 1996 Oct, 12(13), 1339 - 50 Mutations in an Abf1p binding site in the promoter of yeast RPO26 shift the transcription start sites and reduce the level of RPO26 mRNA; Nouraini S et al.; A binding site for the transcription factor Abf1p was identified as an important promoter element of the gene that encodes Rpo26, a subunit common to all three yeast nuclear RNA polymerases (RNAP) . Mutations in the Abf1p binding site were identified among a pool of rpo26 mutant alleles that confer synthetic lethality in combination with a temperature-sensitive mutation (rpo21-4) in the gene that encodes the largest subunit of RNAPII (Rpo21p) . In the presence of the wild-type allele of RPO21 these rpo26 promoter mutations confer a cold-sensitive growth defect . Electrophoretic mobility-shift assays using purified Abf1p demonstrated that Abf1p binds to the RPO26 promoter and that the promoter mutations abolish this binding in vitro . Quantitation of the amount of RPO26 mRNA showed that mutations in the Abf1p binding site reduce the expression of RPO26 by approximately 60% . Mutations that affect Abf1p binding also result in a shift of the RPO26 transcriptional start sites to positions further upstream than normal . These results suggest that binding of the Abf1p transcription factor to the RPO26 promoter is important not only in establishing the level of transcription for this gene, but also in positioning the initiation sites of transcription. J Cell Sci, 1996 Oct, 109 ( Pt 10), 2471 - 81 Isolation and characterization of SYS genes from yeast, multicopy suppressors of the functional loss of the transport GTPase Ypt6p; Tsukada M et al.; In Saccharomyces cerevisiae, the YPT6 gene encodes the homologue of the mammalian Rab6 protein found in the Golgi apparatus . Deletion of YPT6 in yeast produces a phenotype showing temperature-sensitive growth and partial missorting of the vacuolar enzyme, carboxypeptidase Y . To identify proteins that might: (1) interact with Ypt6p; or (2) act in the same pathway, we have isolated four multicopy suppressors, named SYS1, SYS2, SYS3 and SYS5, that can complement the temperature-sensitive growth phenotype of the ypt6 null mutant . On high expression, these genes are also able to partially suppress the missorting of carboxypeptidase Y.SYS2 on a multicopy plasmid suppresses in addition the temperature-sensitive phenotype of sec7-1, a mutant defective in transport between and from the Golgi compartment . Gene disruption of SYS1 and SYS2 did not result in significant growth defects . However, deletion of SYS1 and/or SYS2 in the ypt6 null mutant enhances defects in vacuolar protein sorting and in cell growth . Whereas protein secretion was not significantly affected in these mutants, the processing of alpha-factor precursor by the Kex2 protease was inhibited, suggesting a function of YPT6 and its null mutant suppressors in transport between the late Golgi and a prevacuolar, endosome-like compartment. Biol Chem, 1996 Oct, 377(10), 669 - 73 BZLF1 (ZEBRA, Zta) protein of Epstein-Barr virus selected in a yeast one-hybrid system by binding to a consensus site in the IgH intronic enhancer: a role in immunoglobulin expression? Gstaiger M, Hovens C, Georgiev O, Knoepfel L, Schaffner W. We have used a yeast one hybrid screen to search for factors interacting with a subsegment of the immunoglobulin heavy chain (IgH) intronic enhancer . The 51 bp enhancer segment harbored a so-called E-box and an octamer site, known to bind helix-loop-helix transcription factors and Oct factors, respectively . Mammalian Oct-2A protein was also expressed in yeast, to select for transcription factors possibly cooperating with Oct-2 . Six strongly interacting protein clones were selected from a peripheral blood lymphocyte library . These included a B cell-specific co-activator, termed Bob1, that directly binds to Oct-2 (Gstaiger et al., 1995, Nature 373, 360-362) . Three further clones represent the helix-loop factors ITF-1 and ITF-2, another one the nucleolar protein nucleophosmin, or B23 . Unexpectedly, the sixth clone with strong activity encoded the BZLF1 (= ZLF1, zta, ZEBRA, EB1) protein of Epstein-Barr virus (EBV) . BZLF1 is a leucine zipper-related transcription factor and induces the switch from viral latency to lytic growth . We found that BZLF1 also activated transcription in transiently transfected mammalian cells via a consensus binding site located within the IgH intron enhancer . BZLF1 may thus influence immunoglobulin heavy chain expression in EBV-infected B lymphocytes. Genomics, 1996 Oct 1, 37(1), 87 - 95 Construction of high-resolution physical maps from yeast artificial chromosomes using restriction landmark genomic scanning (RLGS); Hirotsune S et al.; We have established a new system for chromosome-specific yeast artificial chromosome (YAC) contig construction using restriction landmark genomic scanning (RLGS-based YAC contig mapper) . RLGS is a powerful tool for detecting more than 1000 restriction landmarks distributed on an entire genome in one procedure . In this system, RLGS is applied to sorted chromosomes to cover the target chromosome . Using these landmarks as guideposts, chromosome-specific YAC clones are then ordered . In this paper, we report the construction of a map for a human chromosome 21 YAC contig spanning q22.1 using this new approach . Applying RLGS to sorted chromosomes 21 enables detection of approximately 1400 spots (equivalent of 1050 PacI landmarks), covering the entire region of this chromosome . We constructed the 2.5-Mb YAC contig encompassing 21q22.1 with 66 spots (equivalent of 50 PacI landmarks) . With this contig map, we could detect two deleted regions and chimerism in the YAC insert DNA . Our results demonstrated the usefulness of this approach for finding DNA alterations of YACs, such as deletions and chimerism. Trends Genet, 1996 Oct, 12(10), 405 - 12 At the heart of the budding yeast cell cycle; Nasmyth K; It might now seem obvious that the mechanisms regulating cell division would be found to be a highly conserved feature of eukaryotic cells . This was less clear 20 years ago when the pioneering genetic studies of the cell cycle were initiated . This article presents one view as to what lies at the heart of the budding yeast cell cycle . It is written on the premise that most of the key players, such as cyclin-dependent kinases, the anaphase-promoting complex, the origin recognition complex, Cdc6p and Mcm proteins, were performing similar functions in the common ancestor of yeast and man . Ideas about the budding yeast cell cycle might, therefore, have universal significance for other eukaryotic cells. Genome Res, 1996 Oct, 6(10), 935 - 42 An ordered yeast artificial chromosome library covering over half of rice chromosome 6; Umehara Y et al.; Yeast artificial chromosome (YAC) clones carrying DNA marker sequences located on the rice genetic map of chromosome 6 were ordered for physical mapping . A total of 122 restriction fragment length polymorphism markers, 16 sequence-tagged site markers, and five random amplified polymorphic DNA markers located, on average, at 0.9-cM intervals, were used for YAC clone screening by colony/Southern hybridization and PCR screening, respectively . A total of 216 individual YACs were selected from our YAC library of 7000 clones covering six genome equivalents . Each DNA marker could select, on average, 4.8 YAC clones, with 11 clones being the maximum . The YACs localized to the corresponding linkage map positions form 43 contigs and encompass about 60% of rice chromosome 6 . This is the first step in constructing a physical map covering the whole rice genome by chromosome landing with YAC clones . These YACs and data will be used soon to isolate phenotypical trait genes by map-based cloning. Mol Biol Cell, 1996 Oct, 7(10), 1521 - 33 Amino- and carboxy-terminal domains of the yeast Rab escort protein are both required for binding of Ypt small G proteins; Bauer BE et al.; The Rab escort protein (REP) is an essential component of the heterotrimeric enzyme Rab geranylgeranyl transferase that modifies the carboxy-terminal cysteines of the Ras-like small G proteins belonging to the Rab/Ypt family . Deletions in the human CHM locus, encoding one of the two REPs known in humans, result in a retinal degenerative syndrome called choroideremia . The only known yeast homologue of the choroideremia gene product is encoded by an essential gene called MRS6 . Besides three structurally conserved regions (SCRs) previously