Yeasts

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 found that the encoded 14 kDa protein is cell cycle regulated, declining in level during S phase, and that p14spd1 physically associates with p34cdc2 in vivo when overexpressed, suggesting that p14spd1 may regulate S phase progression via an interaction with p34cdc2 . We conclude that p14spd1 is a negative regulator of S phase, and that it may be part of the control ensuring an orderly onset of S phase or part of a G1-->S phase checkpoint control.

EMBO J, 1996 Sep 2, 15(17), 4592 - 602
Spc110p: assembly properties and role in the connection of nuclear microtubules to the yeast spindle pole body; Kilmartin JV et al.; Spc110p is an essential component of the budding yeast spindle pole body (SPB) . It binds calmodulin and contains a long central coiled-coil rod which acts as a spacer element between the central plaque of the SPB and the ends of the nuclear or spindle microtubules . This suggests that the essential function of Spc110p is to connect the nuclear microtubules to the SPB . To confirm this, we examined the phenotype of ts alleles of SPC110, one of which contains a mutation in the calmodulin binding site and was suppressed by overexpression of calmodulin . The alleles fail to form a functional mitotic spindle because spindle microtubules are not properly connected to the SPB . We also examined the phenotype of the toxic overexpression of either the wild-type or a truncated version of Spc110p containing a deletion of most of the coiled-coil domain . Both of these proteins form large ordered spheroidal polymers in the nucleus . The polymerization of the truncated Spc110p appears to be initiated inside the SPB from the position where Spc110p is normally located, and as the polymer grows in size it severs the connection between the nuclear microtubules and the SPB . The polymers were purified and are composed of Spc110p and calmodulin . A model for the structure of the polymer is proposed.

Mutat Res, 1996 Sep 2, 364(1), 33 - 41
A yeast whole cell extract supports nucleotide excision repair and RNA polymerase II transcription in vitro; Wang Z et al.; Nucleotide excision repair (NER) and RNA polymerase II transcription are cellular processes that require the transcription/NER factor TFIIH . We have developed a whole cell extract from the yeast Saccharomyces cerevisiae that simultaneously supports both NER and RNA polymerase II transcription of independent substrates . NER activity in the yeast whole cell extract was readily detected in the absence of further supplementation but was stimulated in the presence of overexpressed Rad2 protein . The repair of N-acetyl-2-aminofluorene (AAF)-damaged DNA was dependent on RAD genes required for NER and deficient repair in rad mutant extracts was complemented by mixing different mutant extracts or by purified Rad proteins . Both the NER and transcription activities were stimulated by 5% polyethylene glycol in the whole cell extracts . Transcription activity from the template pCYC1G- was not affected by the presence of uracil-containing or AAF-damaged pUC18 DNA, which was expected to result in base excision repair (BER) and NER, respectively . An in vitro condition was defined that supported simultaneous NER and transcription independently in different substrates in the yeast whole cell extracts.

Zh Evol Biokhim Fiziol, 1996 Sep-Oct, 32(5), 545 - 55
{A theoretical analysis of the primary structure of the yeast SEC59 gene product . Topology in the membrane, identification of the repetitive sequences and internal symmetry}; Shpakov AO; In this investigation the results of the theoretical analysis of primary structure of yeast gene SEC59 product, which possesses the dolichol kinase activity, were represented . The amino acid sequences (AAS), potentially penetrating the membrane, were identified by using of hydropathic and hydrophobic analyses . The some of these sequences were homologous . The longer symmetrical AAS with centres of symmetry in dolichol-binding region and related sequences were identified by using of comparative analysis of amino acid codon roots . The graphic method of analysis of amino acid codon roots sequences was used for identification of repeating segments in primary structure of SEC59 molecule . These segments contain less than 18 amino acid residues and construct the main part of polypeptide chain (for exclusion of N-terminal region of SEC59).

J Med Vet Mycol, 1996 Sep-Oct, 34(5), 349 - 52
Evaluation of the Biolog MicroStation system for yeast identification; McGinnis MR et al.; One hundred and fifty-nine isolates representing 16 genera and 53 species of yeasts were processed with the Biolog MicroStation System for yeast identification . Thirteen genera and 38 species were included in the Biolog database . For these 129 isolates, correct identifications to the species level were 13.2, 39.5 and 48.8% after 24, 48 and 72 hours incubation at 30 degrees C, respectively . Three genera and 15 species which were not included in the Biolog database were also tested . Of the 30 isolates studied, 16.7, 53.3 and 56.7% of the isolates were given incorrect names from the system's database after 24,48 and 72 h incubation at 30 degrees C, respectively . The remaining isolates of this group were not identified.

J Biochem (Tokyo), 1996 Sep, 120(3), 642 - 6
Identification and characterization of extragenic suppressors of the yeast sec12 ts mutation; Nakano A; The product of the yeast SEC12 gene is the most upstream player as far as we know in the events of vesicle budding from the endoplasmic reticulum membrane . To understand how the Sec12p function could be regulated, I identified three extragenic suppressor mutations of the sec12 ts mutation . RST1DS is a dominant mutation and causes elevated expression of Sec12p . rst2 and rst3 are recessive and give pleiotropic phenotypes including slow growth at low temperatures and heterogeneous modification of Sec12p . Their possible roles are discussed.

J Dairy Sci, 1996 Sep, 79(9), 1654 - 8
Effects of a supplemental liquid yeast product on feed intake, ruminal profiles, and yield, composition, and organoleptic characteristics of milk from lactating Holstein cows; Besong S et al.; A liquid yeast product (about 11% DM), produced as a by-product of riboflavin synthesis, was incorporated into a TMR at three concentrations to determine the maximal inclusion rate and acceptability as a feed supplement in diets of lactating cows . Twelve Holstein cows in midlactation were randomly assigned to TMR treatments containing (as-fed basis) 1) no yeast product (control), 2) 20% dietary yeast product, or 3) 40% dietary yeast product in a 3 x 3 Latin square design with 3-wk periods . Dietary DM contents were 90, 74.2, and 58.4% for the 0, 20, and 40% yeast product TMR, respectively . The control TMR was based on a 45:55 ratio (DM basis) of mixed concentrate and chopped alfalfa hay . The DMI decreased linearly with increasing percentages of dietary yeast product . Milk yield and milk protein percentage were not altered by inclusion of the dietary yeast product . Milk fat tended to respond quadratically to increases in the percentage of dietary yeast product in the TMR . The supplemental dietary yeast product had no effect on ruminal pH; however, the ratio of acetate to propionate decreased, and propionate percentage tended to increase, as supplementation of the yeast product increased . Addition of yeast product to the TMR had no adverse effect on milk flavor . Milk from cows fed yeast product had better flavor than milk from control cows . Results indicate that inclusion of this yeast product at 40% depressed feed intake.

Microbiologia, 1996 Sep, 12(3), 371 - 84
Genetic constitution of industrial yeast; Benitez T et al.; Saccharomyces cerevisiae industrial yeast strains are highly heterogeneous . These industrial strains, including bakers', wine, brewing and distillers', have been compared with respect to their DNA content, number and size of chromosomes, homologies between their genes and those of laboratory strains, and restriction fragment lengths of their mitDNA . A high variability, and the presence of multigenic families, were observed in some industrial yeast groups . The occurrence or the lack of chromosomal polymorphism, as well as the presence of multiple copies of some genes, could be related to a selective process occurring under specific industrial conditions . This polymorphism is generated by reorganization events, that take place mainly during meiosis and are mediated by repetitive Y' and Ty elements . These elements give rise to ectopic and asymmetric recombination and to gene conversion . The polymorphism displayed by the mitDNA could also result from specific industrial conditions . However, in enological strains the selective process is masked by the mutagenic effect that ethanol exerts on this DNA.

Yeast, 1996 Sep, 12(10B Suppl), 1065 - 70
The sequence of 23 kb surrounding the SNF3 locus on the left arm of yeast chromosome IV reveals the location of five known genes and characterizes at least six new open reading frames including putative genes for ribosomal protein L35 and a sugar transport protein; Verhasselt P et al.; The nucleotide sequence of 22,846 bp of the left arm of chromosome IV is described . Twelve open reading frames (ORFs) greater than 100 triplets were detected, one of which extends into an adjacent cosmid . Two of the ORFs may contain an intron . One of these is an L35 ribosomal protein gene . Five ORFs (D1204, D1214, D1219, D1234 and D1244) encode previously sequenced genes (MGT1, SHM1, ASF2, SNF3 and ARF2, respectively) . The nucleotide sequence of a sixth ORF (D1229) is quite similar to the WEB1 gene, which appeared in the DNA databases shortly after finishing the sequence reported here . It is not clear whether or not WEB1 and D1229 represent one and the same gene . The co-linearity of the reported DNA sequences with the genome of strains from Saccharomyces cerevisiae subspecies carlsbergensis, sake and diastaticus was assessed by comparative PCR with overlapping primer sets.

Yeast, 1996 Sep, 12(10B Suppl), 1053 - 8
Sequence analysis of a 12 801 bp fragment of the left arm of yeast chromosome XV containing a putative 6-phosphofructo-2-kinase gene, a gene for a possible glycophospholipid-anchored surface protein and six other open reading frames; Aldea M et al.; The DNA sequence of a 12,801 bp fragment located near the left telomere of chromosome XV has been determined . Sequence analysis reveals eight open reading frames (ORFs) encoding polypeptides larger than 100 residues . ORFs AOE129 and AOAA121 are in opposite strands and they overlap at their 3' ends . AOE397 has similarity with phosphofructokinase genes from other organisms and may code for a second 6-phosphofructo-2-kinase of Saccharomyces cerevisiae . Sequence of AOA471 shows significant similarity with yeast genes coding for glycophospholipid-containing proteins . AOD1341 would code for a 1341 amino acids long protein with a predicted ATP/GTP-binding site and a transmembrane domain.

Yeast, 1996 Sep, 12(10B Suppl), 1047 - 51
Identification of a putative methylenetetrahydrofolate reductase by sequence analysis of a 6.8 kb DNA fragment of yeast chromosome VII; Tizon B et al.; We report the sequence analysis of a 6.8 kb DNA fragment from Saccharomyces cerevisiae chromosome VII . This sequence contains five open reading frames (ORFs) greater than 100 amino acids . There is also an incomplete ORF flanking one of the extremes, G2868, which is the 3' end of the SCS3 gene (Hosaka et al., 1994) . The translated sequence of ORF G2882 shows similarity to the human methylenetetrahydrofolate reductase (Goyette et al., 1994) . ORF G2889 shows no significant homologies with the sequences compiled in databases . ORF G2893 corresponds to the gene SUP44, coding for the yeast ribosomal protein S4 (All-Robin et al., 1990) . G2873 and G2896 are internal ORFs.

Yeast, 1996 Sep, 12(10B Suppl), 1021 - 31
Sequencing of a 35.71 kb DNA segment on the right arm of yeast chromosome XV reveals regions of similarity to chromosomes I and XIII; Pearson BM et al.; In a shotgun approach we sequenced the cosmid pEOA284 containing a fragment derived from the right arm of chromosome XV of Saccharomyces cerevisiae . An analysis of the sequence revealed that it contained open reading frames (ORFs) corresponding to the known genes SLY41, SPS4, COT1, FAA1, PMT3, PRO2 and MYO2 . Of the 18 unknown ORFs, five are contained totally within, and two, O6105 and O6163, partially overlap other ORFs . ORF O6116 and O6139 have putative introns . Regions of similarity with chromosomes I and XIII have been uncovered . Interestingly, most of the paired ORFs encode proteins of the same gene family . The relatedness of these ORFs suggests gene duplication.

J Cell Sci, 1996 Sep, 109 ( Pt 9), 2331 - 42
The fission yeast sts5+ gene is required for maintenance of growth polarity and functionally interacts with protein kinase C and an osmosensing MAP-kinase pathway; Toda T et al.; Cell morphogenesis is a fundamental phenomenon that involves understanding a number of biological processes including the developmental program, polarity and cell division . Fission yeast sts5 mutant cells are round rather than cylindrical with cortical actin randomly dispersed . Genetic analyses demonstrate that the sts5+ gene is required for maintenance of cell shape during interphase when the cell normally exhibits polarised growth . The sts5 mutant is not defective in cell wall integrity . Deletion of ppe1+, which encodes a type 2A-like protein phosphatase, shows similar phenotypes to the sts5 mutant and these two mutations are synthetically lethal . Multicopy plasmids containing either the protein kinase C-like gene pck1+ or the protein tyrosine phosphatase pyp1+, an inhibitor of an osmosensing Sty1/Spc1 MAP-kinase, are capable of suppressing the sts5 mutation . Consistent with this, we have found that the wis1 mutation, which is defective in a MAP-kinase kinase of the pathway, suppresses the sts5 mutation . The predicted sts5+ gene product exhibits sequence similarity to two yeast proteins, Dis3 and Ssd1 and a nematode protein, F46E8.6, where the former two yeast proteins have been shown to be involved in cell cycle control and cell morphogenesis . The sts5+ gene is not essential for cell viability, but is absolutely required for polarised growth as the gene disruption showed the same phenotypes as those of the original mutants . Overexpression of the sts5+ gene resulted in altered cell morphology and, cortical actin in these overproducing cells was also abnormal, fainter and often dispersed . Anti-Sts5 antibody specifically detected a 130 kDa protein by western blotting . A green fluorescent protein-Sts5 fusion protein localised in the cytoplasm with a discrete punctate pattern, suggesting that the Sts5 protein is a component of a novel structure . These results have indicated that the Sts5 protein is a crucial determinant of polarised growth and that it functionally interacts with the serine/threonine phosphatase, protein kinase C, and an osmosensing MAP-kinase to maintain cell morphology.

J Cell Sci, 1996 Sep, 109 ( Pt 9), 2229 - 37
The spacer protein Spc110p targets calmodulin to the central plaque of the yeast spindle pole body; Spang A et al.; Yeast calmodulin (CaM) was found to be localized to the microtubule organizing centre, the spindle pole body . The spindle pole body is a multi-layered structure consisting of outer, central and inner plaques . In this paper, we report that a fraction of CaM is in association with the central plaque of the spindle pole body . This localization is dependent on the calmodulin-binding site of another spindle pole body component, Spc110p, which serves as a spacer connecting the inner plaque with the central plaque . Since the CaM-binding site of Spc110p is located near the carboxy terminus, Spc110p-dependent localization of calmodulin defines the orientation of Spc110p with the carboxy terminus towards the central plaque and the amino terminus towards the inner plaque . This orientation of Spc110p was confirmed using antibodies specific for the amino-terminal end of Spc110p, which predominantly labelled the inner plaque . In addition, synthetic peptides corresponding to the calmodulin-binding site of Spc110p bound to calmodulin with a Kd in the nanomolar range and nearly independent of Ca2+.

Mol Microbiol, 1996 Sep, 21(5), 1061 - 73
Membrane topology of the yeast uracil permease; Garnier C et al.; The uracil permease of Saccharomyces cerevisiae is a 633 residue polytopic plasma membrane protein . Hydropathy profile analysis indicates that this protein has long hydrophilic N- and C-termini and 10-12 potential transmembrane segments . Previous results based on analysis of hybrid proteins allowed identification of the first transmembrane segment of uracil permease, and provided a preliminary indication of the cytoplasmic orientation of its N-terminus . In this work, other experimental approaches were used to confirm this orientation, and to determine that of the C-terminus . Epitopes in the N- and the C-termini of the protein were protected against trypsin degradation on intact protoplasts, but readily digested on permeabilized protoplasts . Immunofluorescent analysis showed that antibodies to the last 10 amino acids of uracil permease bind to detergent-treated protoplasts, but not to intact ones . Carboxypeptidase digested the C-terminus of uracil permease inserted into the sealed dog-pancreas microsomes . These results establish that both N- and C-termini are cytoplasmic, the permease polypeptide spanning the membrane an even number of times . The orientation of several hydrophilic loops with respect to the membrane was investigated by introducing potential glycosylation sites into these regions . We checked whether the resulting mutant proteins were glycosylated when expressed in the presence of dog-pancreas microsomes . Our data show that two loops of the protein are lumenal . Together with previous results, this work indicates that uracil permease is a 10 membrane-spanning protein, with rather small external loops and three main cytoplasmic regions (the N- and C-termini and a central 60-residue loop).