detected in the amino-terminal half of REPs and RabGDIs, three other regions in the carboxy-terminal domain (RCR 1-3) are here identified as being characteristic of REPs alone . We have performed the first mutational analysis of a REP protein to experimentally define the regions functionally important for Rab/Ypt protein binding, making use of the genetic system of the yeast Saccharomyces cerevisiae . This analysis has shown that the SCRs are necessary but not sufficient for Ypt1p binding by the yeast REP, the carboxy-terminal region also being required. EMBO J, 1996 Oct 1, 15(19), 5437 - 48 The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases; Simos G et al.; Arc1p was found in a screen for components that interact genetically with Los1p, a nuclear pore-associated yeast protein involved in tRNA biogenesis . Arc1p is associated with two proteins which were identified as methionyl-tRNA and glutamyl-tRNA synthetase (MetRS and GluRS) by a new mass spectrometry method . ARC1 gene disruption leads to slow growth and reduced MetRS activity, and synthetically lethal arc1- mutants are complemented by the genes for MetRS and GluRS . Recombinant Arc1p binds in vitro to purified monomeric yeast MetRS, but not to an N-terminal truncated form, and strongly increases its apparent affinity for tRNAMet . Furthermore, Arc1p, which is allelic to the quadruplex nucleic acid binding protein G4p1, exhibits specific binding to tRNA as determined by gel retardation and UV-cross-linking . Arc1p is, therefore, a yeast protein with dual specificity: it associates with tRNA and aminoacyl-tRNA synthetases . This functional interaction may be required for efficient aminoacylation in vivo. Plant J, 1996 Oct, 10(4), 625 - 36 Functional analysis of yeast-derived phytochrome A and B phycocyanobilin adducts; Kunkel T et al.; Investigations of phytochrome mutants of Arabidopsis suggested that the expression of chalcone synthase (chs) and anthocyanin accumulation is predominantly controlled by phytochrome A . To test the functionality of phytochrome A and B at the molecular level recombinant, yeast-derived phytochrome-phycocyanobilin adducts (phyA, phyB) and oat phytochrome A (phyA) were microinjected into etiolated aurea tomato seedlings . Subsequent to microinjection anthocyanin and chlorophyll accumulation was monitored as well as beta-glucuronidase (GUS) expression mediated by light-regulated promoters (chs, chlorophyll a/b binding protein (lhcb1) and ferredoxin NADP+ oxidoreductase (fnn) . Microinjection of phyA under white light conditions caused anthocyanin and chlorophyll accumulation and mediated chs-GUS, lhcb 1-GUS and fnr-GUS expression . Microinjection of phyB under identical conditions induced chlorophyll accumulation and mediated lhcb 1-GUS and fnr-GUS expression but neither anthocyanin accumulation nor chs-GUS expression were observed . The characterization of Arabidopsis phytochrome mutants and the microinjection experiments suggested that phyB cannot induce the accumulation of juvenile anthocyanin . Microinjections under far-red light conditions demonstrated that phyA can act independently of other photoreceptors . By contrast, phyB injections under red light conditions indicated that phyB needs interactions with other photoreceptors to mediate a rapid and efficient de-etiolation signal. Genetics, 1996 Oct, 144(2), 479 - 93 In vivo analysis reveals that the interdomain region of the yeast proliferating cell nuclear antigen is important for DNA replication and DNA repair; Amin NS et al.; To identify the regions of the proliferating cell nuclear antigen (PCNA) that are important for function in vivo, we used random mutagenesis to isolate 10 cold-sensitive (Cs-) and 31 methyl methanesulfonate-sensitive (Mmss) mutations of the PCNA gene (POL30) in Saccharomyces cerevisiae . Unlike the Mmss mutations, the Cs- mutations are strikingly clustered in the interdomain region of the three-dimensional PCNA monomer structure . At the restrictive temperature, the Cs- pol30 mutants undergo a RAD9-dependent arrest as large-budded cells with a 2c DNA content . Defects in DNA synthesis are suggested by a significant delay in the progression of synchronized pol30 cells through S phase at the restrictive temperature . DNA repair defects are revealed by the observation that Cs- pol30 mutants are very sensitive to the alkylating agent MMS and mildly sensitive to ultraviolet radiation, although they are not sensitive to gamma radiation . Finally, analysis of the chromosomal DNA in pol30 cells by velocity sedimentation gradients shows that pol30 cells accumulate single-stranded DNA breaks at the restrictive temperature . Thus, our results show that PCNA plays an essential role in both DNA replication and DNA repair in vivo. Genetics, 1996 Oct, 144(2), 445 - 58 Vacuole partitioning during meiotic division in yeast; Roeder AD et al.; We have examined the partitioning of the yeast vacuole during meiotic division . In pulse-chase experiments, vacuoles labeled with the lumenal ade2 fluorophore or the membrane-specific dye FM 4-64 were not inherited by haploid spores . Instead, these fluorescent markers were excluded from spores and trapped between the spore cell walls and the ascus . Serial optical sections using a confocal microscope confirmed that spores did not inherit detectable amounts of fluorescently labeled vacuoles . Moreover, indirect immunofluorescence studies established that an endogenous vacuolar membrane protein, alkaline phosphatase, and a soluable vacuolar protease, carboxypeptidase Y . were also detected outside spores after meiotic division . Spores that did not inherit ade2- or FM 4-64-labeled vacuoles did generate an organelle that could be visualized by subsequent staining with vacuole-specific fluorophores . These data contrast with genetic evidence that a soluble vacuolar protease is inherited by spores . When the partitioning of both types of markers was examined in sporulating cultures, the vacuolar protease activity was inherited by spores while fluorescently labeled vacuoles were largely excluded from spores . Our results indicate that the majority of the diploid vacuole, both soluble contents and membrane-bound components, are excluded from spores formed during meiotic division. Biophys J, 1996 Oct, 71(4), 2192 - 200 Dielectric behavior of wild-type yeast and vacuole-deficient mutant over a frequency range of 10 kHz to 10 GHz; Asami K et al.; Dielectric behavior of Saccharomyces cerevisiae wild-type and vacuole-deficient mutant cells has been studied over a frequency range of 10 kHz to 10 GHz . Both types of cells harvested at the early stationary growth phase showed dielectric dispersion that was phenomenologically formulated by a sum of three separate dispersion terms: beta 1-dispersion (main dispersion) and beta 2-dispersion (additional dispersion) and gamma-dispersion due to orientation of water molecules . The beta 1-dispersion centered at a few MHz, which has been extensively studied so far, is due to interfacial polarization (or the Maxwell-Wagner effect) related to the plasma membrane . The beta 2-dispersion for the vacuole-deficient mutant centered at approximately 50 MHz was explained by taking the cell wall into account, whereas, for the wild-type cells, the beta 2-dispersion around a few tens MHz involved the contributions from the vacuole and cell wall. Nucleic Acids Res, 1996 Oct 1, 24(19), 3685 - 92 Functional analysis of the DNA-stimulated ATPase domain of yeast SWI2/SNF2; Richmond E et al.; The yeast SWI2/SNF2 polypeptide is a subunit of the SWI/SNF protein complex that is required for many transcriptional activators to function in a chromatin context . SWI2 is believed to be the founding member of a new subfamily of DNA-stimulated ATPases/DNA helicases that includes proteins that function in DNA repair (RAD5, RAD16, ERCC6), recombination (RAD54), transcription (MOT1, ISWI, brm, BRG1, hBRM) and cell cycle control (STH1) . We have created a set of 16 mutations within the SWI2 ATPase domain and have analyzed the functional consequences of these mutations in vivo . We have identified residues within each of the seven ATPase motifs that are required for SWI2 function . We have also identified crucial residues that are interspersed between the known ATPase motifs . In contrast, we identify other highly conserved residues that appear to be dispensable for SWI2 function . We also find that single amino acid changes in ATPase motifs IV and VI lead to a dominant negative