Mol Biol Cell, 1996 Sep, 7(9), 1405 - 17
CDC37 is required for p60v-src activity in yeast; Dey B et al.; Mutations in genes encoding the molecular chaperones Hsp90 and Ydj1p suppress the toxicity of the protein tyrosine kinase p60v-src in yeast by reducing its levels or its kinase activity . We describe isolation and characterization of novel p60v-src-resistant, temperature-sensitive cdc37 mutants, cdc37-34 and cdc37-17, which produce less p60v-src than the parental wild-type strain at 23 degrees C . However, p60v-src levels are not low enough to account for the resistance of these strains . Asynchronously growing cdc37-34 and cdc37-17 mutants arrest in G1 and G2/M when shifted from permissive temperatures (23 degrees C) to the restrictive temperature (37 degrees C), but hydroxyurea-synchronized cdc37-34 and cdc37-17 mutants arrest in G2/M when released from the hydroxyurea block and shifted from 23 to 37 degrees C . The previously described temperature-sensitive cdc37-1 mutant is p60v-src-sensitive and produces wild-type amounts of p60v-src at permissive temperatures but becomes p60v-src-resistant at its restrictive temperature, 38 degrees C . In all three cdc37 mutants, inactivation of Cdc37p by incubation at 38 degrees C reduces p60v-src-dependent tyrosine phosphorylation of yeast proteins to low or undetectable levels . Also, p60v-src levels are enriched in urea-solubilized extracts and depleted in detergent-solubilized extracts of all three cdc37 mutants prepared from cells incubated at the restrictive temperature . These results suggest that Cdc37p is required for maintenance of p60v-src in a soluble, biologically active form.

Mol Biol Cell, 1996 Sep, 7(9), 1375 - 89
Multiple classes of yeast mutants are defective in vacuole partitioning yet target vacuole proteins correctly; Wang YX et al.; In Saccharomyces cerevisiae the vacuoles are partitioned from mother cells to daughter cells in a cell-cycle-coordinated process . The molecular basis of this event remains obscure . To date, few yeast mutants had been identified that are defective in vacuole partitioning (vac), and most such mutants are also defective in vacuole protein sorting (vps) from the Golgi to the vacuole . Both the vps mutants and previously identified non-vps vac mutants display an altered vacuolar morphology . Here, we report a new method to monitor vacuole inheritance and the isolation of six new non-vps vac mutants . They define five complementation groups (VAC8-VAC12) . Unlike mutants identified previously, three of the complementation groups exhibit normal vacuolar morphology . Zygote studies revealed that these vac mutants are also defective in intervacuole communication . Although at least four pathways of protein delivery to the vacuole are known, only the Vps pathway seems to significantly overlap with vacuole partitioning . Mutants defective in both vacuole partitioning and endocytosis or vacuole partitioning and autophagy were not observed . However, one of the new vac mutants was additionally defective in direct protein transport from the cytoplasm to the vacuole.

Clin Diagn Lab Immunol, 1996 Sep, 3(5), 614 - 5
False-positive serologic test resulting from a probable yeast infection in a chimpanzee; Heller T et al.; Sera used to identify putative hepatitis E viral proteins expressed in Pischia pastoris produced a false-positive reaction because of antibodies to a yeast protein . This report illustrates a potential problem when serological reagents are used in combination with recombinant proteins expressed in yeast.

Brain Res Mol Brain Res, 1996 Sep 1, 40(2), 240 - 53
Caprine homologue of rodent 5'-AMP-activated protein kinase subunit and yeast SNF4/CAT3 is down-regulated by thyroid hormone; Piosik PA et al.; We isolated a cDNA, B12, that was down-regulated by thyroid hormone (TH) in the goat cerebellum, using a polymerase chain reaction (PCR)-based subtractive hybridization and differential screening procedure . Northern blot analysis of RNA from cerebellum of T4-treated and untreated hypothyroid goats confirmed that clone B12 was TH-regulated with an average reduction in expression of 21% after 4 days of T4 supplementation . Other tissues from a T4-treated and an untreated hypothyroid goat also revealed down-regulation of B12, with the highest reduction in expression found in the thyroid gland (38%) . Steady-state levels of the approximately 1.8 kb B12 mRNA were higher in brain than in peripheral tissues . In situ hybridization showed that B12 mRNA in the brain is mainly present in various layers of the cerebellum and the cerebral cortex, the hippocampus, and the olfactory tubercle and is predominantly expressed in neurons . Sequence analysis of the caprine B12 cDNA clone, and the murine homologue, revealed 61% similarity to SNF4/CAT3, a regulator involved in the transcriptional control of glucose-repressible genes in yeast, and 99% identity to a rat 5'-AMP-activated protein kinase subunit, which is involved in the regulation of fatty acid, glycogen and isoprenoid metabolism . In view of these homologies, B12 might encode a regulator involved in distinct metabolic pathways and therefore, TH might also affect gene expression indirectly by down-regulation of regulators like B12.

Trends Genet, 1996 Sep, 12(9), 345 - 50
A quantitative model for the cdc2 control of S phase and mitosis in fission yeast; Stern B et al.; In this article we consider the role of the cyclin-dependent protein kinase cdc2 in regulating progression through the fission yeast cell cycle . The onset of mitosis is governed by cdc2 in partnership with the B-type cyclin, cdc13 . Recent evidence shows that the cdc2-cdc13 complex can also control the onset of S phase and, in addition, ensures that there is only one S phase per cell cycle . This leads us to propose a novel quantitative model in which different levels of cdc2 activity regulate cell-cycle progression: S phase is initiated when protein kinase activity increases from a very low to a moderate level; maintenance of this moderate level prevents re-initiation of S phase, and a further increase of activity to a high level initiates mitosis . Inactivation of the kinase activity at the end of mitosis resets the cell for a new cell cycle.

Microbiology, 1996 Sep, 142 ( Pt 9), 2655 - 62
Cell cycle studies on the mode of action of yeast K28 killer toxin; Schmitt MJ et al.; The virally encoded K28 killer toxin of Saccharomyces cerevisiae kills sensitive cells by a receptor-mediated process . DNA synthesis is rapidly inhibited, cell viability is lost more slowly and cells eventually arrest, apparently in the S phase of the cell cycle with a medium-sized bud, a single nucleus in the mother cell and a pre-replicated (1n) DNA content . Cytoplasmic microtubules appear normal, and no spindle is detectable . Arrest of a sensitive haploid yeast strain by alpha-factor at START gave complete protection for at least 4 h against a toxin concentration that killed non-arrested cells at the rate of one log each 2.5 h . Cells released from alpha-factor arrest were killed by toxin at a similar rate; arrest occurred with medium-sized buds within the same cell cycle . Cells arrested by hydroxyurea, with unreplicated DNA, or by the spindle poison methylbenzimidazol-2yl-carbamate, with unseparated chromosomes, both arrest at the checkpoint at the G2/M boundary; these arrested cells were not protected against toxin, losing about one log of viability every 4 h . Following release from the cell cycle block, a majority of these toxin-exposed cells progressed through the cell cycle and arrested in the following S-phase, again with medium-sized buds . Killing by K28 toxin apparently requires entry into the nuclear division and bud cycles, but can result from inhibition of either early or late events in these cycles . Morphogenesis in moribund cells is uniformly blocked in early S-phase with an immature bud . Toxin action causes either independent blockage of both DNA synthesis and the budding cycle, or inhibits some unknown step required for both events.

J AOAC Int, 1996 Sep-Oct, 79(5), 1069 - 82
Two-day hydrophobic grid membrane filter method for yeast and mold enumeration in foods using YM-11 agar: collaborative study; Entis P; Twenty laboratories participated in a collaborative study to validate a 2-day hydrophobic grid membrane filter method using YM-11 agar for enumeration of yeast and mold in foods . Six naturally contaminated food products were included in the study: garlic powder, raw ground beef, walnuts, flour/meal, orange juice, and yogurt . The test method produced significantly higher results than the 5-day pour plate reference method for orange juice and significantly lower, though numerically similar, results for walnuts and yogurt . Differences between the test and reference methods were not significant for garlic powder, raw ground beef, or flour/meal . Repeatability and reproducibility were similar for both the test and reference methods in all cases . The hydrophobic grid membrane filter method for enumeration of yeast and mold in foods has been adopted by AOAC INTERNATIONAL.

Plant J, 1996 Sep, 10(3), 451 - 8
Modified plant plasma membrane H(+)-ATPase with improved transport coupling efficiency identified by mutant selection in yeast; Baunsgaard L et al.; Transport across the plasma membrane is driven by an electrochemical gradient of H+ ions generated by the plasma membrane proton pump (H(+)-ATPase) . Random mutants of Arabidopsis H(+)-ATPase AHA1 were isolated by phenotypic selection of growth of transformed yeast cells in the absence of endogenous yeast H(+)-ATPase (PMA1) . A Trp-874-Leu substitution as well as a Trp-874 to Lys-935 deletion in the hydrophilic C-terminal domain of AHA1 conferred growth of yeast cells devoid of PMA1 . A Trp-874-Phe substitution in AHA1 was produced gy site-directed mutagenesis . The modified enzymes hydrolyzed ATP at 200-500% of wild-type level, had a sixfold increase in affinity for ATP (from 1.2 to 0.2 mM; pH 7.0), and had the acidic pH optimum shifted towards neutral pH . AHA1 did not contribute significantly to H+ extrusion by transformed yeast cells . The different specifies of aha1, however, displayed marked differences in initial rates of net H+ extrusion and in their ability to sustain an electrochemical H+ gradient . These results provide evidence that Trp-874 plays an important role in auto-inhibition of the plant H(+)-ATPase and may be involved in controlling the degree of coupling between ATP hydrolysis and H+ pumping . Finally, these results demonstrate the usefulness of yeast as a generalized screening tool for isolating regulatory mutants of plant transporters.

Nucleic Acids Res, 1996 Sep 1, 24(17), 3469 - 71
One-step PCR mediated strategy for the construction of conditionally expressed and epitope tagged yeast proteins; Lafontaine D et al.; With the availability of the complete yeast genomic sequence, techniques which allow the rapid functional analysis of genes of interest are of increasing importance . Here we report a technique which allows the initial characterisation of genes of interest, through the construction of conditionally expressed mutations for functional analyses and the generation of epitope-tagged fusion proteins for immuno-localisation and immuno-purification, entirely by PCR.

Nucleic Acids Res, 1996 Sep 1, 24(17), 3332 - 6
A yeast cap binding protein complex (yCBC) acts at an early step in pre-mRNA splicing; Lewis JD et al.; The function in splicing of a heterodimeric nuclear cap binding complex (yCBC) from the yeast Saccharomyces cerevisiae has been examined . Immunodepletion of splicing extracts with antibodies directed against one component of the complex, yCBP80, results in the efficient co-depletion of the second component, yCBP20, producing CBC-deficient splicing extract . This extract exhibits strongly reduced splicing efficiency and similar reductions in the assembly of both spliceosomes and of the earliest defined precursors to spliceosomes, commitment complexes . The addition of highly purified yCBC substantially restores these defects . These results, together with other data, suggest that CBCs play a highly conserved role in the recognition of pre-mRNA substrates at an early step in the splicing process.

J Appl Bacteriol, 1996 Sep, 81(3), 283 - 7
Brewery yeast as a biosorbent for uranium; Omar NB et al.; Yeast cells are capable of carrying out biosorption with various heavy metals . The biomass deriving from Saccharomyces cerevisiae coming from brewing industries is a by-product that is possible to be used in the purification of water contaminated with these ions . In this paper we show that yeast biomass from one of the city's breweries can adsorb uranium efficiently, up to 2.4 mmol of this metal per gram of dry biomass . It can also be seen that the temperature (between 10 degrees and 37 degrees C) has no effect on the biosorption, while pH does have an influence, 4.5 being the best value . When the concentrations of uranium range between 0.1 and 0.5 mol l-1 the yeast dry biomass is capable of adsorbing between 84% and 98% of this metal in solution.

Curr Genet, 1996 Sep, 30(4), 273 - 8
Organization and characterization of the two yeast ribosomal protein YL19 genes; Song JM et al.; A second copy of the Saccharomyces cerevisiae ribosomal protein YL19 gene was isolated through the use of the RPL19A gene as a probe . The nucleotide sequence of the gene, RPL19B, was determined . RPL19B contains an intron of 384 nucleotides located near its 5'-end . The coding regions of the two yeast genes, RPL19A and RPL19B, differ in only 34 nucleotides, none of which lead to changes in the amino-acid sequences of the predicted protein of 189 amino acids . RPL19B is also closely linked to a mitochondrial ADP/ATP carrier protein gene AAC2 . Yeast cells containing disruption of either RPL19A or RPL19B formed smaller colonies than wild-type strains; however, simultaneous deletion of both genes is lethal.

Mol Cell Biol, 1996 Sep, 16(9), 5139 - 46
A DEAD-box-family protein is required for nucleocytoplasmic transport of yeast mRNA; Liang S et al.; An enormous variety of primary and secondary mRNA structures are compatible with export from the nucleus to the cytoplasm . Therefore, there seems to be a mechanism for RNA export which is independent of sequence recognition . There nevertheless is likely to be some relatively uniform mechanism which allows transcripts to be packaged as ribonucleoprotein particles, to gain access to the periphery of the nucleus and ultimately to translocate across nuclear pores . To study these events, we and others have generated temperature-sensitive recessive mRNA transport (mtr) mutants of Saccharomyces cerevisiae which accumulate poly(A)+ RNA in the nucleus at 37 degrees C . Several of the corresponding genes have been cloned . Upon depletion of one of these proteins, Mtr4p, conspicuous amounts of nuclear poly(A)+ RNA accumulate in association with the nucleolus . Corresponding dense material is also seen by electron microscopy . MTR4 is essential for growth and encodes a novel nuclear protein with a size of approximately 120 kDa . Mtr4p shares characteristic motifs with DEAD-box RNA helicases and associates with RNA . It therefore may well affect RNA conformation . It shows extensive homology to a human predicted gene product and the yeast antiviral protein Ski2p . Critical residues of Mtr4p, including the mtr4-1 point mutation, have been identified . Mtr4p may serve as a chaperone which translocates or normalizes the structure of mRNAs in preparation for export.

Mol Cell Biol, 1996 Sep, 16(9), 5117 - 26
Stable episomal maintenance of yeast artificial chromosomes in human cells; Simpson K et al.; Plasmids carrying the Epstein-Barr virus origin of plasmid replication (oriP) have been shown to replicate autonomously in latently infected human cells (J . Yates, N . Warren, D . Reisman, and B . Sugden, Proc . Natl . Acad . Sci . USA 81:3806-3810, 1984) . We demonstrate that addition of this domain is sufficient for stable episomal maintenance of yeast artificial chromosomes (YACs), up to at least 660 kb, in human cells expressing the viral protein EBNA-1 . To better approximate the latent viral genome, YACs were circularized before addition of the oriP domain by homologous recombination in yeast cells . The resulting OriPYACs were maintained as extrachromosomal molecules over long periods in selection; a 90-kb OriPYAC was unrearranged in all cell lines analyzed, whereas the intact form of a 660-kb molecule was present in two of three cell lines . The molecules were also relatively stable in the absence of selection . This finding indicates that the oriP-EBNA-1 interaction is sufficient to stabilize episomal molecules of at least 660 kb and that such elements do not undergo rearrangements over time . Fluorescence in situ hybridization analysis demonstrated a close association of OriPYACs, some of which were visible as pairs, with host cell chromosomes, suggesting that the episomes replicate once per cell cycle and that stability is achieved by attachment to host chromosomes, as suggested for the viral genome . The wide availability of YAC libraries, the ease of manipulation of cloned sequences in yeast cells, and the episomal stability make OriPYACs ideal for studying gene function and control of gene expression.

Mol Cell Biol, 1996 Sep, 16(9), 4818 - 23
Agonist-specific conformational changes in the yeast alpha-factor pheromone receptor; Bukusoglu G et al.; The yeast alpha-factor pheromone receptor is a member of the G-protein-coupled receptor family . Limited trypsin digestion of yeast membranes was used to investigate ligand-induced conformational changes in this receptor . The agonist, alpha-factor, accelerated cleavage in the third intracellular loop, whereas the antagonist, desTrp1,Ala3-alpha-factor, reduced the cleavage rate . Thus, the enhanced accessibility of the third intracellular loop is specific to the agonist . alpha-Factor inhibited cleavage weakly at a second site near the cytoplasmic terminus of the seventh transmembrane helix, whereas the antagonist showed a stronger inhibition of cleavage at this site and at another site in the C-terminal domain of the receptor . The alpha-factor-induced conformational changes appeared to be inherent properties of the receptor, as they were retained in G-protein-deficient mutants . Moreover, a mutant receptor (ste2-L236H) that affects the third loop and is defective for G-protein coupling retained the ability to undergo the agonist-induced conformational changes . These results are consistent with a model in which G-protein activation is limited by the availability of specific contacts between the G protein and the third intracellular loop of the receptor . The antagonist appears to promote a distinct conformational state that differs from either the unoccupied or the agonist-occupied state.