phenotype . None of the 12 SWI2 mutations that disrupt SWI2 activity in vivo alter the assembly of the SWI/SNF complex . These studies provide an invaluable framework for biochemical analysis of the SWI2 ATPase and for functional analysis of other SWI2 family members. Mol Pharmacol, 1996 Oct, 50(4), 829 - 37 Pharmacological characterization of the rat A2a adenosine receptor functionally coupled to the yeast pheromone response pathway; Price LA et al.; The rat A2a adenosine receptor, a G protein-coupled receptor, was functionally expressed in the yeast Saccharomyces cerevisiae . High affinity binding sites for A2a adenosine agonists were detected in yeast membranes containing the endogenous Grx protein Gpa1 . Agonist saturation binding isotherms using {3H}5'-N-ethylcarboxamidoadenosine indicated that the A2a adenosine receptor expressed in yeast cell membranes displays pharmacological properties equivalent to those observed when the receptor is expressed in human embryonic kidney 293 cell membranes . The rank order of potency of various agonists in {3H}5'-N-ethylcarboxamidoadenosine competition binding assays performed with yeast cell membranes was comparable to that seen for the receptor expressed in mammalian cell membranes . Adenosine agonist-dependent growth response of yeast strains expressing the A2a adenosine receptor was elicited via activation of the yeast pheromone-response pathway . Induction of a pheromone-responsive FUS1-HIS3 reporter gene in far1 his3 cells permits cell growth in medium lacking histidine . The sensitivity of the bioassay was increased by deletion of the STE2 gene, which encodes the yeast alpha-mating pheromone receptor . The growth response was dose dependent, and agonists of varying affinities displayed a rank order of potency comparable to that observed in competition binding assays . Agonist-activated growth assays performed in liquid culture gave ED50 values for various adenosine agonists consistent with reported Kd alpha values . Yeast strains expressing a single receptor/G protein complex will be useful as a model system for the study of receptor/G protein interactions in vivo. Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10928 - 32 Mammalian orthologues of a yeast regulator of nonsense transcript stability; Perlick HA et al.; All eukaryotes that have been studied to date possess the ability to detect and degrade transcripts that contain a premature signal for the termination of translation . This process of nonsense-mediated RNA decay has been most comprehensively studied in the yeast Saccharomyces cerevisiae where at least three trans-acting factors (Upf1p through Upf3P) are required . We have cloned cDNAs encoding human and murine homologues of Upf1p, termed rent1 (regulator of nonsense transcripts) . Rent1 is the first identified mammalian protein that contains all of the putative functional elements in Upf1p including zinc finger-like and NTPase domains, as well as all motifs common to members of helicase superfamily I . Moreover, expression of a chimeric protein, N and C termini of Upf1p, complements the Upf1p-deficient phenotype in yeast . Thus, despite apparent differences between yeast and mammalian nonsense-mediated RNA decay, these data suggest that the two pathways use functionally related machinery. Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10729 - 34 DNA strand annealing is promoted by the yeast Rad52 protein; Mortensen UH et al.; The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination . Here we demonstrate that yeast Rad52 is a DNA binding protein . To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed . The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA . Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein . More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA . Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction . How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed. Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 10718 - 22 Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome; Habraken Y et al.; Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors . Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors . Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA . Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site . It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome . Here we examine whether Rad2 is part of a higher order subassembly . Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2 . By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant < 3.3 x 10(-9) M . These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3 . Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision . Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome. Chromosoma, 1996 Oct, 105(4), 250 - 60 Inactivation of topoisomerase I or II may lead to recombination or to aberrant replication termination on both SV40 and yeast 2 micron DNA; Levac P et al.; Topoisomerase I is believed to be sufficient for early replication of circular viral genomes such as those of SV40 and of yeast plasmids . Topoisomerase II is required for the decatenation of the daughter genomes and probably also for fork elongation during the later stages of SV40 replication . Using the neutral-neutral two-dimensional gel system, we have followed the progression of replication of both SV40 and the yeast 2 micron plasmid under various conditions of topoisomerase inhibition . During SV40 replication, inhibition of topoisomerase II by VP16, VM26 or hypertonic shock (but not by merbarone), and inhibition of topoisomerase I by camptothecin all led to the accumulation of aberrant DNA structures containing two almost completely replicated genomes . These aberrant structures resembled either recombination intermediates or late Cairns structures in which the site of replication termination had shifted and now mapped to a continuum of sites throughout the genome . Replication of the 2 micron plasmid in a topoisomerase II- but not a topoisomerase I-deficient yeast gave rise to very similar structures . The data suggest that inactivation of topoisomerase I or II either stimulates recombination or, by differentially affecting replication fork progression, leads to aberrant replication termination. RNA, 1996 Oct, 2(10), 1058 - 70 Characterization of yeast U1 snRNP A protein: identification of the N-terminal RNA binding domain (RBD) binding site and evidence that the C-terminal RBD functions in splicing; Tang J et al.; The yeast U1A protein is a U1 snRNP-specific protein . Like its human counterpart (hU1A), it has two conserved RNA binding domains (RBDs) . The N-terminal RBD is quite different from the human protein, and a binding site on yeast U1 snRNA is not readily apparent . The C-terminal RBD is of unknown function . Using in vivo dimethyl sulfate (DMS) protection of mutant strains, we defined a region in yeast U1 snRNA as the likely U1A N-terminal RBD binding site . This was confirmed by direct in vitro binding assays . The site is very different from its vertebrate counterpart, but its location within yeast U1 snRNA suggests a conserved structural relationship to other U1 snRNP components . Genetic studies and sensitive in vivo splicing measurements indicate that the yeast U1A C-terminal RBD also functions in pre-mRNA splicing . We propose that the N-terminal RBD serves to tether the splicing-relevant C-terminal RBD to the snRNP. RNA, 1996 Oct, 2(10), 995 - 1010 Site-specific crosslinks of yeast U6 snRNA to the pre-mRNA near the 5' splice site; Kim CH et al.; We have introduced a single photochemical crosslinking reagent into specific sites in the central domain of U6 to identify the sites that are in close proximity to the pre-mRNA substrate . Four distinct U6 snRNAs were synthesized with a single 4-thiouridine (4-thioU) at positions 46, 51, 54, and 57, respectively . Synthetic U6 RNA containing the 4-thioU modifications can functionally reconstitute splicing activity in cell-free yeast splicing extracts depleted of endogenous U6 snRNA . Upon photoactivation with UV (>300 nm), 4-thioU at position 46 forms crosslinks to pre-mRNA near the 5' splice site at nt +4, +5, +6, and +7 in the intron, whereas 4-thioU at position 51 crosslinks to the pre-mRNA at positions -2, -1, +1, +2, +3, and at the invariant G in the lariat intermediate . All crosslinks are dependent on the presence of ATP and the splicing substrate . The two crosslinks to the pre-mRNA from position 46 and 51 of U6 can also occur in