Mol Cell Biol, 1996 Sep, 16(9), 4782 - 9
Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase; Chanet R et al.; Suppressors of the methyl methanesulfonate sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase turned out to contain semidominant mutations in Rad5l, a homolog of the bacterial RecA protein . The nature of these mutations was determined by direct sequencing . The 26 mutations characterized were single base substitutions leading to amino acid replacements at 18 different sites . The great majority of these sites (75%) are conserved in the family of RecA-like proteins, and 10 of them affect sites corresponding to amino acids in RecA that are probably directly involved in ATP reactions, binding, and/or hydrolysis . Six mutations are in domains thought to be involved in interaction between monomers; they may also affect ATP reactions . By themselves, all the alleles confer a rad5l null phenotype . When heterozygous, however, they are, to varying degrees, negative semidominant for radiation sensitivity; presumably the mutant proteins are coassembled with wild-type Rad51 and poison the resulting nucleofilaments or recombination complexes . This negative effect is partially suppressed by an SRS2 deletion, which supports the hypothesis that Srs2 reverses recombination structures that contain either mutated proteins or numerous DNA lesions.

Hum Genet, 1996 Sep, 98(3), 321 - 2
Yeast artificial chromosome mapping of the cystinosis locus on chromosome 17p by fluorescence in situ hybridization; Stec I et al.; The gene locus for cystinosis has been mapped between markers D17S1583 and D17S1584 on the short arm of chromosome 17 . Using markers encompassing the cystinosis region, we assigned different yeast artificial chromosome (YAC) clones previously identified by sequence tagged site (STS) screening to 17p13.3 . Three of the clones hybridized to the target 17p gene region; one of these was chimeric, hybridizing both to chromosomes 3p and 5q; two of the YACs did not contain sequences of 17p13.3 . Our physical mapping has identified candidate YACs as a first step towards a positional cloning approach.

J Biol Chem, 1996 Aug 30, 271(35), 21062 - 7
RRN11 encodes the third subunit of the complex containing Rrn6p and Rrn7p that is essential for the initiation of rDNA transcription by yeast RNA polymerase I; Lalo D et al.; A new gene, RRN11, has been defined by certain rrn mutants of Saccharomyces cerevisiae which are defective specifically in the transcription of 35 S rRNA gene by RNA polymerase I (pol I) . We have cloned the gene and found that it encodes a protein of 507 amino acids . We have used a strain with the chromosomal RRN11 deleted and carrying HA1 epitope-tagged RRN11 on a plasmid to isolate a protein complex containing the protein encoded by RRN11 . This protein complex complemented rrn6 mutant extracts, which were previously shown to be deficient in the essential pol I transcription factor called Rrn6/7 complex or core factor (CF) . The CF complex was previously shown to consist of three proteins, the 102- and 60-kDa subunits encoded by RRN6 and RRN7, respectively, and the 66-kDa subunit . The results of the above complementation experiments combined with mobility of Rrn11p in SDS-polyacrylamide gel electrophoresis analysis relative to Rrn6p and Rrn7p led to the conclusion that RRN11 encodes the 66-kDa subunit of CF . Glutathione S-transferase-Rrn11p fusion protein was found to bind strongly to 35S-labeled Rrn6p and Rrn7p but only weakly to 35S-labeled TATA-binding protein . Similarly, glutathione S-transferase-Rrn7p fusion protein bound strongly to 35S-labeled Rrn6p and Rrn11p but only weakly to 35S-labeled TATA-binding protein . These results are consistent with the fact that one can purify CF consisting of Rrn6p, Rrn7p, and Rrn11p from yeast cell extracts, but the purified complex does not contain TATA-binding protein . RRN11 was shown to be an essential gene, and {3H}uridine pulse experiments demonstrated directly that RRN11 is essential for rDNA transcription by pol I in vivo . Thus all three subunits of CF are essential for rDNA transcription . Because of the resemblance of CF to mammalian essential pol I transcription factor SL1, the amino acid sequences of Rrn11p and the other two subunits of CF were compared with those of the three TATA-binding protein-associated factors (TAFs) in the human SL1, TAFI48, TAFI63, and TAFI110 . No significant similarity was detected between two sets of the proteins . Similarity as well as differences between CF and SL1 are discussed.

Mol Gen Genet, 1996 Aug 27, 252(1-2), 79 - 86
Human DNA topoisomerases II alpha and II beta can functionally substitute for yeast TOP2 in chromosome segregation and recombination; Jensen S et al.; The ability of the human DNA topoisomerase II alpha and II beta isozymes to complement functional defects conferred by conditional top2 mutations in Saccharomyces cerevisiae has been investigated . At the restrictive temperature, top2 strains show multiple abnormalities, including an inability to complete mitotic and meiotic division owing to a defect in chromosome segregation, and hyper-recombination within the repetitive rDNA gene cluster . We show that the human topoisomerases II alpha and II beta can each support both vegetative growth and the production of viable spores in a top2-4 mutant at the restrictive temperature . Similarly, both human isozymes can rescue a strain carrying a top2 gene disruption, and suppress hyper-recombination within the rDNA gene cluster . We conclude that the human topoisomerase II alpha and II beta isozymes are functionally interchangeable with yeast topoisomerase II and suggest that any isozyme-specific roles in human cells are likely to be dependent upon factors other than inherent differences in catalytic ability between the alpha and beta isozymes.

FEBS Lett, 1996 Aug 26, 392(2), 194 - 200
Elevated cytosolic free Ca2+ concentrations and massive Ca2+ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca2+ -ATPase; Halachmi D et al.; The Ca2+ -ATPase homolog of Saccharomyces cerevisiae, PMR1, cloned by Rudolph et al . (Cell 58 (1989) 133-145) is required for normal Golgi functions . We have investigated the role of Pmr1-protein in maintaining homeostasis of cytosolic free Ca2+ concentration ({Ca2+}i) . It was found that exposure to moderately high Ca2+ concentrations led to elevated levels of {Ca2+}i in cells of pmr1 null mutant, in comparison with cells of pmr2 isogenic mutant (defective in cell-membrane Na+ - ATPase) and of an isogenic wild type . In addition, we showed that PMR1 deletion causes massive accumulation of Ca2+ in the vacuoles and affects the rates of Ca2+ influx and efflux.

Cell, 1996 Aug 23, 86(4), 679 - 89
The invariant U5 snRNA loop 1 sequence is dispensable for the first catalytic step of pre-mRNA splicing in yeast; O'Keefe RT et al.; We have developed an in vitro reconstitution system to investigate the role of U5 snRNA in the two catalytic steps of pre-mRNA splicing . The invariant U5 loop 1 is known to interact with exon sequences at the 5' splice site before the first catalytic step . Remarkably, analysis of U5 mutations in vitro reveals that the first transesterification occurs accurately in the absence of the U5 loop . Therefore this sequence is not an essential component of the spliceosomal active site for the first catalytic step . The second catalytic step, although strongly dependent on the presence of a U5 loop to tether the exon 1 splicing intermediate, is surprisingly tolerant of mutations in the invariant sequence.

J Biol Chem, 1996 Aug 23, 271(34), 20307 - 14
Mutations in the PAY5 gene of the yeast Yarrowia lipolytica cause the accumulation of multiple subpopulations of peroxisomes; Titorenko VI et al.; We previously reported the cloning of the PAY5 gene of the yeast Yarrowia lipolytica by complementation of the peroxisome assembly mutant pay5-1 (Eitzen, G . A., Titorenko, V . I., Smith, J . J., Veenhuis, M., Szilard, R . K., and Rachubinski, R . A . (1996) J . Biol . Chem . 271, 20300-20306) . The peroxisomal integral membrane protein Pay5p is a homologue of mammalian PAF-1 proteins, which are essential for peroxisome assembly and whose mutation in humans results in peroxisome biogenesis disorders . Mutations in the PAY5 gene result in the accumulation of three distinct peroxisomal subpopulations . These subpopulations are characterized by differences in 1) buoyant density, 2) the relative distribution of peroxisomal matrix and membrane proteins, 3) the efficiency of import of several peroxisomal matrix proteins, and 4) the phospholipid levels of peroxisomal membranes . These data, together with the analysis of temporal changes in the relative abundance of individual peroxisomal subpopulations in pay5 mutants, suggest that these subpopulations represent intermediates in a multistep peroxisome assembly pathway normally operating in yeast cells.

J Biol Chem, 1996 Aug 23, 271(34), 20284 - 90
ATP synthase of yeast mitochondria . Isolation of the subunit h and disruption of the ATP14 gene; Arselin G et al.; A new subunit of the yeast ATP synthase (termed subunit h) has been isolated . Amino acid composition and N-terminal sequencing were determined by chemical methods . These data were in agreement with the sequence of the hypothetical protein L8003.20 whose primary structure was deduced from DNA sequencing of the yeast chromosome XII . The amino acid sequence encoded by ATP14 gene is 32 amino acids longer than the mature protein, which contains 92 amino acids corresponding to a calculated mass of 10,408 Da . The protein is hydrophilic and acidic with a calculated pHi of 4.08 . It is not apparently related to any subunit described in other ATP synthases . A null mutant was constructed . The mutation was recessive and the mutant strain was unable to grow on glycerol medium . A high percentage of rho- cells arose spontaneously . The mutant mitochondria had no detectable oligomycin-sensitive ATPase activity, but still contained ATPase activity with a catalytic sector dissociated from the membranous components . The mutant mitochondria did not contain subunit h, and the mitochondrially encoded hydrophobic subunit 6 was not present.

J Biol Chem, 1996 Aug 23, 271(34), 20273 - 83
Regulation of membrane and subunit interactions by N-myristoylation of a G protein alpha subunit in yeast; Song J et al.; Initiation of the mating process in yeast Saccharomyces cerevisiae requires the action of secreted pheromones and G protein-coupled receptors . As in other eukaryotes, the yeast G protein alpha subunit undergoes N-myristoylation (GPA1 gene product, Gpa1p) . This modification appears to be essential for function, since a myristoylation site mutation exhibits the null phenotype in vivo (gpa1(G2A)) . Here we examine how myristoylation affects Gpa1p activity in vitro . We show that the G2A mutant of Gpa1p, when fused with glutathione S-transferase, can still form a complex with the G protein betagamma subunits . The complex is stabilized by GDP and is dissociated upon treatment with guanosine 5'-O-(thiotriphosphate) . In addition, there is no apparent difference in the relative binding affinity of Gbetagamma for mutant and wild-type Gpa1p . Using sucrose density gradient fractionation of cell membranes, Gpa1p associates normally with the plasma membrane whereas Gpa1pG2A is mislocalized to a microsomal membrane fraction . A portion of Gbetagamma is also mislocalized in these cells, as it is in a gpa1Delta strain . In contrast, wild-type Gpa1p reaches the plasma membrane in cells that do not express Gbetagamma or cell surface receptors . These findings indicate that mislocalization of Gpa1pG2A is not caused by a redistribution of Gbetagamma, nor is it the result of any difference in Gbetagamma binding affinity . These data suggest that myristoylation is required for specific targeting of Gpa1p to the plasma membrane, where it is needed to interact with the receptor and to regulate the release of Gbetagamma.

Biochemistry, 1996 Aug 20, 35(33), 10938 - 48
Mutational analysis of the catalytic subunit of the yeast vacuolar proton-translocating ATPase; Liu J et al.; In order to generate a set of tools for probing structure-function relationships in the catalytic subunit of the yeast vacuolar H(+)-ATPase, the gene encoding this subunit (VMA1) was randomly mutagenized . Mutant plasmids unable to complement the growth defects of yeast cells lacking an intact VMA1 gene were isolated and sequenced . Eight different mutant alleles of VMA1 were examined for levels of the catalytic subunit and other subunits of the enzyme, assembly of the ATPase complex, targeting to the vacuolar membrane, and concanamycin A-sensitive ATPase activity . The mutations S811P and E740D resulted in mutant enzymes that assembled fully but were incapable of ATP hydrolysis, and the mutation E785G generated a similar but somewhat less severe phenotype (17% of the ATPase activity of wild-type vacuoles) . When MgATP-dependent stripping of the peripheral subunits by 100 mM KNO3 was examined in these three mutants, only the E785G mutant exhibited significant stripping, suggesting that ATP hydrolysis, even at relatively low levels, generates a conformation susceptible to dissociation . Plasmids containing the mutations E751G and F752S partially complemented the growth defects and resulted in partial defects in ATPase activity that appear to reflect reduced catalytic efficiency . Partial defects in growth and ATPase activity were also seen in the Y797H mutant, but this mutation caused an assembly defect manifested as a preferential loss of two of the peripheral subunits of the enzyme . The phenotypes of these mutants are interpreted in the context of homologies with other V-type and F-type ATPases.

Biochemistry, 1996 Aug 20, 35(33), 10727 - 34
Cleavages induced by different metal ions in yeast tRNA(Phe) U59C60 mutants; Michalowski D et al.; The U59 and C60 residues, which form the strong Pb(II) ion binding site in yeast tRNA(Phe), were systematically mutated, and the effects of individual mutations on cleavages induced by various metal ions were analyzed . It turned out that the presence of C60 is essential for efficient cleavage of the D-loop by Pb(II), Eu(III), and Mg(II)ions . On the other hand, manganese ions were capable of cleaving these mutants with an efficiency similar to that observed for the wild type transcript . Moreover, in all C60 mutants, the main Mn(II) cleavage site was shifted by one phosphate from P16 to P17 . Mutations of U59 did not affect so dramatically the efficiency and specificity of the D-loop hydrolysis induced by all studied metal ions . In the G59C60 mutant cleaved by Pb(II) ions, new cuts took place in the T-stem at P63-P65 . Also, the C60 mutants were cleaved more strongly at P49 by Pb(II) ions . In G59C60 and A59C60 as well as in all C60 mutants, the Mg(II) and Mn(II) cleavage at P61 was suppressed . Nevertheless, the changes in overall tRNA structure resulting from U59 and C60 mutations were rather subtle . The studied mutants showed S1 and V1 nuclease digestion patterns practically indistinguishable from those observed in the wild type transcript . The metal ions are shown to be well-suited for monitoring the local changes in the structure of the investigated tRNA variants and when used as a set of probes can give a more complete picture of changes that occur in transcripts as a result of a mutation.

Science, 1996 Aug 16, 273(5277), 953 - 6
Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption; Hardwick KG et al.; The spindle assembly checkpoint keeps cells with defective spindles from initiating chromosome segregation . The protein kinase Mps1 phosphorylates the yeast protein Mad1p when this checkpoint is activated, and the overexpression of Mps1p induces modification of Mad1p and arrests wild-type yeast cells in mitosis with morphologically normal spindles . Spindle assembly checkpoint mutants overexpressing Mps1p pass through mitosis without delay and can produce viable progeny, which demonstrates that the arrest of wild-type cells results from inappropriate activation of the checkpoint in cells whose spindle is fully functional . Ectopic activation of cell-cycle checkpoints might be used to exploit the differences in checkpoint status between normal and tumor cells and thus improve the selectivity of chemotherapy.

Eur J Biochem, 1996 Aug 15, 240(1), 104 - 11
Three alpha-galactosidase genes of Trichoderma reesei cloned by expression in yeast; Margolles-Clark E et al.; Three alpha-galactosidase genes, agl1, agl2 and agl3, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside . The genes agl1, agl2 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences . The deduced amino acid sequences of AGLI and AGLIII showed similarity with the alpha-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial alpha-galactosidases of family 36 . The enzymes produced by yeast were analysed for enzymatic activity against different substrates . AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-alpha-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose . They liberated galactose from polymeric galacto(gluco)mannan with different efficiencies . The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-1,4-beta-mannanase of T . reesei . AGLII and AGLIII showed synergy in galacto(gluco)mannan hydrolysis with the endo-1,4-beta-mannanase of T . reesei and a beta-mannosidase of Aspergillus niger . The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the alpha-galactosidase previously purified from T . reesei.

Blood, 1996 Aug 15, 88(4), 1321 - 9
Human platelet cGI-PDE: expression in yeast and localization of the catalytic domain by deletion mutagenesis; Cheung PP et al.; Cyclic adenosine monophosphate (cAMP) is an important modulator of platelet responses to agonists . Cyclic nucleotide phosphodiesterase (PDE) controls intracellular cAMP concentrations by hydrolyzing it to AMP . The major PDE activity in platelets is PDE3A (cyclic guanosine monophosphate {cGMP}-inhibited PDE) . To obtain structural information on platelet PDE3A, we cloned the enzyme cDNA from a human erythroleukemia cell (HEL) library since the cell line expresses many platelet proteins . This clone consists of 87% of the full-length human myocardial PDE3A cDNA, spanning from nucleotides 456 to 4606, and is identical in sequence . The nucleotide coding for the N terminal 179 amino acid sequence (nt 1-536) as well as four other cDNAs (nt 1459-1632, nt 1765-1986, nt 2152-2538, and nt 2978-3375) obtained by RT-PCR of platelet RNA are also identical to the myocardial sequences, indicating that the HEL, myocardial, and platelet PDE3As are the same . Northern blot analysis of HEL cell RNA detected two mRNAs of 7.5 and 4.4 kb . Four new deletion mutants are reported . PDE 3A delta 1 and PDE 3A delta 2, encoding amino acids 665 to 1141 and amino acids 679 to 1141, respectively, were expressed in a PDE-deficient yeast . They displayed PDE activities of 172 and 79 pmol/mg/min, respectively . PDE 3A delta 3 and PDE 3A delta 4, encoding amino acids 686 to 1141 and 700 to 1141, had no detectable PDE activity . All mutant proteins were expressed as determined by Western blot analysis . These findings localize the PDE3A catalytic domain to within amino acid residues 679 to 1141.

Biochim Biophys Acta, 1996 Aug 14, 1308(2), 133 - 41
Involvement of lysine 270 and lysine 271 of yeast 5S rRNA binding protein in RNA binding and ribosome assembly; Yeh LC et al.; Contributions of the highly conserved K270 and its neighboring K271 in the C-terminal region of the yeast ribosomal protein L1 to 5S rRNA binding and ribosome assembly were examined by in vivo and in vitro studies on the consequences of 14 substitution mutations . All mutant proteins with a single amino-acid substitution at either position were able to bind 5S rRNA in vitro to an extent comparable to the wild-type . Yeast cells expressing these mutant proteins, except the K270G mutant, grew at nearly normal rates . Mutations of K270 appeared to produce more demonstrable effects than those of K271 . The double mutant K270,271G bound RNA poorly and yeast cells expressing the mutant protein grew 30% slower . Double mutants K270,271E and K270,271R were lethal, although the mutant protein was assembled into the 60S ribosomal subunits . The resultant subunits were not stable leading eventually to cell death . The in vitro RNA binding ability of the respective protein was reduced by 60% and 20% . Taken together, the present data identified K270 and K271 as important amino-acid residues in the function of the yeast ribosomal protein L1.

Biochemistry, 1996 Aug 13, 35(32), 10249 - 55
Three of four cysteines, including that responsible for substrate activation, are ionized at pH 6.0 in yeast pyruvate decarboxylase: evidence from Fourier transform infrared and isoelectric focusing studies; Baburina I et al.; Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at three of the four cysteines (152, 221, and 222), the fourth (69) being buried according to X-ray crystallographic results {Arjunan et al . (1996) J . Mol . Biol . 256, 590-600} . All of the variants still retained significant activity, and all could be purified to homogeneity . FT-IR experiments were run on the C221S, C222S, C221S/C222S and C152A variants, as well as on the wild-type enzyme . There is a band present at 2557 cm-1 in the spectra of all variants and the wild-type enzyme, except in the spectrum of the C152A variant . This frequency is appropriate to a cysteine S-H stretching mode . It was therefore concluded that C152 is the only undissociated cysteine on the enzyme at pH 6.0, the pH optimum of this enzyme, whereas C221, C222, and C69 are all ionized . Isoelectric focusing experiments were carried out on all of these variants, as well as on the H92A variant (H92 is across the domain divide on the alpha domain, from C221 located on the beta domain) . The variation in isoelectric points deduced from the data was consistent with removal of negative charges concomitant with the C221S, C222S, and C221S/C222S substitutions and removal of a positive charge with the H92A substitution when compared to that of the wild-type enzyme . The results of these two types of experiments are in good accord and suggest that the site of substrate activation at C221 {Baburina et al . (1994) Biochemistry 33, 5630-5635} is comprised of a Cys221S- +HHis92 ion pair, not unlike that found in papain and glyceraldehyde-3-phosphate dehydrogenase . This finding suggests that the regulatory site of this enzyme has been optimized for nucleophilic reactivity between the thiolate of C221 and the keto carbon of the 2-oxoacid.

FEBS Lett, 1996 Aug 12, 391(3), 252 - 6
Four Arabidopsis thaliana 14-3-3 protein isoforms can complement the lethal yeast bmh1 bmh2 double disruption; van Heusden GP et al.; The 14-3-3 proteins comprise a family of highly conserved proteins with multiple functions, most of which are related to signal transduction . Four isoforms from the plant Arabidopsis thaliana were able to complement the lethal disruption of the two Saccharomyces cerevisiae genes encoding 14-3-3 proteins; one complemented very poorly and one did not complement . However, the expression of the latter two isoforms was very low . These results show that at least four of the six A . thaliana isoforms are able to perform the same function(s) as the yeast 14-3-3 proteins.

J Biol Chem, 1996 Aug 9, 271(32), 19592 - 8
Wild-type and mutant vacuolar membranes support pH-dependent reassembly of the yeast vacuolar H+-ATPase in vitro; Parra KJ et al.; Treatment of the yeast vacuolar proton-translocating ATPase (H+-ATPase) with 300 mM KI in the presence of 5 mM MgATP results in a 90% inhibition of ATPase activity accompanied by removal of at least five of the peripheral subunits of the enzyme from the membrane . Functional reassembly of the enzyme, as indicated by reattachment of the peripheral subunits and a partial (30-70%) recovery of ATPase activity, could be achieved by dialysis of the stripped wild-type membranes to remove the KI and MgATP, but proved to be strongly pH-dependent, with optimal reassembly and recovery of activity occurring after dialysis at pH 5.5 . Vacuolar membranes isolated from vma2Delta mutants, which lack one of the peripheral subunits of the enzyme, do not contain any of the peripheral subunits but are shown to contain assembled membrane (Vo) complexes . The vma2Delta mutant vacuoles are demonstrated to be competent for attachment of KI-stripped peripheral subunits and reactivation of ATPase activity . The results indicate that previously assembled Vo complexes are capable of inducing assembly of the peripheral subunits, both with each other and with the membrane subunits, and of activating the ATPase activity that resides in the peripheral subunits in a pH-dependent manner.

J Biol Chem, 1996 Aug 9, 271(32), 19376 - 84
Comparison of the rat nucleolar protein nopp140 with its yeast homolog SRP40 . Differential phosphorylation in vertebrates and yeast; Meier UT; Rat Nopp140, a nonribosomal protein of the nucleolus and coiled bodies, was characterized as one of the most highly phosphorylated proteins in the cell . Based on its subcellular location, its nuclear localization signal binding capacity, and its shuttling between the nucleolus and the cytoplasm, Nopp140 was proposed to function as a chaperone in ribosome biogenesis . This study shows that casein kinase II phosphorylates Nopp140 to its unusual high degree and identifies the yeast SRP40 gene product as immunologically and structurally related to rat Nopp140 . SRP40 encodes an acidic (pI = 3 . 9), serine-rich (49%) protein of 41 kDa whose carboxyl terminus exhibits 59% sequence identity to that of Nopp140 . SRP40 localizes to the yeast nucleolus and is required at a specific cellular concentration for optimal growth as indicated by the negative effect on cell growth of both overexpression and deletion of its gene . Like Nopp140, SRP40 is phosphorylated by casein kinase II, but to a much lesser extent . While the parallels between these two proteins suggest that SRP40 is the bona fide yeast Nopp140 homolog, their disparities reflect the differences in nucleolar dynamics and regulation of ribosome biogenesis between yeast and vertebrates.

J Biol Chem, 1996 Aug 9, 271(32), 19318 - 23
Cell cycle arrest promotes trans-hammerhead ribozyme action in yeast; Ferbeyre G et al.; A hammerhead ribozyme designed to cleave the yeast ADE1 mRNA has been expressed in yeast under the control of a galactose-inducible promoter . RNA prepared from the galactose-induced yeast cultures possesses an activity that cleaves ADE1 mRNA in vitro . However, in spite of high expression levels of the ribozyme, no cleavage activity could be demonstrated in vivo . On the other hand, when the yeast cells expressing hammerhead RNA were treated with the alpha-factor mating pheromone, the level of ADE1 mRNA was reduced by 50% . Similar reductions were observed when this strain was cultured in the presence of lithium acetate or in nitrogen-free medium . Moreover, control experiments in which disabled hammerhead genes were expressed showed no such reductions . Extension of the length of the flanking recognition arms of the ribozyme from a total of 10 to 16 or 24 nucleotides diminished the inhibitory effect of the ribozyme . These data suggest that ribozymes are able to cleave a trans-RNA target in yeast.

Proc Natl Acad Sci U S A, 1996 Aug 6, 93(16), 8274 - 7
Association of yeast SIN1 with the tetratrico peptide repeats of CDC23; Shpungin S et al.; The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes . In addition, sin1 mutants are defective in proper mitotic chromosome segregation . In an effort to understand the basis for these phenotypes, we employed the yeast two-hybrid system to identify proteins that interact with SIN1 in vivo . Here we demonstrate that CDC23, a protein known to be involved in sister chromatid separation during mitosis, is able to directly interact with SIN1 . Furthermore, using recombinant molecules in vitro, we show that the N terminal of SIN1 is sufficient to bind a portion of CDC23 consisting solely of tetratrico peptide repeats . Earlier experiments identified the C-terminal domain of SIN1 to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SIN1 . Taken together with the results presented here, we suggest that SIN1 is a chromatin protein having at least a dual function: The N terminal of SIN1 interacts with the tetratrico peptide repeat domains of CDC23, a protein involved in chromosome segregation, whereas the C terminal of SIN1 binds proteins involved in transcriptional regulation.

Science, 1996 Aug 2, 273(5275), 622 - 6
Support for the prion hypothesis for inheritance of a phenotypic trait in yeast; Patino MM et al.; A cytoplasmically inherited genetic element in yeast, {PSI+}, was confirmed to be a prionlike aggregate of the cellular protein Sup35 by differential centrifugation analysis and microscopic localization of a Sup35-green fluorescent protein fusion . Aggregation depended on the intracellular concentration and functional state of the chaperone protein Hsp104 in the same manner as did {PSI+} inheritance . The amino-terminal and carboxy-terminal domains of Sup35 contributed to the unusual behavior of {PSI+} . {PSI+} altered the conformational state of newly synthesized prion proteins, inducing them to aggregate as well, thus fulfilling a major tenet of the prion hypothesis.

J Electron Microsc (Tokyo), 1996 Aug, 45(4), 321 - 4
Translocation of hepatitis B virus core particles through nuclear pores in transformed yeast cells; Yamaguchi M et al.; In our previous transmission electron microscopic study of hepatitis B virus core antigen in transformed yeast cells, we observed core particles passing through the nuclear pores . We have now analyzed 1,421 nuclear pores in transformed yeast, and conclude that 1) translocation of core particles from the nucleus to the cytoplasm occurs through the nuclear pores; 2) translocation sites are located in the center of nuclear pores; 3) at least 95% of pores are involved in the translocation process; 4) proteins as large as 28 nm in diameter can cross the envelope; 5) translocation does not stop, but rather becomes more active during nuclear division in yeast cells.

Yeast, 1996 Aug, 12(10), 917 - 23
Flavin adenine dinucleotide binding is the crucial step in alcohol oxidase assembly in the yeast Hansenula polymorpha; Evers ME et al.; We have studied the role of flavin adenine dinucleotide (FAD) in the in vivo assembly of peroxisomal alcohol oxidase (AO) in the yeast Hansenula polymorpha . In previous studies, using a riboflavin (Rf) autotrophic mutant, an unequivocal judgement could not be made, since Rf-limitation led to a partial block of AO import in this mutant . This resulted in the accumulation of AO precursors in the cytosol where they remained separated from the putative peroxisomal AO assembly factors . In order to circumvent the peroxisomal membrane barrier, we have now studied AO assembly in a peroxisome-deficient/Rf-autotrophic double mutant (delta per1.rif1) of H . polymorpha . By sucrose density centrifugation and native gel electrophoresis, three conformations of AO were detected in crude extracts of delta per1.rif1 cells grown under Rf-limitation, namely active octameric AO and two inactive, monomeric forms . One of the latter forms lacked FAD; this form was barely detectable in extracts wild-type and delta per1 cells, but had accumulated in the cytosol of rif1 cells . The second form of monomeric AO contained FAD; this form was also present in delta per1 cells but absent/very low in wild-type and rif1 cells . In vivo only these FAD-containing monomers associate into the active, octameric protein . We conclude that in H . polymorpha FAD binding to the AO monomer is mediated by a yet unknown peroxisomal factor and represents the crucial and essential step to enable AO oligomerization; the actual octamerization and the eventual crystallization in peroxisomes most probably occurs spontaneously.

Lipids, 1996 Aug, 31(8), 785 - 802
Phosphatidate phosphatases of mammals, yeast, and higher plants; Kocsis MG et al.; Phosphatidate phosphatase (EC 3.1.3.4) catalyzes the hydrolysis of phosphatidate to yield sn-1,2-diacylglycerol and inorganic phosphate . In mammalian systems, forms of phosphatidate phosphatase involved in glycerolipid synthesis and signal transduction have been identified . Forms of the enzyme involved in signal transduction have been purified and partially characterized . In yeast, phosphatidate phosphatases associated with the endoplasmic reticulum and mitochondria have also been purified and partially characterized . Information on phosphatidate phosphatases from mammals and yeast is useful in characterizing the enzyme from plant systems . This review examines progress on the characterization of phosphatidate phosphatases from mammals, yeast, and higher plants . The purification and characterization of the phosphatidate phosphatase involved in glycerolipid synthesis in developing oilseeds may lead to the identification of the encoding gene . Increasing our understanding of the enzymes of lipid synthesis in developing seeds will aid in the development of biotechnological strategies for seed oil modification.

Mol Biol Cell, 1996 Aug, 7(8), 1195 - 208
Abnormal kinetochore structure activates the spindle assembly checkpoint in budding yeast; Pangilinan F et al.; Saccharomyces cerevisiae cells containing one or more abnormal kinetochores delay anaphase entry . The delay can be produced by using centromere DNA mutations present in single-copy or kinetochore protein mutations . This observation is strikingly similar to the preanaphase delay or arrest exhibited in animal cells that experience spontaneous or induced failures in bipolar attachment of one or more chromosomes and may reveal the existence of a conserved surveillance pathway that monitors the state of chromosome attachment to the spindle before anaphase . We find that three genes (MAD2, BUB1, and BUB2) that are required for the spindle assembly checkpoint in budding yeast (defined by antimicrotubule drug-induced arrest or delay) are also required in the establishment and/or maintenance of kinetochore-induced delays . This was tested in strains in which the delays were generated by limited function of a mutant kinetochore protein (ctf13-30) or by the presence of a single-copy centromere DNA mutation (CDEII delta 31) . Whereas the MAD2 and BUB1 genes were absolutely required for delay, loss of BUB2 function resulted in a partial delay defect, and we suggest that BUB2 is required for delay maintenance . The inability of mad2-1 and bub1 delta mutants to execute kinetochore-induced delay is correlated with striking increases in chromosome missegregation, indicating that the delay does indeed have a role in chromosome transmission fidelity . Our results also indicated that the yeast RAD9 gene, necessary for DNA damage-induced arrest, had no role in the kinetochore-induced delays . We conclude that abnormal kinetochore structures induce preanaphase delay by activating the same functions that have defined the spindle assembly checkpoint in budding yeast.

Genetics, 1996 Aug, 143(4), 1533 - 42
Mutations in the yeast SRB2 general transcription factor suppress hpr1-induced recombination and show defects in DNA repair; Piruat JI et al.; We have obtained genetic and molecular evidence that the hrs2-1 mutation, isolated as a suppressor of the hyperrecombination phenotype of hpr1 delta, is in the SRB2 gene, which encodes a component of the RNA polII holoenzyme . A newly constructed srb2 delta allele restores the wild-type levels of deletions in hpr1 delta cells, indicating that the lack of a functional SRB2 transcription factor suppresses recombination between direct repeats . These results suggest a direct connection between transcription and recombination between DNA repeats . On the other hand, the hrs2-1 mutation (renamed srb2-101), in which Gly150 has been changed to Asp, makes cells sensitive to long MMS treatments, a phenotype observed for the srb2 delta null allele only in a hpr1 delta background . This indicates that mutations in the basal transcription factor SRB2 impair DNA repair of MMS-induced damage, which adds a new connection between transcription and DNA repair . We discuss the possibility that hpr1-induced deletions occurred as a consequence of a SRB2-dependent stalled or blocked transcription complex.