prp2 heat-inactivated yeast splicing extracts blocked immediately prior to the first chemical step . Significantly, the crosslink from position 51 can undergo subsequent splicing when the mutant extract is complemented with functional Prp2 protein in a chase experiment, indicating that the crosslink reflects a functional interaction that is maintained during the first step . The crosslink to lariat intermediate appears when the mutant spliceosomes are complemented with functional Prp2 protein added exogenously . This experiment is a paradigm for future studies in which different mutant extracts are used to establish the stage in assembly at which particular RNA-RNA interactions defined by unique crosslinks occur. Biometals, 1996 Oct, 9(4), 345 - 9 Copper-release from yeast Cu(I)-thionein by hypothiocyanite (OSCN-); Hartmann HJ et al.; In the course of an oxidative burst oxygen free radicals and hypothiocyanite (OSCN-), a transiently abundant derivative of thiocyanate (SCN-), are formed in the presence of activated polymorphonuclear leukocytes (PMNs) . At the same time Cu(I)-thionein is present and the question arose whether or not thiocyanate and its oxidized form may transiently release highly Fenton active copper to improve the efficacy of the above mentioned oxidative burst . Thus, the reaction of yeast Cu-thionein with OSCN- was examined . Indeed, a release of copper from the Cu(I)-thiolate clusters of the protein was observed ex vivo . Both the chiroptic and luminescence emission signals of Cu-thionein essentially levelled off in the presence of a 15-fold molar excess of OSCN- expressed per equivalent of thionein-copper . The effective copper-releasing activity of this reagent was confirmed by equilibrium dialysis . The demetallized protein could be reconstituted under reductive conditions . SCN- did not affect the copper-thiolate bonding . It rather acts as a potent metabolic source for the transient copper release from Cu-thionein in the presence of activated PMNs. Appl Environ Microbiol, 1996 Oct, 62(10), 3614 - 9 Malt-yeast extract-sucrose agar, a suitable medium for enumeration and isolation of fungi from silage; Skaar I et al.; A general medium named malt-yeast extract-sucrose agar (MYSA) containing oxgall was designed . The medium was intended for the enumeration and isolation of molds and yeasts in routine examinations of animal feed stuffs . In this study MYSA was tested as a general medium for mycological examination of silage . The medium was compared with dichloran-rose bengal medium (DRBC) in an examination of more than 500 specimens of big bale grass silage . Selected characteristics of known fungal species commonly isolated from feeds were examined after growth on MYSA and DRBC and on malt extract agar, used as a noninhibitory control medium . MYSA suppressed bacterial growth, without affecting the growth of fungi common in feeds . The fungi growing on MYSA were easily recognized, and the medium seemed to slow radial growth of fungal colonies, which permitted, easy counting . The number of species found was higher on MYSA than on DRBC . When we compared MYSA with DRBC for mycological examination of grass silage samples, MYSA was found to be the medium of choice. Mol Cell Biol, 1996 Oct, 16(10), 5536 - 45 Two different repressors collaborate to restrict expression of the yeast glucose transporter genes HXT2 and HXT4 to low levels of glucose; Ozcan S et al.; Transcription of the yeast HXT2 and HXT4 genes, which encode glucose transporters, is induced only by low levels of glucose . This low-glucose-induced expression is mediated by two independent repression mechanisms: in the absence of glucose, transcription of both genes is prevented by Rgt1p, a C6 zinc cluster protein; at high levels of glucose, expression of HXT2 and HXT4 is repressed by Mig1p . Only at low glucose concentrations are both repressors inactive, leading to a 10- to 20-fold induction of gene expression . Mig1p and Rgt1p act directly on HXT2 and HXT4 by binding to their promoters . This transcriptional regulation is physiologically very important to the yeast cell because it causes these glucose transporters to be expressed only in low-glucose media, in which they are required for growth. Mol Cell Biol, 1996 Oct, 16(10), 5375 - 85 Prenylated isoforms of yeast casein kinase I, including the novel Yck3p, suppress the gcs1 blockage of cell proliferation from stationary phase; Wang X et al.; The GCS1 gene of the budding yeast Saccharomyces cerevisiae mediate the resumption of cell proliferation from the starved, stationary-phase state . Here we identify yeast genes that, in increased dosages, overcome the growth defect of gcs1 delta mutant cells . Among these are YCK1 (CK12) and YCK2 (CKI1), encoding membrane-associated casein kinase I, and YCK3, encoding a novel casein kinase I isoform . Some Yck3p gene product was found associated with the plasma membrane, like Yck1p and Yck2p, but most confractionated with the nucleus, like another yeast casein kinase I isoform, Hrr25p . Genetic studies showed that YCK3 and HRR25 constitute an essential gene family and that Yck3p can weakly substitute for Yck1p-Yck2p . For gcs1 delta suppression, both a protein kinase domain and a C-terminal prenylation motif were shown to be necessary . An impairment in endocytosis was found for gcs1 delta mutant cells, which was alleviated by an increased YCK2 gene dosage . The ability of an increased casein kinase I gene dosage to suppress the effects caused by the absence of Gcs1p suggests that Gcs1p and Yck1p-Yck2p affect parallel pathways. Yeast, 1996 Sep 30, 12(12), 1263 - 72 Reconstitution of lactate proton symport activity in plasma membrane vesicles from the yeast Candida utilis; Geros H et al.; Lactic acid transport was studied in plasma membrane vesicles from the yeast Candida utilis IGC 3092 which were fused with liposomes containing cytochrome c oxidase . After the addition of an electron donor system, these hybrid membrane vesicles were able to generate a proton-motive force of about--150 mV, inside alkaline and negative . In vesicles prepared from lactic acid-grown cells, the uptake of labelled lactic acid, at pH 6.2, under energized conditions, was expressed by a kinetics consistent with the involvement of a mediated transport system . This carrier exhibited a substrate specificity pattern identical to the one found for the lactate-proton symport in intact cells . The transport of labelled lactic acid was accumulative and strongly sensitive to the effects of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, consistent with the involvement of the proton-motive force in acid uptake, hence with the presence of a proton symport for lactate . Dissipation of the transmembrane electric potential by valinomycin did not have a significant effect on lactate accumulation, whereas abolishing the transmembrane pH gradient (delta pH) by nigericin prevented the accumulation and led to a rapid efflux of the accumulated acid . The data support that the delta pH is the main component of the proton-motive force involved in the transport of the acid and its accumulation . The lactate-proton symport stoichiometry was 1:1, being independent of the pH . Vesicles prepared from glucose-grown cells did not display the capacity to transport and accumulate lactate . However, activity for the carrier was also reconstituted in vesicles obtained from glucose-grown cells after incubation in buffer containing lactic acid . These results were consistent with those obtained in intact cells, which demonstrated that the lactate-proton symport of the yeast C . utilis is inducible. Yeast, 1996 Sep 30, 12(12), 1229 - 38 N-Glycosylation affects endoplasmic reticulum degradation of a mutated derivative of carboxypeptidase yscY in yeast; Knop M et al.; The endoplasmic reticulum (ER) of eukaryotic cells contains a quality control system, that is required for the proteolytic removal of aberrantly folded proteins that accumulate in this organelle . We used genetic and biochemical methods to analyse the involvement of N-glycosylation in the degradation of a mutant derivative of carboxypeptidase yscY in the ER of the yeast Saccharomyces cerevisiae . Our results demonstrate that N-glycosylation of this protein is required for its degradation since an unglycosylated species is retained stably in the ER . Cells that were devoid of the ER-processing alpha 1,2-mannosidase showed reduced degradation of the glycosylated substrate protein . Disruption of