Int J Biol Macromol, 1996 Aug, 19(2), 113 - 9
Comparison of inactivation and unfolding of yeast alcohol dehydrogenase during denaturation in urea solutions; Zhang T et al.; It has been reported that inactivation occurs before noticeable conformational changes can be detected during the denaturation of a number of enzymes by denaturants . Therefore, Tsou suggested that the conformation of the enzyme active sites is more easily perturbed and hence more flexible than the molecule as a whole (Tsou, Trends in Biochemical Science, 1986, 11, 427-429; Tsou, Science, 1993, 262, 380-381) . In this study, inactivation and unfolding of yeast alcohol dehydrogenase containing zinc ions in urea solutions of different concentrations are compared . The results show that much lower concentrations of urea are required to bring about inactivation than significant unfolding of the enzyme molecule . At the same urea concentration, inactivation rate is much faster than that of the unfolding of the enzyme molecule as a whole . The results suggest that active sites of yeast alcohol dehydrogenase containing zinc ions also display more conformational flexibility than the enzyme molecules as a whole.

Biophys J, 1996 Aug, 71(2), 868 - 77
Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation; Neumann E et al.; Detailed kinetic data suggest that the direct transfer of plasmid DNA (YEp 351, 5.6 kbp, supercoiled, Mr approximately 3.5 x 10(6)) by membrane electroporation of yeast cells (Saccharomyces cerevisiae, strain AH 215) is mainly due to electrodiffusive processes . The rate-limiting step for the cell transformation, however, is a bimolecular DNA-binding interaction in the cell interior . Both the adsorption of DNA, directly measured with {32P}dCTP DNA, and the number of transformants are collinearly enhanced with increasing total concentrations {Dt} and {Cat} of DNA and of calcium, respectively . At {Cat} = 1 mM, the half-saturation or equilibrium constant is KD = 15 +/- 1 nM at 293 K (20 degrees C) . The optimal transformation frequency is TFopt = 4.1 +/- 0.4 X 10(-5) if a single exponential pulse of initial field strength E0 = 4 kV cm-1 and decay time constant tauE = 45 ms is applied at {Dt} = 2.7 nM and 10(8) cells in 0.1 ml . The dependence of TF on {Cat} yields the equilibrium constants KCazero = 1.8 +/- 0.2 mM (in the absence of DNA) and K'Ca (at 2.7 nM DNA), comparable with and derived from electrophoresis data . In yeast cells, too, the appearance of a DNA molecule in its whole length in the cell interior is clearly an after-field event . At Eo = 4.0 kV cm-1 and T = 293 K, the flow coefficient of DNA through the porous membrane patches is Kto = 7.0 +/- 0.7 x 10(3)S-1 and the electrodiffusion of DNA is approximately 10 times more effective than simple diffusion: D/D0 approximately 10.3 . The mean radius of these pores is rp = 0.39 +/- 0.05 nm, and the mean number of pores per cell (of size o approximately 5.5 microns) is Np = 2.2 +/- 0.2 x 10(4) . The maximal membrane area that is involved in the electrodiffusive penetration of adsorbed DNA into the outer surface of the electroporated cell membrane patches is only 0.023% of the total cell surface . The surface penetration is followed either by additional electrodiffusive or by passive (after-field) diffusive translocation of the inserted DNA into the cell interior . For practical purposes of optimal transformation efficiency, 1 mM calcium is necessary for sufficient DNA binding and the relatively long pulse duration of 20-40 ms is required to achieve efficient electrodiffusive transport across the cell wall and into the outer surface of electroporated cell membrane patches.

Protein Expr Purif, 1996 Aug, 8(1), 85 - 90
A novel collection of accessory factors associated with yeast RNA polymerase II; Wade PA et al.; A relatively simple subset of general transcription factors is sufficient for transcript initiation by RNA polymerase II . However, a recently identified "holoenzyme" contains additional accessory proteins required for mediating signals from some activators (Y-J . Kim et al., 1994, Cell 77, 599-608; A . Koleske and R . Young, 1994, Nature 368, 466-469) . By immobilizing RNA polymerase II and associated proteins (RAPs) from a transcriptionally active yeast extract, we have identified a novel collection of proteins distinct from those found in the holoenzyme . The eluted RAP fraction did not contain the holoenzyme components Srb2,4,5 + 6p, Gal11p, or Sug1p, but did include the known transcription factors TFIIB and TFIIS and the three subunits of yeast TFIIF (Ssu71p/Tfg1p, Tfg2p, and Anc1p/Tfg3p) . Also isolated as RAPs are two proteins (Cdc73p and Paf1p) with interesting connections to gene expression . Mutations in CDC73 and PAF1 affect cell growth and the abundance of transcripts from a subset of yeast genes (X . Shi et al., Mol . Cell . Biol., 1996 16, 669-676) . The RAP fraction may therefore define one or more functional forms of RNA polymerase II distinct from the activator-mediating holoenzyme.

Curr Opin Cell Biol, 1996 Aug, 8(4), 557 - 65
Generation of cell polarity in yeast; Chant J; Yeast cells constitute an excellent system for studying cell polarity . They polarize by means of internally programmed patterns of cell division; they polarize chemotropically towards a partner during mating; and they utilize polarity to segregate cell-fate determinants during division . In the past year, considerable progress has been made towards increasing our understanding of the molecular mechanisms underlying each of these processes.

Plant Cell, 1996 Aug, 8(8), 1437 - 46
Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant; Rentsch D et al.; A yeast mutant lacking SHR3, a protein specifically required for correct targeting of plasma membrane amino acid permeases, was used to study the targeting of plant transporters and as a tool to isolate new SHR3-independent amino acid transporters . For this purpose, an shr3 mutant was transformed with an Arabidopsis cDNA library . Thirty-four clones were capable of growth under selective conditions, but none showed homology with SHR3 . However, genes encoding eight different amino acid transporters belonging to three different transporter families were isolated . Five of these are members of the general amino acid permease (AAP) gene family, one is a member of the NTR family, encoding an oligopeptide transporter, and two belong to a new class of transporter genes . A functional analysis of the latter two genes revealed that they encode specific proline transporters (ProT) that are distantly related to the AAP gene family . ProT1 was found to be expressed in all organs, but highest levels were found in roots, stems, and flowers . Expression in flowers was highest in the floral stalk phloem that enters the carpels and was downregulated after fertilization, indicating a specific role in supplying the ovules with proline . ProT2 transcripts were found ubiquitously throughout the plant, but expression was strongly induced under water or salt stress, implying that ProT2 plays an important role in nitrogen distribution during water stress, unlike members of the AAP gene family whose expression was repressed under the same conditions . These results corroborate the general finding that under water stress, amino acid export is impaired whereas proline export is increased.

Plant Cell, 1996 Aug, 8(8), 1261 - 75
A pollen-, ovule-, and early embryo-specific poly(A) binding protein from Arabidopsis complements essential functions in yeast; Belostotsky DA et al.; Poly(A) tails of eukaryotic mRNAs serve as targets for regulatory proteins affecting mRNA stability and translation . Differential mRNA polyadenylation and deadenylation during gametogenesis and early development are now widely recognized as mechanisms of translational regulation in animals, but they have not been observed in plants . Here, we report that the expression of the PAB5 gene encoding one of the poly(A) binding proteins (PABPs) in Arabidopsis is restricted to pollen and ovule development and early embryogenesis . Furthermore, PAB5 is capable of rescuing a PABP-deficient yeast strain by partially restoring both poly(A) shortening and translational initiation functions of PABP . However, PAB5 did not restore the linkage of deadenylation and decapping, thus demonstrating that this function of PABP is not essential for viability . Also, like endogenous PABP, PAB5 expressed in yeast demonstrated genetic interaction with a recently characterized yeast protein SIS1, which is also involved in translational initiation . We propose that PAB5 encodes a post-transcriptional regulatory factor acting through molecular mechanisms similar to those reported for yeast PABP . This factor may have evolved further to post-transcriptionally regulate plant sexual reproduction and early development.

J Cell Biol, 1996 Aug, 134(4), 949 - 61
Aberrant mitosis in fission yeast mutants defective in fatty acid synthetase and acetyl CoA carboxylase; Saitoh S et al.; Two fission yeast temperature-sensitive mutants, cut6 and lsd1, show a defect in nuclear division . The daughter nuclei differ dramatically in size (the phenotype designated lsd, large and small daughter) . Fluorescence in situ hybridization (FISH) revealed that sister chromatids were separated in the lsd cells, but appeared highly compact in one of the two daughter nuclei . EM showed asymmetric nuclear elongation followed by unequal separation of nonchromosomal nuclear structures in these mutant nuclei . The small nuclei lacked electron-dense nuclear materials and contained highly compacted chromatin . The cut6+ and lsd1+ genes are essential for viability and encode, respectively, acetyl CoA carboxylase and fatty acid synthetase, the key enzymes for fatty acid synthesis . Gene disruption of lsd1+ led to the lsd phenotype . Palmitate in medium fully suppressed the phenotypes of lsd1 . Cerulenin, an inhibitor for fatty acid synthesis, produced the lsd phenotype in wild type . The drug caused cell inviability during mitosis but not during the G2-arrest induced by the cdc25 mutation . A reduced level of fatty acid thus led to impaired separation of non-chromosomal nuclear components . We propose that fatty acid is directly or indirectly required for separating the mother nucleus into two equal daughters.

J Virol, 1996 Aug, 70(8), 5548 - 56
A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag protein; Merkulov GV et al.; Cleavage of the Gag and Gag-Pol polyprotein precursors is a critical step in proliferation of retroviruses and retroelements . The Ty1 retroelement of Saccharomyces cerevisiae forms virus-like particles (VLPs) made of the Gag protein . Ty1 Gag is not obviously homologous to the Gag proteins of retroviruses . The apparent molecular mass of Gag is reduced from 58 to 54 kDa during particle maturation . Antibodies raised against the C-terminal peptide of Gag react with the 58-kDa polypeptide but not with the 54-kDa one, indicating that Gag is proteolytically processed at the C terminus . A protease cleavage site between positions 401 and 402 of the Gag precursor was defined by carboxy-terminal sequencing of the processed form of Gag . Certain deletion and substitution mutations in the C terminus of the Gag precursor result in particles that are two-thirds the diameter of the wild-type VLPs . While the Ty1 protease is active in these mutants, their transposition rates are decreased 20-fold compared with that of wild-type Ty1 . Thus, the Gag C-terminal portion, released in the course of particle maturation, probably plays a significant role in VLP morphogenesis and Ty1 transposition.

Microbiology, 1996 Aug, 142 ( Pt 8), 2289 - 97
LAG2, a gene that determines yeast longevity; Childress AM et al.; Saccharomyces cerevisiae has a limited life span, measured by the reproductive capacity of the individual cell . Several genes that are differentially expressed during the yeast life span have been isolated . One of these genes, LAG2, has been characterized for its role in longevity . LAG2 is preferentially expressed in young cells . It encodes a predicted 680 amino acid protein with a putative transmembrane helix . The sequences does not show significant similarity to any other DNA or protein sequences in the databases . Deletion of LAG2 in a haploid strain did not affect growth, but it resulted in a 50% decrease in the mean and maximum life span . When LAG2 was overexpressed, the mean and maximum life span of the yeasts was extended by about 36% and 54%, respectively . These results indicate that this is a longevity-assurance gene in yeast.

Nucleic Acids Res, 1996 Aug 1, 24(15), 2885 - 93
Transcriptional terminators of RNA polymerase II are associated with yeast replication origins; Chen S et al.; The compact organization of the Saccharomyces cerevisiae genome necessitates that non-coding regulatory sequences reside in close proximity to one another . Here we show there is an intimate association between transcription terminators and DNA replication origins . Four replication origins were analyzed in a reporter gene assay that detects sequences that direct 3' end formation of mRNA transcripts . All four replication origins function as orientation-independent transcription terminators in this system, producing truncated polyadenylated mRNAs . Despite this close association, the cis-acting elements that confer replication origin function are genetically separable from those required for transcription termination . Several models are explored in an attempt to address how and why the signals specifying transcription termination and replication initiation overlap.

Invest Ophthalmol Vis Sci, 1996 Aug, 37(9), 1877 - 83
Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis . Sequence homology with yeast nucleopore complex protein; Bora NS et al.; PURPOSE . Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation . The current studies were undertaken to determine the primary structure of this protein . METHODS . A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library . The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced . Southern blot analysis was used to study the genomic hybridization pattern . p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells . RESULTS . The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively . The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100 . Southern blot analysis of human genomic DNA revealed a simple hybridization pattern . Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein . CONCLUSIONS . The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library . Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein . Thus, we refer to this protein as nup36.

Genes Dev, 1996 Aug 1, 10(15), 1917 - 29
A widespread transposable element masks expression of a yeast copper transport gene; Knight SA et al.; The trace element copper (Cu) is essential for cell growth . In this report we describe the identification of a new component of the high-affinity Cu transport machinery in yeast, encoded by the CTR3 gene . Ctr3p is a small intracellular cysteine-rich integral membrane protein that restores high-affinity Cu uptake, Cu, Zn superoxide dismutase activity, ferrous iron transport, and respiratory proficiency to strains lacking the CTR1 (Cu transporter 1) gene . In most commonly used Saccharomyces cerevisiae laboratory strains, expression of CTR3 is abolished by a Ty2 transposon insertion that separates the CTR3 promoter from the transcriptional start sites by 6 kb . In strains that do not possess a Ty2 transposon at the CTR3 locus, expression of CTR3 is repressed by copper and activated by copper starvation . In such strains inactivation of both CTR1 and CTR3 is required to generate lethal copper-deficient phenotypes . Although Ctr1p and Ctr3p can function independently in copper transport, the expression of both proteins provides maximal copper uptake and growth rate under copper-limiting conditions . These results underscore the importance of mobile DNA elements in the alteration of gene function and phenotypic variation.

J Bacteriol, 1996 Aug, 178(15), 4420 - 8
A novel autonomously replicating sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-1; Sohn JH et al.; Several autonomously replicating sequences of Hansenula polymorpha DL-1 (HARSs) with the characteristics of tandem integration were cloned by an enrichment procedure and analyzed for their functional elements to elucidate the mechanism of multiple integration in tandem repeats . All plasmids harboring newly cloned HARSs showed a high frequency of transformation and were maintained episomally before stabilization . After stabilization, the transforming DNA was stably integrated into the chromosome . HARS36 was selected for its high efficiency of transformation and tendency for integration . Several tandemly repeated copies of the transforming plasmid containing HARS36 (pCE36) integrated into the vicinity of the chromosomal end . Bal 31 digestion of the total DNA from the integrants followed by Southern blotting generated progressive shortening of the hybridization signal, indicating the telomeric localization of the transforming plasmids on the chromosome . The minimum region of HARS36 required for its HARS activity was analyzed by deletion analyses . Three important regions, A, B, and C, for episomal replication and integration were detected . Analysis of the DNA sequences of regions A and B required for the episomal replication revealed that region A contained several AT-rich sequences that showed sequence homology with the ARS core consensus sequence of Saccharomyces cerevisiae . Region B contained two directly repeated sequences which were predicted to form a bent DNA structure . Deletion of the AT-rich core in region A resulted in a complete loss of ARS activity, and deletion of the repeated sequences in region B greatly reduced the stability of the transforming plasmid and resulted in retarded cell growth . Region C was required for the facilitated chromosomal integration of transforming plasmids.

Mol Cell Biol, 1996 Aug, 16(8), 4414 - 25
A double-strand break within a yeast artificial chromosome (YAC) containing human DNA can result in YAC loss, deletion or cell lethality; Bennett CB et al.; Human chromosomal DNA contains many repeats which might provide opportunities for DNA repair . We have examined the consequences of a single double-strand break (DSB) within a 360-kb dispensable yeast artificial chromosome (YAC) containing human DNA (YAC12) . An Alu-URA3-YZ sequence was targeted to several Alu sites within the YAC in strains of the yeast Saccharomyces cerevisiae; the strains contained a galactose-inducible HO endonuclease that cut the YAC at the YZ site . The presence of a DSB in most YACs led to deletion of the URA3 cassette, with retention of the telomeric markers, through recombination between surrounding Alus . For two YACs, the DSBs were not repaired and there was a G2 delay associated with the persistent DSBs . The presence of persistent DSBs resulted in cell death even though the YACs were dispensable . Among the survivors of the persistent DSBs, most had lost the YAC . By a pullback procedure, cell death was observed to begin at least 6 h after induction of a break . For YACs in which the DSB was rapidly repaired, the breaks did not cause cell cycle delay or lead to cell death . These results are consistent with our previous conclusion that a persistent DSB in a plasmid (YZ-CEN) also caused lethality (C . B . Bennett, A . L . Lewis, K . K . Baldwin, and M . A . Resnick, Proc . Natl . Acad . Sci . USA 90:5613-5617, 1993) . However, a break in the YZ-CEN plasmid did not induce lethality in the strain (CBY) background used in the present study . The differences in survival levels appear to be due to the rapid degradation of the plasmid in the CBY strain . We, therefore, propose that for a DSB to cause cell cycle delay and death by means other than the loss of essential genetic material, it must remain unrepaired and be long-lived.

Mol Cell Biol, 1996 Aug, 16(8), 3990 - 4002
Kar4p, a karyogamy-specific component of the yeast pheromone response pathway; Kurihara LJ et al.; Karyogamy is the process whereby two haploid nuclei fuse to form a diploid nucleus during mating in Saccharomyces cerevisiae . Here, we describe the characterization of the KAR4 gene, previously identified in a screen for new nuclear fusion-defective mutants . During mating, kar4 mutants were defective for the microtubule-dependent movement of nuclei, a phenotype identical to that of mutations in KAR3 and CIK1 . Consistent with its mutant phenotype, we found that the kar4 mutation resulted in failure to induce KAR3 and CIK1 mRNA during mating . Expression of KAR3 and CIK1 under independent regulatory control suppressed the kar4 defect, indicating that KAR4 is required primarily for the induction of KAR3 and CIK1 . KAR4 was also required for meiosis, during which it may regulate KAR3; however, mitotic expression of KAR3 and CIK1 during S/G2 phase was independent of KAR4 . A 30-bp region upstream of KAR3 conferred both KAR4- and STE12-dependent induction by mating pheromone . This region contained one moderate and two weak matches to the consensus pheromone response element to which the Ste12p transcriptional activator binds and five repeats of the sequence CAAA(A) . Overproduction of Ste12p suppressed the kar4 defect in KAR3 induction and nuclear fusion . In contrast, Ste12p-independent expression of Kar4p did not alleviate the requirement for Ste12p during KAR3 induction . We propose that Kar4p assists Ste12p in the pheromone-dependent expression of KAR3 and CIK1 . KAR4 defines a novel level of regulation for the pheromone response pathway, acting at a subset of Stel2p-inducible genes required for karyogamy.

RNA, 1996 Aug, 2(8), 835 - 48
Identification and characterization of yeast mutants that overcome an experimentally introduced block to splicing at the 3' splice site; Lin J et al.; An experimentally introduced secondary structure in exon 2 adjacent to the 3' splice site of a yeast ACT-Escherichia coli lacZ fusion gene abolishes splicing in vivo and inhibits beta-galactosidase production . We have devised a genetic screen to isolate both cis and trans-acting mutants that restore beta-galactosidase activity . Two cis-acting mutants potentially destabilize the stem in the region close to the 3' splice site . One trans-acting mutant, designated rss1-1, partially restores beta-galactosidase activity by both increasing the splicing efficiency and stabilizing the precursor and lariat intermediate . The trans-acting suppression activity of rss1-1 is specific for a particular structure because another artificially introduced secondary structure, which also blocks splicing, is not suppressed by this mutant allele . We have cloned the gene encoding the trans-acting mutant protein . The RSS1 gene is located on Saccharomyces cerevisiae chromosome V and is a single copy, essential gene . The predicted RSS1 protein has marked similarity to members of the putative ATP-dependent RNA helicase family . At the nonpermissive temperature, the rss1-1 mutant allele decreases the steady-state levels of several endogenous messenger RNAs and increases the ratio of pre-mRNA to mRNA of specific messages . RSS1 is likely to play an interesting role in RNA processing.

J Cell Biol, 1996 Aug, 134(3), 661 - 74
Ubiquitination of the yeast a-factor receptor; Roth AF et al.; The a-factor receptor (Ste3p) is one of two pheromone receptors in the yeast Saccharomyces cerevisiae that enable the cell-cell communication of mating . In this report, we show that this receptor is subject to two distinct covalent modifications-phosphorylation and ubiquitination . Phosphorylation, evident on the unstimulated receptor, increases upon challenge by the receptor's ligand, a-factor . We suggest that this phosphorylation likely functions in the adaptive, negative regulation of receptor activity . Removal of phosphorylation by phosphatase treatment uncovered two phosphatase-resistant modifications identified as ubiquitination using a myc-epitope-tagged ubiquitin construct . Ste3p undergoes rapid, ligand-independent turnover that depends on vacuolar proteases and also on transport of the receptor from surface to vacuole (i.e., endocytosis) (Davis, N.G., J.L.Horecka, and G.F . Sprague, Jr., 1993 J . Cell Biol . 122:53-65) . An end4 mutation, isolated for its defect in the endocytic uptake of alpha-factor pheromone (Raths, S., J . Rohrer, F . Crausaz, and H . Riezman . 1993 . J . Cell Biol . 120:55-65), blocks constitutive endocytosis of the a-factor receptor, yet fails to block ubiquitination of the receptor . In fact, both phosphorylation and ubiquitination of the surfacebound receptor were found to increase, suggesting that these modifications may occur normally while the receptor is at the cell surface . In a mutant strain constructed to allow for depletion of ubiquitin, the level of receptor ubiquitination was found to be substantially decreased . Correlated with this was an impairment of receptor degradative turnover-receptor half-life that is normally approximately 20 min at 30 degrees C was increased to approximately 2 h under these ubiquitin-depletion conditions . Furthermore, surface residency, normally of short duration in wild-type cells (terminated by endocytosis to the vacuole), was found to be prolonged; the majority of the receptor protein remained surface localized fully 2 h after biosynthesis . Thus, the rates of a-factor receptor endocytosis and consequent vacuolar turnover depend on the available level of ubiquitin in the cell . In cells mutant for two E2 activities, i.e., ubc4 delta ubc5 delta cells, the receptor was found to be substantially less ubiquitinated, and in addition, receptor turnover was slowed, suggesting that Ubc4p and Ubc5p may play a role in the recognition of the receptor protein as substrate for the ubiquitin system . In addition to ligand-independent uptake, the a-factor receptor also undergoes a ligand-dependent form of endocytosis (Davis, N.G., J.L . Horecka, and G.F . Sprague, Jr . 1993 . J . Cell . Biol . 122:53-65) . Concurrent with ligand-dependent uptake, we now show that the receptor undergoes ligand-induced ubiquitination, suggesting that receptor ubiquitination may function in the ligand-dependent endocytosis of the a-factor receptor as well as in its constitutive endocytosis . To account for these findings, we propose a model wherein the covalent attachment of ubiquitin to surface receptor triggers endocytic uptake.

Fertil Steril, 1996 Aug, 66(2), 335 - 7
Transfer of embryos from yeast-colonized dishes; Ben-Chetrit A et al.; OBJECTIVE: To report yeast colonization in IVF dishes, where ET was carried out, and the IVF outcome was not compromised . DESIGN: Retrospective study of patients who underwent IVF cycle during the last 4 years . SETTING: In vitro fertilization program at the Shaare-Zedek hospital in Jerusalem . PATIENTS: Five couples who underwent standard IVF cycles and whose dishes were colonized with yeast . After thorough discussion ET was carried out . RESULTS: Although colonized with yeast, the quality of the embryos was not compromised . One to three of these embryos were transferred . All five women conceived . CONCLUSIONS: In vitro fertilization outcome is not necessarily compromised by yeast colonization . Nevertheless, the possible teratogenic effect of yeast on embryos has not been investigated and research is required to address this concern.

EMBO J, 1996 Aug 1, 15(15), 3974 - 85
All four core histone N-termini contain sequences required for the repression of basal transcription in yeast; Lenfant F et al.; Nucleosomes prevent the recognition of TATA promoter elements by the basal transcriptional machinery in the absence of induction . However, while Saccharomyces cerevisiae histones H3 and H4 contain N-terminal regions involved in the activation and repression of GAL1 and in the expression of heterochromatin-like regions, the sequences involved in repressing basal transcription have not yet been identified . Here, we describe the mapping of new N-terminal domains, in all four core histones (H2A, H2B, H3 and H4), required for the repression of basal, uninduced transcription . Basal transcription was monitored by the use of a GAL1 promoter-URA3 reporter construct whose uninduced activity can be detected through cellular sensitivity to the drug, 5-fluoroorotic acid . We have found for each histone that the N-terminal sequences repressing basal activity are in a short region adjacent to the structured alpha-helical core . Analysis of minichromosome DNA topology demonstrates that the basal domains are required for the proper folding of DNA around the chromosomal particle . Deletion of the basal domain at each histone significantly decreases plasmid superhelical density, which probably reflects a release of DNA from the constraints of the nucleosome into the linker region . This provides a means by which basal factors may recognize otherwise repressed regulatory elements.

Biochim Biophys Acta, 1996 Jul 31, 1275(3), 204 - 12
Control of frequency and amplitudes is shared by all enzymes in three models for yeast glycolytic oscillations; Teusink B et al.; The three main existing models for glycolytic oscillations in yeast were re-examined to investigate how these oscillations are controlled . We implemented the operational definitions provided by metabolic control analysis to quantify the control properties of enzymes with regard to glycolytic oscillations . In all three models, the control of the frequency and that of the amplitudes of the metabolites were distributed among the enzymes . There was no obvious correlation between the control of the average flax and the control of the frequency . Most importantly, the so-called 'oscillophore' of the system, traditionally the enzyme primarily held responsible for the generation of the oscillation, was not the only controlling step . We conclude that just like steady-state flux control is not necessarily limited to a rate-limiting step, oscillations are not dictated by a single 'oscillophore'.

Curr Genet, 1996 Jul 31, 30(2), 126 - 34
Amphimeric mitochondrial genomes of petite mutants of yeast . I . Flip-flop amphimers make up the mitochondrial genomes of "palindromic" petite mutants of yeast; Rayko E et al.; The mitochondrial (mt) genomes of three spontaneous cytoplasmic "palindromic" petite mutants of yeast were studied by restriction-enzyme analysis . These mt genomes were shown to be made up of an amplified "master basic unit" consisting of two inverted segments (a and A) and of two different unique segments (d and t) separating them . The basic unit was called "amphimeric", this term having been first proposed for certain lambda-phage mutants . We propose that in the mt genomes of the petite mutants studied, the four possible variants of the amphimeric basic unit form two - "flip" and "flop" - tetra-amphimeric repeat units datA-datA-DaTA-DaTA and DatA-DatA-daTA-daTA, respectively . These repeat units make two types of "amphimeric" mt genomes which exist in equal proportions in the cell . In each mt genome, the duplicated segment regularly alternates in its direct and inverted orientation (a...A...a...A...), whereas the unique segments are arranged twice in tandem fashion and twice in inverted fashion (d...d...D...D...d...d...andt...t...T...T...t...t...) . The only difference between flip and flop amphimeric mt petite genomes is the different relative orientation of the unique segments in the mono-amphimers . In the mono-amphimers of flip mt genomes, both unique segments are arranged in the same direction (d...t and D...T), whereas in the mono-amphimers of flop mt genomes, both unique segments are arranged in opposite directions (D...t and d...T) . Control experiments on one spontaneous petite mutant (which was an ancestor of the mutants studied here) and on three independent, previously investigated, EtBr-induced mutants showed that all of them were, in fact, organized in the same way . Analysing our experimental data and the results published by others, we conclude that amphimeric organization is a general feature of mt petite genomes of yeast previously called "palindromic" or "rearranged".

Biochemistry, 1996 Jul 30, 35(30), 9821 - 31
Probing the protein-DNA contacts of a yeast RNA polymerase III transcription complex in a crude extract: solid phase synthesis of DNA photoaffinity probes containing a novel photoreactive deoxycytidine analog; Lannutti BJ et al.; A novel photoreactive deoxycytidine analog, 4-{N-(p-azidobenzoyl)-2-aminoethyl}-dCTP (ABdCTP), has been synthesized and incorporated at specific sites within the SUP4 tRNA(Tyr) gene . Immobilized single-stranded DNA was annealed to specific oligonucleotides and AB-dCMP incorporated into DNA by primer extension . DNA photoaffinity labeling with AB-dCMP was used to survey protein-DNA contacts in initiation and elongation complexes of RNA polymerase III (Pol III), and compared to DNA photoaffinity labeling using the previously described photoreactive deoxyuridine analog, 5-{N-(pazidobenzoyl)-3-aminoallyl}-dUMP (AB-dUMP) {Bartholomew et al . (1993) Mol . Cell.Biol . 13,942-952} . In contrast to previous studies, we have used a crude protein fraction rather than highly purified preparations of Pol III and transcription factors TFIIIC and TFIIIB to examine if some component of the transcription complex is lost upon purification . Eleven nucleotide positions from bp-17 to bp +17 (+1 being the start site of transcription) on the nontranscribed strand were modified and shown to have little or no effect on transcription complex formation, initiation, or elongation as determined by multiple-round transcription assays . Efficient photoaffinity labeling by DNA containing AB-dCMP gave results comparable to that with AB-dUMP at proximal nucleotide positions and provided new evidence for the placement of the 160 and 31 kDa subunits of Pol III near the 5' end of the transcriptional bubble in an elongation complex . A novel 40 kDa protein was cross-linked at bps -17, -9, and -8 in a TFIIIC-dependent manner that had not been previously detected.

Int J Cancer, 1996 Jul 29, 67(3), 447 - 50
Clonality and stability of the p53 gene in human astrocytic tumor cells: quantitative analysis of p53 gene mutations by yeast functional assay; Tada M et al.; Mutation of the p53 gene is found in about one third of astrocytic brain tumors, and expansion of tumor cell clones containing mutant p53 has been implicated in astrocytic tumor progression . However, admixture of normal cells in astrocytic tumor specimens limits the power of traditional studies of tumor cell clonality . To address this problem we have employed a yeast p53 functional assay that scores the content of mutant p53 alleles in tumors and cell lines quantitatively . We have analyzed 17 cases where matching tumor material and derived cell lines were available . The yeast assay gave > 20% red (i.e., mutant p53-containing) yeast colonies in 7 out of 17 cases . One case had no mutations in the primary tumor but gave 76% red colonies in a recurrence, clearly demonstrating tumor overgrowth by a mutant clone . During early passages of cultured tumor cells, mutant p53 content increased rapidly with passage due to outgrowth of mutant clones from a heterogeneous starting population . In addition, de novo p53 mutations appeared during culture in 2 cases . This indicates that there is stronger selective pressure for mutation during the establishment of cell lines in vitro than during tumor growth in vivo . Our results demonstrate the utility of the p53 functional assay for studies of clonality and support the hypothesis of clonal progression of brain tumors in vivo.

Mol Gen Genet, 1996 Jul 26, 251(6), 682 - 91
Protein-protein interactions in the yeast PKC1 pathway: Pkc1p interacts with a component of the MAP kinase cascade; Paravicini G et al.; The two-hybrid system for the identification of protein-protein interactions was used to screen for proteins that interact in vivo with the Saccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C . Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in the PKC1-mediated signalling pathway . Subsequently, Pkc1p and the other PKC1 pathway components encoding members of a MAP kinase cascade, Bck1p (a MEKK), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP kinase), were tested pairwise for interaction in the two-hybrid assay . Pkc1p interacted specifically with small N-terminal deletions of Mkk1p, and no interaction between Pkc1p and any of the other known pathway components could be detected . Interaction between Pkc1p and Mkk1p, however, was found to be independent of Mkk1p kinase activity . Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction required only the predicted C-terminal catalytic domain of Mkk1p . Furthermore, we detected protein-protein interactions between two Bck1p molecules via their N-terminal regions . Finally, Mkk2p and Mpk1p also interacted in the two-hybrid assay . These results suggest that the members of the PKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component of this pathway.