CNE1, a gene encoding a putative yeast homologue for calnexin, did not exhibit any effects on the degradation of this substrate protein in vivo . Also, the alpha 1,2-mannosidase-dependent reduction in the degradation rate did not show any correlation with the function of the CNE1 gene product . Our results suggest that the ER of yeast contains a glycosylation-dependent quality control system, as has been shown for higher eukaryotic cells. Yeast, 1996 Sep 30, 12(12), 1209 - 17 A set of genetic markers for the chromosomes of the imperfect yeast Arxula adeninivorans; Samsonova IA et al.; The nuclear genome of the anamorphic yeast Arxula adeninivorans was analysed by benomyl-induced haploidization of parasexual hybrids marked with 32 auxotrophic mutations and pulsed field gel electrophoresis followed by DNA hybridization . Twenty-seven genes have been arranged into four linkage groups by haploidization, 15 genes belong to group 1, six to group 2, and three each to groups 3 and 4 . Five genes could be localized by DNA hybridization on three out of four separated chromosomes . The gene LYS2 of the largest linkage group 1 and the 25S rDNA were identified on the largest chromosome, the GAA and the TEF1 gene on chromosome 2, and the ILV1 gene of linkage group 4 on the smallest chromosome. J Biol Chem, 1996 Sep 27, 271(39), 23657 - 60 Yeast-derived recombinant DG42 protein of Xenopus can synthesize hyaluronan in vitro; DeAngelis PL et al.; We demonstrate in this report that the Xenopus DG42 gene product made in the yeast Saccharomyces cerevisiae can synthesize authentic high molecular weight hyaluronan (hyaluronic acid; HA) in vitro . Saccharomyces are eukaryotes that do not naturally produce HA or any other molecules known to contain glucuronic acid . Therefore bakers' yeast is a good model system to determine the enzymatic activity of the DG42 protein, which is the topic of recent debate . Membrane extracts prepared from cells expressing DG42 encoded on a plasmid incorporated {14C}glucuronic acid and N-{3H}acetylglucosamine from exogenously supplied UDP-sugar nucleotides into a high molecular mass (10(6) to 10(7) Da) polymer in the presence of magnesium ions . Both sugar precursors were simultaneously required for elongation . Control extracts prepared from cells with the vector plasmid alone or the DG42 cDNA in the antisense orientation did not display this activity . The double-labeled polysaccharide product synthesized in vitro was deemed to be HA by enzymatic analyses; specific HA lyase could degrade the polymer, but it was unaffected by protease or chitinase treatments . The fragments generated by HA lyase were identical to fragments derived from authentic vertebrate HA as compared by high performance liquid chromatography . We conclude that DG42 is a membrane-associated HA synthase capable of transferring both glucuronic acid and N-acetylglucosamine groups. Mol Gen Genet, 1996 Sep 25, 252(4), 420 - 8 Yeast chitin synthases 1 and 2 consist of a non-homologous and dispensable N-terminal region and of a homologous moiety essential for function; Ford RA et al.; Predicted protein sequences of fungal chitin synthases can be divided into a non-homologous N-terminal region and a C-terminal region that shows significant homology among the various synthases . We have explored the function of these domains by constructing a series of nested deletions, extending from either end, in the CHS1 and CHS2 genes of Saccharomyces cerevisiae . In both cases, most or all of the sequences encoding the non-homologous N-terminal region (one-third of the protein for Chs1p and about one-fourth for Chs2p) could be excised, with little effect on the enzymatic activity in vitro of the corresponding synthase or on its function in vivo . However, further small deletions (20-25 amino acids) into the homologous region were deleterious to enzymatic activity and function, and often led to changes in the zymogenic character of the enzymes . Similarly, relatively small (about 75 amino acids) deletions from the C-terminus resulted in loss of enzymatic activity and function of both synthases . Thus, it appears that all the information necessary for membrane localization, enzymatic activity and function resides in the homologous regions of Chs1p and Chs2p, a situation that may also apply to other chitin synthases. J Biol Chem, 1996 Sep 20, 271(38), 23357 - 62 Yeast respond to hypotonic shock with a calcium pulse; Batiza AF et al.; We have used the transgenic AEQUORIN calcium reporter system to monitor the cytosolic calcium ({Ca2+}cyt) response of Saccharomyces cerevisiae to hypotonic shock . Such a shock generates an almost immediate and transient rise in {Ca2+}cyt which is eliminated by gadolinium, a blocker of stretch-activated channels . In addition, this transient rise in {Ca2+}cyt is initially insensitive to 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an extracellular calcium chelator . However, BAPTA abruptly attenuates the maintenance of that transient rise . These data show that hypotonic shock generates a stretch-activated channel-dependent calcium pulse in yeast . They also suggest that the immediate calcium influx is primarily generated from intracellular stores, and that a sustained increase in {Ca2+}cyt depends upon extracellular calcium. J Biol Chem, 1996 Sep 20, 271(38), 23061 - 7 Activated calcineurin confers high tolerance to ion stress and alters the budding pattern and cell morphology of yeast cells; Mendoza I et al.; The PP2B protein phosphatase, also known as calcineurin, is a regulator of ion homeostasis in yeast cells . We have investigated the physiological consequences of constitutive expression of a recombinant form of calcineurin in which the Ca2+/calmodulin-binding and autoinhibitory domains of the catalytic subunit were deleted . The concomitant expression of the regulatory subunit along with the truncated catalytic subunit resulted in high tolerance to toxic levels of Na+ and Li+ . This activated form of calcineurin substituted for the Na+ stress signal to promote the expression of the ENA1 gene, encoding a P-ATPase pump, and to induce the transition of the K+ uptake system to the high affinity mode that restricts influx of Na+ and Li+ . In addition, the transcriptional responsiveness of ENA1 to Na+ stress was enhanced . These results demonstrate that calcineurin has a pivotal role in a signaling cascade activated by ion stress in yeast . Moreover, we found that changes in the level of calcineurin activity affected budding pattern and cell morphology . Cells expressing the truncated calcineurin were elongated and budded in an unipolar pattern, whereas calcineurin-deficient mutants budded randomly . These results suggest that calcineurin may also act in the establishment of cell polarity. J Biol Chem, 1996 Sep 20, 271(38), 22999 - 3005 Alanine-scanning mutagenesis along membrane segment 4 of the yeast plasma membrane H+-ATPase . Effects on structure and function; Ambesi A et al.; Membrane segment 4 of P-type cation pumps has been suggested to play a critical role in the coupling of ATP hydrolysis to ion translocation . In this study, structure-function relationships in M4 of the yeast (Saccharomyces cerevisiae) plasma membrane H+-ATPase have been explored by alanine-scanning mutagenesis . Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles, as described previously (Nakamoto, R . K., Rao, R . , and Slayman, C . W . (1991) J . Biol . Chem . 266, 7940-7949) . One substitution (I329A) led to arrest of the enzyme at an early stage of biogenesis, and three others (G333A, L338A, G349A) reduced ATP hydrolysis to near-background levels . The remaining 26 mutants were expressed well enough in secretory vesicles (44-121% of wild type) and had sufficient ATPase activity (16-123% of wild type) to be characterized in detail . When acridine orange fluorescence quenching was used to measure rates of ATP-dependent proton pumping over a range of ATP concentrations, only minor changes were seen . In kinetic studies, however, seven of the mutant enzymes (I331A, I332A, V334A, V336A, V341A, V342A, and M346A) were resistant to vanadate inhibition, and three of them (I332A, V336A, and V341A) also had a decreased Km and increased pH optimum for ATP hydrolysis . Limited trypsinolysis was used to probe the structure of two different Val-336 substitutions, V336A, described above, and V336R, which displayed little or no ATPase activity . Both were cleaved at a relatively normal rate to give a pattern of fragments essentially identical to that seen with the wild-type enzyme . However, while vanadate, ADP, and ATP were able to protect the wild-type and V336A enzymes against trypsinolysis, the V336R ATPase was protected only by ADP and ATP . Taken together, the data suggest that key residues in the M4 segment may help to communicate the E1-E2 conformational change to ion-binding sites in the membrane. J Biol Chem, 1996 Sep 20, 271(38), 22983 - 9 Functional and physical interactions between partial molecules of STE6, a yeast ATP-binding cassette protein; Berkower C et al.; The Saccharomyces cerevisiae a-factor transporter, STE6, is a member of the ATP binding cassette (ABC) transporter superfamily . ABC proteins consist of four modular units that comprise two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs) . Like many ABC proteins, STE6 contains these four domains in a single polypeptide; certain other ABC proteins are encoded as pairs of "half-molecules" or are further subdivided . Our previous studies demonstrated that STE6 can be expressed as two half-molecules that are functional when co-expressed . Here we dissect the interactions between modules of STE6 in greater detail . We show by co-immunoprecipitation that STE6 half-molecules interact physically, supporting the view that they co-assemble in vivo to form a functional transporter . We also demonstrate a physical interaction between a STE6 half-molecule and full-length STE6; such complexes appear to be functional, based on the striking finding that the defective activity of full-length STE6 mutated in one of its NBDs can be corrected by co-expression of the corresponding "wild-type" half-molecule . We also show that a quarter-molecule consisting solely of the N-terminal MSD of STE6 can interact physically and functionally with a C-terminal three-quarter molecule of STE6, indicating that information directing the assembly of STE6 from partial molecules is contained, at least in part, within its membrane spans. Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10321 - 6 Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system; Vidal M et al.; Many biological processes rely upon protein-protein interactions . Hence, detailed analysis of these interactions is critical for their understanding . Due to the complexities involved, genetic approaches are often needed . In yeast and phage, genetic characterizations of protein complexes are possible . However, in multicellular organisms, such characterizations are limited by the lack of powerful selection systems . Herein we describe genetic selections that allow single amino acid changes that disrupt protein-protein interactions to be selected from large libraries of randomly generated mutant alleles . The strategy, based on a yeast reverse two-hybrid system, involves a first-step negative selection for mutations that affect interaction, followed by a second-step positive selection for a subset of these mutations that maintain expression of full-length protein (two-step selection) . We have selected such mutations in the transcription factor E2F1 that affect its ability to heterodimerize with DP1 . The mutations obtained identified a putative helix in the marked box, a region conserved among E2F family members, as an important determinant for interaction . This two-step selection procedure can be used to characterize any interaction domain that can be tested in the two-hybrid system. EMBO J, 1996 Sep 16, 15(18), 5001 - 13 Coordinated regulation of gene expression by the cell cycle transcription factor Swi4 and the protein kinase C MAP kinase pathway for yeast cell integrity; Igual JC et al.; Specific transcription in late G1, mediated by the transcription factors SBF (Swi4p-Swi6p) and MBF (Mbp1p-Swi6p), is crucial for cell cycle progression in budding yeast . In order to better understand the G1/S transition, we initiated a search for conditional mutations synthetic lethal with swi4delta . One of the isolated mutants, rsf8swi4delta, showed a growth defect due to cell lysis . rsf8 is allelic to PKC1, encoding a protein kinase C homologue which controls cell integrity . In the presence of the rsf8/(pkc1-8) mutation, a functional SBF but not MBF is required for viability . Importantly, swi4delta and swi6delta strains are hypersensitive to calcofluor white and SDS, indicating that they possess a weakened cell wall . Overexpression or ectopic expression of CLN did not suppress the pkc1-8swi4delta mutant phenotype, thus SBF must control cell integrity independently, rather than acting through CLN expression . We found that at least six genes involved in cell wall biosynthesis are periodically expressed at the G1/S phase boundary . In all six cases, cell cycle-regulated expression is due mainly to Swi4p . Finally, we found that the PKC1 MAP kinase pathway is a positive regulator of five of these cell wall genes, these genes being novel targets of regulation by this pathway . We suggest that SBF and the PKC1 MAP kinase pathway act in concert to maintain cell integrity during bud formation. Yeast, 1996 Sep 15, 12(11), 1163 - 78 A computer filtering method to drive out tiny genes from the yeast genome; Barry C et al.; The authors of the first yeast chromosome sequence defined a minimum threshold requirement of 100 codons, above which an open reading frame (ORF) is retained as a putative coding sequence . However, at least 58 yeast genes shorter than 100 codons have an assigned protein function . Therefore, the yeast genome may contain other tiny but functionally important genes that are discarded from analyses by this simple filtering rule . We have established discriminant functions from the in-phase hexamer frequencies of functional genes and of simulated ORFs derived from a stationary Markov chain model . Fifty-two out of the 58 genes were recognized as coding ORFs by our discriminating method . The test was also applied to all the small ORFs (36 to 100 codons) found in the intergenic regions of published chromosomes . It retained 140 new potential tiny coding sequences, among which we identified seven new genes by similarity searches . Our method, used conjointly with similarity searches, can also highlight sequencing errors resulting from the disruption of the coding frame of longer ORFs . This method, by its ability to detect potential coding ORFs, can be a very useful tool for functional analysis. Eur J Biochem, 1996 Sep 15, 240(3), 798 - 806 Transbilayer phosphatidylethanolamine movements in the yeast plasma membrane . Evidence for a protein-mediated, energy-dependent mechanism; Balasubramanian K et al.; Aminophospholipid movements in the plasma membrane of higher eukaryotic cells seem to be regulated by an ATP-dependent, protein-mediated process . To examine whether similar mechanisms exist in yeast cells, we have analysed phosphatidylethanolamine (PtdEtn) distributions in Saccharomyces cerevisiae (A184D) cells under a variety of conditions, with trinitrobenzenesulfonic acid and fluorescamine as the external membrane probes . The levels of external PtdEtn in the intact cells were reduced to about 50% by pretreatment of the cells with inhibitors of mitochondrial ATP synthesis, ATPase inhibitors or protein-sulfhydryl-group-modifying reagents, or by depletion of the cells of ATP by metabolic starvation . The levels of external PtdEtn could be restored to normal by repletion of the energy-depleted cells with ATP . Furthermore, treatment of the energy-depleted cells with sulfhydryl-modifying reagents did not cause further reduction in the external PtdEtn levels but decreased the accessibility of PtdEtn to fluorescamine after restoration of the cellular ATP levels to normal in these cells . These results demonstrate an involvement of an ATP-dependent, protein-mediated process(es) in the regulation of the PtdEtn distribution across the plasma-membrane bilayer of yeast cells . The results are discussed with regard to possible models that can generate and maintain the transbilayer phospholipid asymmetry in the yeast plasma membrane. Eur J Biochem, 1996 Sep 15, 240(3), 526 - 31 Phenylalanyl-tRNA synthetase from yeast and its discrimination of 19 amino acids in aminoacylation of tRNA(Phe)-C-C-A and tRNA(Phe)-C-C-A(3'NH2); Freist W et al.; For discrimination between phenylalanine and 18 other naturally occurring non-cognate amino acids by the class II aminoacyl-tRNA synthetase specific for phenylalanine, discrimination factors, D, of 190-6300 have been determined from kcal