Cell, 1996 Jul 26, 86(2), 275 - 85
Budding yeast SKP1 encodes an evolutionarily conserved kinetochore protein required for cell cycle progression; Connelly C et al.; The budding yeast SKP1 gene, identified as a dosage suppressor of a known kinetochore protein mutant, encodes an intrinsic 22.3 kDa subunit of CBF3, a multiprotein complex that binds centromere DNA in vitro . Temperature-sensitive mutations in SKP1 define two distinct phenotypic classes . skp1-4 mutants arrest predominantly as large budded cells with a G2 DNA content and short mitotic spindle, consistent with a role in kinetochore function . skp1-3 mutants, however, arrest predominantly as multiply budded cells with a G1 DNA content, suggesting an additional role during the G1/S phase . Identification of Skp1p homologs from C . elegans, A . thaliana, and H . sapiens indicates that SKP1 is evolutionarily highly conserved . Skp1p therefore represents an intrinsic kinetochore protein conserved throughout eukaryotic evolution and may be directly involved in linking kinetochore function with the cell cycle-regulatory machinery.

J Biol Chem, 1996 Jul 26, 271(30), 18243 - 52
Two separate functions are encoded by the carboxyl-terminal domains of the yeast cyclase-associated protein and its mammalian homologs . Dimerization and actin binding; Zelicof A et al.; The yeast adenylyl cyclase-associated protein, CAP, was identified as a component of the RAS-activated cyclase complex . CAP consists of two functional domains separated by a proline-rich region . One domain, which localizes to the amino terminus, mediates RAS signaling through adenylyl cyclase, while a domain at the carboxyl terminus is involved in the regulation of cell growth and morphogenesis . Recently, the carboxyl terminus of yeast CAP was shown to sequester actin, but whether this function has been conserved, and is the sole function of this domain, is unclear . Here, we demonstrate that the carboxyl-terminal domains of CAP and CAP homologs have two separate functions . We show that carboxyl-terminals of both yeast CAP and a mammalian CAP homolog, MCH1, bind to actin . We also show that this domain contains a signal for dimerization, allowing both CAP and MCH1 to form homodimers and heterodimers . The properties of actin binding and dimerization are mediated by separate regions on the carboxyl terminus; the last 27 amino acids of CAP being critical for actin binding . Finally, we present evidence that links a segment of the proline-rich region of CAP to its localization in yeast . Together, these results suggest that all three domains of CAP proteins are functional.

FEBS Lett, 1996 Jul 22, 390(2), 191 - 5
Regulation of transcription in mammalian cells by yeast Leu3p and externally supplied inducer; Guo H et al.; The Leu3 protein of yeast is a dual-function regulator, stimulating transcription when the inducer alpha-isopropylmalate (alpha-IPM) is present and suppressing transcription when the inducer is absent . Here we show that Leu3p retains both its positive and negative regulatory properties when expressed in mammalian cells or when added to a mammalian nuclear extract . Alpha-IPM stimulates reporter gene expression 15-20-fold, both in vivo and in vitro . The concentration of alpha-IPM required for half-maximal stimulation in vitro is 2.5 x 10(-4) M . No yeast-specific factors other than Leu3p itself are required for up- or down-regulation . Since alpha-IPM is not metabolized in mammalian cells, the Leu3p-alpha-IPM system might be useful in gene therapy and other studies as a highly specific, externally controlled on/off switch of gene expression.

Mol Gen Genet, 1996 Jul 19, 251(5), 613 - 8
Genetic evidence for functional specificity of the yeast GCN2 kinase; Tavernarakis N et al.; In yeast the GCN2 kinase mediates translational control of GCN4 by phosphorylating the alpha subunit of eIF-2 in response to extracellular amino acid limitation . Although phosphorylation of eIF-2 alpha has been shown to inhibit global protein synthesis, amino acid starvation results in a specific activation effect on GCN4 mRNA translation . Under the same conditions, translation of other mRNAs appears only slightly affected . The mechanism responsible for the observed selectivity of the GCN2 kinase is not clear . Here, we present genetic evidence that suggests that locally restricted action of the GCN2 kinase facilitates GCN4-specific translational regulation.

Mol Gen Genet, 1996 Jul 19, 251(5), 556 - 64
cAMP inhibits bud growth in a yeast strain compromised for Ca2+ influx into the Golgi; Hartley AD et al.; Biochemical and physiological studies have implicated cAMP and cAMP-dependent protein kinase (PKA) in a plethora of essential cellular processes . Here we show that yeast cells partially depleted of PKA activity (due to a tpkw mutation) and bearing a lesion in a Golgi-localized Ca2+ pump (Pmr1), arrest division with a small bud . The bud morphology of the arrested tpk1w pmr1 mutant cells is characteristic of cells in S phase; however, the terminal phenotype of processes such as DNA replication and nuclear division suggests arrest at the G2/M boundary . This small bud, G2-arrest phenotype is similar to that of strains with a defect in cell wall biosynthesis (pkc1) or membrane biogenesis (och1); however, the biochemical defect may be different since the tpk1w pmr1 double mutants retain viability . The growth defect of the tpk1w pmr1 mutant can be alleviated by preventing the increase in cellular cAMP levels that is known to be associated with a decrease in PKA activity, or by supplementing the medium with millimolar amounts of Ca2+ . Although the biochemical consequences of this increase in cAMP concentration are not known, the small-bud phenotype of the double mutant and the known protein processing defect of the pmr1 lesion suggest that the localization or function of some membrane component might be compromised and susceptible to perturbations in cellular cAMP levels . One candidate for such a protein is the cAMP-binding membrane ectoprotein recently described in yeast.

Biochim Biophys Acta, 1996 Jul 18, 1275(1-2), 133 - 7
The yeast ADP/ATP carrier . Mutagenesis and second-site revertants; Nelson DR; Results of mutagenesis and selection of spontaneous second-site revertants of the yeast ADP/ATP carrier AAC2 is described . Currently, 50 mutants have been made in AAC2 at 35 locations . Yeast carrying mutations at K38, K48, R96, D149, R152, R204, D249, R252, R253, R254 and R294 are all unable to grow on glycerol . Seven of these mutants have yielded second-site revertants when plated on rich yeast media containing glycerol and ethanol . The R96 mutants and the R254 and R253 mutants produce similar changes in the AAC2 molecule because the same sites are affected by their revertant mutations . This system of mutations and revertants is now poised to yield insights into the dynamics of ADP and ATP transport, and mitochondrial carrier structure in general.

Genes Dev, 1996 Jul 15, 10(14), 1796 - 811
Evidence for silencing compartments within the yeast nucleus: a role for telomere proximity and Sir protein concentration in silencer-mediated repression; Maillet L et al.; Transcriptional repression at the silent mating-type loci in yeast requires the targeting of silent information regulator (Sir) proteins through specific interactions formed at cis-acting silencer elements . We show here that a reporter gene flanked by two functional silencers is not repressed when integrated at >200 kb from a telomere . Repression is restored by creation of a new telomere 13 kb from the integrated reporter or by elevated expression of SIR1, SIR3, and/or SIR4 . Coupled expression represses in an additive manner, suggesting that all three factors are in limiting concentrations . When overexpressed, Sir3 and Sir4 are dispersed throughout the nucleoplasm, in contrast to wild-type cells where they are clustered in a limited number of foci together with telomeres . Efficient silencer function thus seems to require either proximity to a pool of concentrated Sir proteins, that is, proximity to telomeres, or delocalization of the silencing factors.

Cell, 1996 Jul 12, 86(1), 95 - 101
Chromosomal inheritance of epigenetic states in fission yeast during mitosis and meiosis; Grewal SI et al.; Inheritance of the active and inactive states of gene expression by individual cells is crucial for development . In fission yeast, mating-type region consists of three loci called mat1, mat2, and mat3 . Transcriptionally silent mat2 and mat3 loci are separated by a 15 kb interval, designated the K-region, and serve as donors of information for transcriptionally active mat1 interconversion . In a strain carrying replacement of 7.5 kb of the K-region with the ura4 gene, we discovered that ura4 silencing and efficiency of mating-type switching were covariegated and were regulated by an epigenetic mechanism . Genetic analyses demonstrated that epigenetic states were remarkably stable not only in mitosis but also in meiosis and were linked to the mating-type region . This study indicates that different epigenetic states are heritable forms of chromatin organization at the mat region.

J Biol Chem, 1996 Jul 12, 271(28), 16927 - 33
Characterization of yeast methyl sterol oxidase (ERG25) and identification of a human homologue; Li L et al.; A yeast mutant (LT06) was isolated that showed no growth on iron-limited medium but normal growth on iron-replete medium . A gene cloned from a genomic yeast library complemented the defect, allowing growth on low iron medium . Allelic segregation analysis demonstrated that the cloned gene was the normal allele rather than a high copy suppressor . A disruption mutant was nonviable, indicating that the gene was essential . Sequence analysis and functional assays indicated that the cloned gene was identical to ERG25, a gene that codes for methyl sterol oxidase . Incubation of LT06 in low iron medium resulted in marked changes in lipid metabolism, including the accumulation of fatty acids, triglycerides, methyl sterols, and other sterol precursors . A human homologue of ERG25 was cloned, sequenced, and mapped to human chromosome 4q32-34 . Analysis of the data base with both ERG25 and the human homologue resulted in the identification of a putative set of metal binding motifs with similarity to that seen in a family of membrane desaturases-hydroxylases . Western analysis using antibodies to an Erg25-GST fusion protein detected two proteins of 34 and 75 kDa . Both proteins are membrane bound and contain one N-glycosyl unit . Immunofluorescence data suggest that the proteins are present in the endoplasmic reticulum and plasma membrane . Although ERG25 transcripts are not iron regulated, there is a large increase in the concentration of transcript in the mutant LT06 grown in low iron medium . These results suggest that the enzyme is regulated not by iron but by an end product of the ergosterol pathway.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7143 - 8
ECA39, a conserved gene regulated by c-Myc in mice, is involved in G1/S cell cycle regulation in yeast; Schuldiner O et al.; The c-myc oncogene has been shown to play a role in cell proliferation and apoptosis . The realization that myc oncogenes may control the level of expression of other genes has opened the field to search for genetic targets for Myc regulation . Recently, using a subtraction/coexpression strategy, a murine genetic target for Myc regulation, called EC439, was isolated . To further characterize the ECA39 gene, we set out to determine the evolutionary conservation of its regulatory and coding sequences . We describe the human, nematode, and budding yeast homologs of the mouse ECA39 gene . Identities between the mouse ECA39 protein and the human, nematode, or yeast proteins are 79%, 52%, and 49%, respectively . Interestingly, the recognition site for Myc binding, located 3' to the start site of transcription in the mouse gene, is also conserved in the human homolog . This regulatory element is missing in the ECA39 homologs from nematode or yeast, which also lack the regulator c-myc . To understand the function of ECA39, we deleted the gene from the yeast genome . Disruption of ECA39 which is a recessive mutation that leads to a marked alteration in the cell cycle . Mutant haploids and homozygous diploids have a faster growth rate than isogenic wild-type strains . Fluorescence-activated cell sorter analyses indicate that the mutation shortens the G1 stage in the cell cycle . Moreover, mutant strains show higher rates of UV-induced mutations . The results suggest that the product of ECA39 is involved in the regulation of G1 to S transition.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7048 - 52
Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast; Sugimoto K et al.; The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint . In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis . In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1 . The RFC5 gene encodes a small subunit of replication factor C complex . At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability . Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint . Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect . Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.

Biochem Biophys Res Commun, 1996 Jul 5, 224(1), 126 - 33
The putative membrane anchor protein for yeast Sec7p recruitment; Wolf JR et al.; Proteins required for yeast secretory pathway function have been identified by genetic selection and characterization of the temperature-sensitive secretory (sec) mutants . The use of genetic and biochemical approaches has expanded the catalog of components of the secretory pathway, yet many proteins, especially membrane and lumenal proteins, remain to be identified . Sec7p, one of the original SEC gene products to be described, is required at multiple stages of the yeast secretory pathway in the coating of transport vesicles . A chemical cross-linking approach was used to identify proteins associated with Sec7p protein complexes from yeast cell lysates . A 90 kDa integral membrane protein (p90) was isolated whose interactions with Sec7p were reproduced in the absence of chemical cross-linking . Further biochemical analysis indicated that p90 may act as the anchor protein for Sec7p membrane recruitment in transport vesicle assembly.

Mutat Res, 1996 Jul 5, 354(1), 69 - 75
Hydroxyurea induces recombination in dividing but not in G1 or G2 cell cycle arrested yeast cells; Galli A et al.; Hydroxyurea, a chemotherapeutic and radiosensitizing agent, inhibits ribonucleotide reductase, arrests cells in the S-phase and is mutagenic and recombinagenic . In this paper we investigated whether the recombinagenic activity of hydroxyurea is due to the same activity that leads to arrest in the S-phase or to a more direct action on DNA . The effect of hydroxyurea on intrachromosomal and interchromosomal recombination was investigated in dividing and in G1 or G2 cell cycle-arrested cells of the yeast Saccharomyces cerevisiae . Treatment of dividing cells with hydroxyurea resulted in a large increase in recombination frequencies, even at low non-toxic doses . In contrast, in cells arrested in the G1 or G2 phase, hydroxyurea failed to induce recombination, even at 60-fold higher toxic doses . The presence of metabolic activation (S9 mix) did not change the effects of hydroxyurea on recombination . The data suggest that the recombinagenic activity of hydroxyurea may not be due to any direct effect of hydroxyurea on DNA, but may be linked to the inhibition of ribonucleotide reductase causing inhibition of DNA synthesis leading to S-phase arrest and possibly causing recombinagenic lesions.

J Biol Chem, 1996 Jul 5, 271(27), 16104 - 10
The yeast transcription terminator for RNA polymerase I is designed to prevent polymerase slippage; Jeong SW et al.; A transcription terminator for RNA polymerase I (polI) in the yeast, Saccharomyces cerevisiae, is composed of two essential elements, the 11bp binding site for Reb1p and an upstream T-rich element coding for the last 10-12 nucleotides of the terminated transcript . We now show that, if the upstream element is changed to homopolymer T residues, polI undergoes iterative slippage, long poly(U) tails are added to the transcript, and termination is impaired . Reinsertion of one or two non-T residues within a critical region prevents iterative slippage and reinstates termination . A survey of naturally occurring terminators reveals that many contain T-rich upstream regions with non-T residues situated appropriately to prevent slippage . We discuss the possibility that the first step in slippage, backward sliding of both the transcript and the catalytic center of the polymerase, may be an obligatory step in the normal termination process.

J Biol Chem, 1996 Jul 5, 271(27), 15928 - 33
A yeast phosphofructokinase insensitive to the allosteric activator fructose 2,6-bisphosphate . Glycolysis/metabolic regulation/allosteric control; Heinisch JJ et al.; In this work we used in vitro mutagenesis to modify the allosteric properties of the heterooctameric yeast phosphofructokinase . Specifically, we identified two amino acids involved in the binding of the most potent allosteric activator fructose 2,6-bisphosphate . Thus, Ser724 was replaced by an aspartate and His859 by a serine in each of the enzyme subunits . Whereas the substitutions had no drastic effects when introduced only in one of the two types of subunits, kinetic parameters were modified when both subunits carried the mutation . Thus, the enzyme with His859 --> Ser showed an increase in Ka for binding of the activator, whereas the one with Ser724 --> Asp failed to react to the addition of fructose 2, 6-bisphosphate, at all . The enzymes still responded to other allosteric activators, such as AMP . Stabilities of the mutant subunits were not significantly altered in vivo, as judged from Western blot analysis . Phenotypically, strains expressing the mutant PFK genes showed a pronounced effect on the level of intermediary metabolites after growth on glucose . Mutants not responding to the activator at all (Ser724 --> Asp) also displayed higher generation times on glucose medium . This could be suppressed by increasing the gene dosage of the mutant alleles . These results indicate that fructose 2,6-bisphosphate through its activation of phosphofructokinase plays an important role in regulation of the glycolytic flux.

J Biol Chem, 1996 Jul 5, 271(27), 15884 - 90
Transcriptional activation by yeast PDR1p is inhibited by its association with NGG1p/ADA3p; Martens JA et al.; NGG1p/ADA3p forms a coactivator/repressor complex (ADA complex) in association with at least two other yeast proteins, ADA2p and GCN5p, that is involved in regulating transcriptional activator proteins including GAL4p and GCN4p . Using a two-hybrid analysis, we found that the carboxyl-terminal transcriptional activation domain of PDR1p, the primary regulatory protein involved in yeast pleiotropic drug resistance, interacts with the amino-terminal 373 amino acids of NGG1p (NGG1p1-373) . This interaction was confirmed by coimmunoprecipitation of epitope-tagged derivatives of NGG1p and PDR1p from crude extracts . An overlapping region of the related transcriptional activator PDR3p was also found to interact with NGG1p . Amino acids 274-307 of NGG1p were required for interaction with PDR1p . This same region is required for inhibition of transcriptional activation by GAL4p . The association between NGG1p1-373 and PDR1p may be indirect, possibly mediated by the ADA complex since the two-hybrid interaction required the presence of full-length NGG1 . A partial requirement for ADA2 was also found . This suggests that an additional component of the ADA complex, regulated by ADA2p, may mediate the interaction . Transcriptional activation by a GAL4p DNA binding domain fusion of PDR1p was enhanced in ngg1 and ada2 disruption strains . Similar to its action on GAL4p, the ADA complex acts to inhibit the activation domain of PDR1p.