and K(m) values . Generally, phenylalanyl-tRNA synthetase is more specific than the class II enzymes specific for Lys and Thr, but works with lower accuracy than the class I enzymes specific for IIe, Tyr, and Arg . In aminoacylation of tRNA(Phe)-C-C-A(3'NH2) discrimination factors D1 vary between 80-1610 . Pre-transfer proof-reading factors II1 are in the range 2.3-74, post-transfer proof-reading factors II2 in the range 1.0-4.6, showing that pre-transfer proof-reading is the main correction step, post-transfer proofreading is less effective or negligible . Initial discrimination factors (I1 and I2) caused by differences in Gibbs free energies of binding between phenylalanine and non-cognate amino acids have been calculated assuming a two-step binding process . Factors I1 can be related to hydrophobic-interaction forces depending on accessible surface areas of the amino acids, factors I2 scatter about a low mean value and do not show any relation to amino acid structures or surfaces, indicating less checking of amino acid side chains in the putative second binding step. Nucleic Acids Res, 1996 Sep 15, 24(18), 3533 - 7 The 70 kDa subunit of replication protein A is required for the G1/S and intra-S DNA damage checkpoints in budding yeast; Longhese MP et al.; The rfa1-M2 and rfa1-M4 Saccharomyces cerevisiae mutants, which are altered in the 70 kDa subunit of replication protein A (RPA) and sensitive to UV and methyl methane sulfonate (MMS), have been analyzed for possible checkpoint defects . The G1/S and intra-S DNA damage checkpoints are defective in the rfa1-M2 mutant, since rfa1-M2 cells fail to properly delay cell cycle progression in response to UV irradiation in G1 and MMS treatment during S phase . Conversely, the G2/M DNA damage checkpoint and the S/M checkpoint are proficient in rfa1-M2 cells and all the checkpoints tested are functional in the rfa1-M4 mutant . Preventing S phase entry by alpha-factor treatment after UV irradiation in G1 does not change rfa1-M4 cell lethality, while it allows partial recovery of rfa1-M2 cell viability . Therefore, the hypersensitivity to UV and MMS treatments observed in the rfa1-M4 mutant might only be due to impairment of RPA function in DNA repair, while the rfa1-M2 mutation seems to affect both the DNA repair and checkpoint functions of Rpa70. J Biotechnol, 1996 Sep 13, 50(1), 75 - 87 Inactivation of MET2 in brewer's yeast increases the level of sulfite in beer; Hansen J et al.; Brewer's yeasts sometimes produce inadequate or excessive amounts of sulfite, an antioxidant and flavour stabilizer, so means of controlling the sulfite production are desired . Understanding the physiology and regulation of the sulfur assimilation pathway of Saccharomyces yeasts is the key to change sulfite production . The MET2 gene of Saccharomyces yeasts encodes homoserine O-acetyl transferase, which catalyzes the conversion of homoserine to O-acetyl homoserine which in turn combines with hydrogen sulfide to form homocysteine, the immediate precursor of methionine . We expected that inactivation of MET2 would lead to accumulation of sulfide and derepression of the entire sulfur assimilation pathway and, therefore, possibly also to sulfite accumulation . Brewer's yeasts were constructed in which several of the four MET2 gene copies were inactivated . Sulfite production was increased in strains with one remaining MET2 gene and even more so when no active MET2 was present . In both cases, hydrogen sulfide production was also increased . To the extent that excess sulfide can be removed, this strategy may be applied to control sulfite accumulation by brewer's yeast in beer production. Mutat Res, 1996 Sep 13, 370(2), 81 - 9 Enhanced cell permeability increases the sensitivity of a yeast test for mutagens; Staleva L et al.; ts1 is a mutation which causes a general increase in permeability of Sacharomyces cerevisiae cells in an unspecific manner . The introduction of the ts1 mutation under homozygous conditions into the D7 diploid strain enhanced the sensitivity of the test system described by Zimmermann et al . (1975) . The newly constructed strain D7ts1 responded with a four to six times higher frequency compared to the D7 strain for all genetic end-points induced with chemical mutagens (ethyl methanesulfonate, methyl methanesulfonate, hydroxyurea, benzpyrene) . The increased sensitivity of D7ts1 is specific only for mutagens active in yeast, since treatment of D7ts1 cells with 5-bromouracil or 5-bromouridine, known to be non-mutagenic in yeast, did not result in the induction of any of the measured genetic alterations . Five out of 14 water samples taken from the environment induced recombinogenic events in D7ts1, whereas all 14 water samples were without effect in the D7 test system . We concluded that D7ts1 cells show a higher sensitivity in the detection of mutagenic or carcinogenic action because of their generally enhanced permeability due to the ts1 mutation. J Biol Chem, 1996 Sep 13, 271(37), 22487 - 93 Site-directed mutagenesis of the 100-kDa subunit (Vph1p) of the yeast vacuolar (H+)-ATPase; Leng XH et al.; Vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for acidification of intracellular compartments in eukaryotic cells . V-ATPases possess a subunit of approximate molecular mass 100 kDa of unknown function that is composed of an amino-terminal hydrophilic domain and a carboxyl-terminal hydrophobic domain . To test whether the 100-kDa subunit plays a role in proton transport, site-directed mutagenesis of the VPH1 gene, which is one of two genes that encodes this subunit in yeast, has been carried out in a strain lacking both endogenous genes . Ten charged and twelve polar residues located in the seven putative transmembrane helices in the COOH-terminal domain of the molecule were individually changed, and the effects on proton transport, ATPase activity, and assembly of the yeast V-ATPase were measured . Two mutations (R735L and Q634L) in transmembrane helix 6 and at the border of transmembrane helix 5, respectively, showed greatly reduced levels of the 100-kDa subunit in the vacuolar membrane, suggesting that these mutations affected stability of the 100-kDa subunit . Two mutations, D425N and K538A, in transmembrane helix 1 and at the border of transmembrane helix 3, respectively, showed reduced assembly of the V-ATPase, with the D425N mutation also reducing the activity of V-ATPase complexes that did assemble . Two mutations, H743A and K593A, in transmembrane helix 6 and at the border of transmembrane helix 4, respectively, have significantly greater effects on activity than on assembly, with proton transport and ATPase activity inhibited 40-60% . One mutation, E789Q, in transmembrane helix 7, virtually completely abolished proton transport and ATPase activity while having no effect on assembly . These results suggest that the 100-kDa subunit may be required for activity as well as assembly of the V-ATPase complex and that several charged residues in the last four putative transmembrane helices of this subunit may play a role in proton transport. J Biol Chem, 1996 Sep 13, 271(37), 22453 - 61 Characterization of green alga, yeast, and human centrins . Specific subdomain features determine functional diversity; Wiech H et al.; Centrins are a subfamily within the superfamily of Ca2+-modulated proteins that play a fundamental role in centrosome duplication and contraction of centrin-based fiber systems . We examined the individual molecular properties of yeast, green alga, and human centrins . Circular dichroism spectroscopy revealed a divergent influence of Ca2+ binding on the alpha-helical content of these proteins . Ca2+-free centrins were elongated in shape as determined by size exclusion chromatography . The presence of Ca2+ and binding peptide resulted in more spherical shaped centrins . In contrast to yeast calmodulin, centrins formed multimers in the Ca2+-bound state . This oligomerization was significantly reduced in the absence of Ca2+ and in the presence of binding peptide . The Ca2+-dependent polymerization of the green alga Scherffelia dubia centrin (SdCen) resulted in a filamentous network . This molecular property was mainly dependent on the amino-terminal subdomain and the peptide-binding site of SdCen . Finally, we analyzed whether SdCen and Cdc31p-SdCen hybrid proteins functionally substitute for the Saccharomyces cerevisiae centrin Cdc31p . Only hybrid proteins containing the amino-terminal subdomain or the third EF-hand of SdCen and the other subdomains from Cdc31p were functional in vivo. J Biol Chem, 1996 Sep 13, 271(37), 22434 - 40 Emopamil-binding protein, a mammalian protein that binds a series of structurally diverse neuroprotective agents, exhibits delta8-delta7 sterol isomerase activity in yeast; Silve S et al.; Delta8-delta7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes . This endoplasmic reticulum-resident membrane protein catalyzes the conversion of delta8-sterols to their corresponding delta7-isomers . No sequence data for high eukaryote sterol isomerase being available so far, we have cloned a murine sterol isomerase-encoding cDNA by functional complementation of the corresponding deficiency in the yeast Saccharomyces cerevisiae . The amino acid sequence deduced from the cDNA open reading frame is highly similar to human emopamil-binding protein (EBP), a protein of unknown function that constitutes a molecular target for neuroprotective drugs . A yeast strain in which the sterol isomerase coding sequence has been replaced by that of human EBP or its murine homologue recovers the ability to convert delta8-sterol into delta7-sterol, both in vivo and in vitro . In these recombinant strains, both cell proliferation and the sterol isomerization reaction are inhibited by the high affinity EBP ligand trifluoperazine, as is the case in mammalian cells but not in wild type yeast cell . In contrast, the recombinant strains are much less susceptible to the sterol inhibition effect of haloperidol and fenpropimorph, as compared with wild type yeast strains . Our results strongly suggest that EBP and delta8-delta7 sterol isomerase are identical proteins in mammals. Nature, 1996 Sep 12, 383(6596), 188 - 91 TBP-associated factors are not generally required for transcriptional activation in yeast; Moqtaderi Z et al.; The transcription factor TFIID, a central component of the eukaryotic RNA polymerase II (Pol II) transcription apparatus, comprises the TATA-binding protein (TBP) and approximately ten TBP-associated factors (TAFs) . Although the essential role of TBP in all eukaryotic transcription has been extensively analysed in vivo and in vitro, the function of the TAFs is less clear . In vitro, TAFs are dispensable for basal transcription but are required for the response to activators . In addition, specific TAFs may act as molecular bridges between particular activators and the general transcription machinery . In vivo, TAFS are required for yeast and mammalian cell growth, but little is known about their specific transcriptional functions . Using conditional alleles created by a new double-shutoff method, we show here that TAF depletion in yeast cells can reduce transcription from some promoters lacking conventional TATA elements . However, TAF depletion has surprisingly little effect on transcriptional enhancement by several activators, indicating that TAFs are not generally required for transcriptional activation in yeast. J Biol Chem, 1996 Sep 6, 271(36), 21842 - 7 A yeast transcriptional stimulatory protein similar to human PC4; Henry NL et al.; A yeast protein has been identified that stimulates basal transcription by RNA polymerase II, binds both single- and double-stranded DNA, and interacts with both a general transcription factor and a transcriptional activator . Phosphorylation appears to regulate these interactions . The gene for the transcriptional stimulatory protein, termed TSP1, was cloned and found to be dispensable for yeast cell viability . The deduced amino acid sequence is similar to that of mammalian coactivator protein PC4. Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9942 - 7 The pfmdr1 gene of Plasmodium falciparum confers cellular resistance to antimalarial drugs in yeast cells; Ruetz S et al.; The exact role of the pfmdr1 gene in the emergence of drug resistance in the malarial parasite Plasmodium falciparum remains controversial . pfmdr1 is a member of the ATP binding cassette (ABC) superfamily of transporters that includes the mammalian P-glycoprotein family . We have introduced wild-type and mutant variants of the pfmdr1 gene in the yeast Saccharomyces cerevisiae and have analyzed the effect of pfmdr1 expression on cellular resistance to quinoline-containing antimalarial drugs . Yeast transformants expressing either wild-type or a mutant variant of mouse P-glycoprotein were also analyzed . Dose-response studies showed that expression of wild-type pfmdr1 causes cellular resistance to quinine, quinacrine, mefloquine, and halofantrine in yeast cells . Using quinacrine as substrate, we observed that increased resistance to this drug in pfmdr1 transformants was associated with decreased cellular accumulation and a concomitant increase in drug release from preloaded cells . The introduction of amino acid polymorphisms in TM11 of Pgh-1 (pfmdr1 product) associated with drug resistance in certain field isolates of P . falciparum abolished the capacity of this protein to confer drug resistance . Thus, these findings suggest that Pgh-1 may act as a drug transporter in a manner similar to mammalian P-glycoprotein and that sequence variants associated with drug-resistance pfmdr1 alleles behave as loss of function mutations. Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9488 - 92 The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function; Sakurai H et al.; The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast . The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II . Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II . We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE . The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE . Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast . In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell. EMBO J, 1996 Sep 2, 15(17), 4654 - 64 Distinct activated and non-activated RNA polymerase II complexes in yeast; Akhtar A et al.; We used a transcriptional run-on assay in permeabilized yeast cells to study the distribution of RNA polymerase II (pol II) complexes before and after activation by Gal4 . Polymerases were found engaged on the gene at the 5' end before activation, but only appeared at the 3' end after activation . Mutations of the pol II C-terminal domain (CTD), the CTD kinase Kin28 and the holoenzyme subunit Srb2 all inhibited the formation of 3' polymerases in response to activator . However, these mutations did not inhibit the establishment of polymerases at the 5' end . The differences between 3' and 5' ternary complexes suggest that they represent qualitatively distinct 'activated' and 'non-activated' forms of polymerase . The results implicate CTD phosphorylation in a switch from 'non-activated' transcription, which is confined to the 5' end, to an 'activated' mode that traverses the length of the gene. EMBO J, 1996 Sep 2, 15(17), 4643 - 53 Localization of yeast RNA polymerase I core subunits by immunoelectron microscopy; Klinger C et al.; Immunoelectron microscopy was used to determine the spatial organization of the yeast RNA polymerase I core subunits on a three-dimensional model of the enzyme . Images of antibody-labeled enzymes were compared with the native enzyme to determine the localization of the antibody binding site on the surface of the model . Monoclonal antibodies were used as probes to identify the two largest subunits homologous to the bacterial beta and beta' subunits . The epitopes for the two monoclonal antibodies were mapped using subunit-specific phage display libraries, thus allowing a direct correlation of the structural data with functional information on conserved sequence elements . An epitope close to conserved region C of the beta-like subunit is located at the base of the finger-like domain, whereas a sequence between conserved regions C and D of the beta'-like subunit is located in the apical region of the enzyme . Polyclonal antibodies outlined the alpha-like subunit AC40 and subunit AC19 which were found co-localized also in the apical region of the enzyme . The spatial location of the subunits is correlated with their biological activity and the inhibitory effect of the antibodies. EMBO J, 1996 Sep 2, 15(17), 4603 - 12 A novel S phase inhibitor in fission yeast; Woollard A et al.; We have cloned a novel fission yeast gene, spd1, which causes G1 arrest when overexpressed . Deleting the gene results in cells being accelerated through G1 into S phase in certain circumstances when the G1-->S phase control is compromised . We have fou