Genes Cells, 1996 Jul, 1(7), 645 - 61
Sensing of DNA non-homology lowers the initiation of meiotic recombination in yeast; Rocco V et al.; BACKGROUND: Meiotic recombination between homologous chromosomes in the yeast Saccharomyces cerevisiae is initiated by the formation of DNA double-strand breaks (DSBs) . The mechanism of DSB formation and the factors that determine their frequency and location have yet to be elucidated . Current studies of meiotic recombination are also concerned with the question of the functional relationship between DSB formation and the other meiotic processes of homology searching, pairing and synapsis of homologues . RESULTS: To test if DNA identity is required for high levels of DSBs and recombination, we have asked whether small DNA heterologies (140-547 bp) located within the well characterized ARG4 initiator of meiotic recombination, can affect DSB formation and gene conversion events in the ARG4 locus . The present physical and genetic analyses show that some heterologies reduced recombination frequencies without altering DSB formation, whereas others reduced both DSB and gene conversion frequencies . CONCLUSIONS: These results suggest that DNA heterologies overlapping a recombination initiator impair meiotic gene conversion at two levels . First, some heterologies affect the level of DSB formation, revealing the existence of an anti-initiation process sensing the presence of sequence non-homology between the homologous chromosomes . Second, heterologies can impair the successful processing of the recombination intermediates once DSBs are made . We present a model for interhomologue cross-talks involving chromosomal and DNA/DNA interactions.

Genes Cells, 1996 Jul, 1(7), 633 - 44
Dissection of fission yeast microtubule associating protein p93Dis1: regions implicated in regulated localization and microtubule interaction; Nakaseko Y et al.; BACKGROUND: Fission yeast microtubule associating protein (MAP) p93Dis1 functions for sister chromatid separation: dis1 mutants fail to separate chromosomes, while the spindle elongates but without cyclin destruction . p93Dis1 localizes along microtubules in interphase cytoplasm, but shifts to the spindle pole body (SPB) and spindle microtubules upon the entry into mitosis . In this study, regions of p93Dis1 were dissected to examine their role . RESULTS: Nitrocellulose filter blotting shows that recombinant Dis1 binds to bovine brain microtubules in vitro . A basic central region rich in S, T and P is essential for this association . However, the whole p93Dis1 with N- and C-termini containing a conserved repeat motif and heptad repeats, respectively, is necessary for normal microtubule association in vivo . The N-truncated region also binds to microtubules but only to the portions near the SPBs . Overproduction phenotypes indicate that p93Dis1 greatly affects spindle formation and cell morphogenesis . The central region is essential but, by itself, not sufficient for generating such effects . CONCLUSIONS: We propose that p93Dis1 consists of three regions which carry distinct properties for localization: the N-region for cell cycle dependent localization, the central region for direct microtubule association, and the C-region for SPB and nuclear localization . The essential role of p93Dis1 is carried out in the C-region, while the N-region acts as a regulator.

Mol Biol Cell, 1996 Jul, 7(7), 1083 - 94
A connection between pre-mRNA splicing and the cell cycle in fission yeast: cdc28+ is allelic with prp8+ and encodes an RNA-dependent ATPase/helicase; Lundgren K et al.; The fission-yeast gene cdc28+ was originally identified in a screen for temperature-sensitive mutants that exhibit a cell-division cycle arrest and was found to be required for mitosis . We undertook a study of this gene to understand more fully the general requirements for entry into mitosis . Cells carrying the conditional lethal cdc28-P8 mutation divide once and arrest in G2 after being shifted to the restrictive temperature . We cloned the cdc28+ gene by complementation of the temperature-sensitive growth arrest in cdc28-P8 . DNA sequence analysis indicated that cdc28+ encodes a member of the DEAH-box family of putative RNA-dependent ATPases or helicases . The Cdc28 protein is most similar to the Prp2, Prp16, and Prp22 proteins from budding yeast, which are required for the splicing of mRNA precursors . Consistent with this similarity, the cdc28-P8 mutant accumulates unspliced precursors at the restrictive temperature . Independently, we isolated a temperature-sensitive pre-mRNA splicing mutant prp8-1 that exhibits a cell-cycle phenotype identical to that of cdc28-P8 . We have shown that cdc28 and prp8 are allelic . These results suggest a connection between pre-mRNA splicing and progression through the cell cycle.

Histochem Cell Biol, 1996 Jul, 106(1), 115 - 30
Immunogold labeling of yeast cells: an efficient tool for the study of protein targeting and morphological alterations due to overexpression and inactivation of genes; Binder M et al.; Immunogold labeling on Lowicryl HM20 resin sections is a valuable complement to biochemical methods as well as methods of molecular biology in the study of basic mechanisms in the yeast system . This contribution presents an overview of the state of the art . Emphasis is put on the explanation of caveats and pitfalls rather than on detailed bench protocols . In the Applications section the morphological aspect of genetic manipulation is accentuated and links to human pathology are indicated . The morphological consequences of genetic manipulations may gain importance in view of the efforts made to establish gene therapies . In particular, the contribution of immunoelectron microscopy to the elucidation of peroxisomal targeting signals and to the detection and identification of morphological alteration due to overexpressed, mutated or deleted genes in the context of peroxisome biogenesis is described.

Mol Microbiol, 1996 Jul, 21(1), 171 - 9
Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation; Grant CM et al.; Glutathione (GSH) is an abundant cellular thiol which has been implicated in many cellular processes including protection against xenobiotics, carcinogens and free radicals . Utilization of GSH in both enzymic and non-enzymic defence mechanisms results in its conversion to the oxidized form (GSSG), and it must be recycled to GSH to maintain the high intracellular ratio of GSH to GSSG . Glutathione reductase (GLR) is a flavoenzyme, which catalyses reduction of GSSG to GSH using the reducing power of NADPH . We show that yeast mutants deleted for GLR1, encoding glutathione reductase, lack GLR activity and accumulate increased levels of GSSG . In addition, the glr1 mutant strain was unaffected in the inducible adaptive response to hydrogen peroxide, but showed increased sensitivity to oxidants including both peroxides and superoxide, indicating a requirement for GLR in protection against oxidative stress . Furthermore, GLR1 expression was elevated two to threefold in the presence of oxidants, and regulation was dependent upon the yAP-1 transcriptional activator protein . Thus, GLR1 is one of a growing number of genes involved in the protection of yeast cells against oxidative stress and regulated by yAP-1.

Yeast, 1996 Jul, 12(9), 833 - 8
Enrichment of yeast protein tyrosine kinase activity by substrate affinity chromatography; Adamikova L et al.; The direct biochemical analysis of protein tyrosine kinases from yeast has been difficult due to their very low activity in crude cell lysates . Here we present a procedure for the enrichment and partial purification of protein tyrosine kinases from Saccharomyces cerevisiae based on single-step substrate affinity chromatography using a synthetic random co-polymer of glutamic acid and tyrosine . Fractionation of cell lysates on a poly-glutamic acid:tyrosine (4:1)-Sepharose affinity column resulted in a 4000-fold increase in tyrosine kinase activity . Active fractions contain at least six potential protein kinases as judged by in situ phosphorylation assay and Western blot analysis using anti-phosphotyrosine . We propose that this protocol may also be useful for the initial identification and purification of tyrosine kinases from other organisms exhibiting low levels of this enzymatic activity in cell lysates.

Bone Marrow Transplant, 1996 Jul, 18(1), 93 - 102
Antifungal effects of yeast-derived rhu-GM-CSF in patients receiving high-dose chemotherapy given with or without autologous stem cell transplantation: a retrospective analysis; Peters BG et al.; Systemic fungal infections (SFI) in patients receiving high-dose chemotherapy (HDC) are a frequent cause of morbidity and mortality . Preclinical studies have reported augmented antifungal activity of monocytes, macrophage cells, and neutrophils exposed to certain colony-stimulating factors (CSF), including GM-CSF . We conducted a retrospective descriptive epidemiologic study to examine the characteristics of 145 consecutive patients receiving HDC administered with or without autologous stem cell transplantation (ASCT) and who subsequently received either GM-CSF and G-CSF, G-CSF alone, GM-CSF +/- IL-3 or no CSF . The analysis of this patient population sought to define the incidence of SFI and its relationship to therapy with monocyte/macrophage-stimulating (MMS group) cytokines (GM-CSF and G-CSF; GM-CSF +/- IL-3) or to cytokines which do not result in monocyte/macrophage stimulation (NMMS group, G-CSF alone or no CSF) . Risk factors for the development of SFI were balanced between the MMS (n = 70) and NMMS (n = 75) groups . Two patients (2.9%) in the MMS and nine patients (12%) in the NMMS groups developed SFI . The risk ratio for developing SFI in the NMMS group compared to the MMS group was 4.20 (P = 0.023) . This relationship was confounded, however, by the diagnosis of hematologic tumor or solid tumor (RR = 3.15, P = 0.082) . SFI was the primary cause or major contributing factor in five of the 10 total deaths in our study population . Four SFI-related deaths occurred in the NMMS group and one SFI-related death occurred in the MMS group . Our data suggest a protective role for GM-CSF, IL-3 or other MMS cytokines in preventing SFI in patients receiving HDC . This should be further investigated as a potential complementary approach to conventional strategies in antifungal prophylaxis for patients receiving HDC.

Genetics, 1996 Jul, 143(3), 1115 - 25
Fine-structure mapping of meiosis-specific double-strand DNA breaks at a recombination hotspot associated with an insertion of telomeric sequences upstream of the HIS4 locus in yeast; Xu F et al.; Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand DNA breaks (DSBs) . Using two approaches, we mapped the position of DSBs associated with a recombination hotspot created by insertion of telomeric sequences into the region upstream of HIS4 . We found that the breaks have no obvious sequence specificity and localize to a region of approximately 50 bp adjacent to the telomeric insertion . By mapping the breaks and by studies of the exonuclease III sensitivity of the broken ends, we conclude that most of the broken DNA molecules have blunt ends with 3'-hydroxyl groups.

J Clin Microbiol, 1996 Jul, 34(7), 1846 - 8
Comparison of the new API Candida system to the ID 32C system for identification of clinically important yeast species; Fricker-Hidalgo H et al.; API Candida was evaluated in comparison with the ID 32C system for the identification of 619 yeast isolates . The sensitivity of API Candida for the identification of the 15 species it claims to identify with and without additional tests was 97.4% (593 of 609) and 75.2% (458 of 609), respectively . The API Candida system is easy to use and rapid (result in 18 to 24 h).

Biosci Biotechnol Biochem, 1996 Jul, 60(7), 1207 - 9
Oxidative stress response in yeast: purification and characterization of glutathione reductase from Hansenula mrakii; Miki T et al.; Glutathione reductase was purified from a yeast . Hansenula mrakii IFO 0895, to approximately 3500-fold with 59% activity yield . The enzyme was homogeneous on polyacrylamide gel electrophoresis . The molecular weight of the enzyme was estimated to be 56 kDa by SDS-polyacrylamide gel electrophoresis, and 123 kDa by gel filtration using a calibrated Sephadex G-150 column . The Km values for glutathione disulfide and NADPH were 21.3 microM and 14.3 microM, respectively . The enzyme was most active at pH 7.5, 55 degrees C . The enzyme was stable up to 40 degrees C, and between pHs 4 and 10 . The enzyme was inhibited by p-chloromercuribenzoate and metal ions such as Fe3+, Cd2+, Cu2+, and Zn2+.

RNA, 1996 Jul, 2(7), 707 - 17
Requirement for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3' splice site; Brys A et al.; Yeast pre-mRNA splicing factors SLU7 and PRP16 are required for cleavage of the 3' splice site and exon ligation in vitro . Using natural and model precursor RNAs, we found that SLU7 is dispensable for splicing of RNAs in which the 3' splice site is in close proximity to the branchpoint . SLU7 is only required when the interval between the branchpoint and the 3' splice site is greater than 7 nt . In contrast, PRP16 is essential for splicing of all pre-mRNAs tested . Immunoprecipitation of the products of step 1 by anti-SLU7 antibodies demonstrates that SLU7 is a component of the spliceosome . Recruitment of SLU7 to the spliceosome is greatly enhanced by prior addition of PRP16 . PRP16 is liberated from the spliceosome after completion of step 2, whereas SLU7 remains bound to the excised intron and spliced mature RNA until the spliceosome disassembles, in a reaction that requires ATP.

J Cell Biol, 1996 Jul, 134(2), 443 - 54
Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function; Marschall LG et al.; gamma-Tubulin is a conserved component of microtubule-organizing centers and is thought to be involved in microtubule nucleation . A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin that is related to, but divergent from, gamma-tubulins . TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is localized to the spindle pole body (Sobel, S.G., and M . Snyder . 1995.J . Cell Biol . 131:1775-1788) . We have characterized the expression of TUB4, the association of Tub4p with the spindle pole body, and its role in microtubule organization . Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner . Wild-type Tub4p is localized to the spindle pole body, and a Tub4p-green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p . Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body . The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle . EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules . These results offer insight into the role of gamma-tubulin in microtubule-organizing center function.

J Cell Biol, 1996 Jul, 134(2), 413 - 27
The BUD4 protein of yeast, required for axial budding, is localized to the mother/BUD neck in a cell cycle-dependent manner; Sanders SL et al.; A and alpha cells of the yeast Saccharomyces cerevisiae exhibit an axial budding pattern, whereas a/alpha diploid cells exhibit a bipolar pattern . Mutations in BUD3, BUD4, and AXL1 cause a and alpha cells to exhibit the bipolar pattern, indicating that these genes are necessary to specify the axial budding pattern (Chant, J., and I . Herskowitz . 1991 . Cell . 65:1203-1212; Fujita, A., C . Oka, Y . Arikawa, T . Katagi, A . Tonouchi, S . Kuhara, and Y . Misumi . 1994 . Nature (Lond.) . 372:567-570) . We cloned and sequenced BUD4, which codes for a large, novel protein (Bud4p) with a potential GTP-binding motif . Bud4p is expressed and localized to the mother/bud neck in all cell types . Most mitotic cells contain two apparent rings of Bud4 immunoreactive staining, as observed for Bud3p (Chant, J., M . Mischke, E . Mitchell, I . Herskowitz, and J.R . Pringle . 1995 . J . Cell Biol . 129: 767-778) . Early G1 cells contain a single ring of Bud4p immunoreactive staining, whereas cells at START and in S phase lack these rings . The level of Bud4p is also regulated in a cell cycle-dependent manner . Bud4p is inefficiently localized in bud3 mutants and after a temperature shift of a temperature-sensitive mutant, cdc12, defective in the neck filaments . These observations suggest that Bud4p and Bud3p cooperate to recognize a spatial landmark (the neck filaments) during mitosis and support the hypothesis that they subsequently become a landmark for establishing the axial budding pattern in G1.

Eur J Biochem, 1996 Jul 1, 239(1), 220 - 8
X-ray structure of yeast inorganic pyrophosphatase complexed with manganese and phosphate; Harutyunyan EH et al.; The three-dimensional structure of the manganese-phosphate complex of inorganic pyrophosphatase from Saccharomyces cerevisiae has been refined to an R factor of 19.0% at 2.4-A resolution . X-ray data were collected from a single crystal using an imaging plate scanner and synchrotron radiation . There is one dimeric molecule in the asymmetric unit . The upper estimate of the root-mean-square coordinate error is 0.4 A using either the delta A plot or the superposition of the two crystallographically independent subunits . The good agreement between the coordinates of the two subunits, which were not subjected to non-crystallographic symmetry restraints, provides independent validation of the structure analysis . The active site in each subunit contains four manganese ions and two phosphates . The manganese ions are coordinated by the side chains of aspartate and glutamate residues . The phosphate groups, which were identified on the basis of their local stereochemistry, interact either directly or via water molecules with manganese ions and lysine, arginine, and tyrosine side chains . The phosphates are bridged by two of the manganese ions . The outer phosphate is exposed to solvent . The inner phosphate is surrounded by all four manganese ions . The ion-binding sites are related to the order of binding previously established from kinetic studies . A hypothesis for the transition state of the catalytic reaction is put forward.

Eur J Biochem, 1996 Jul 1, 239(1), 124 - 31
N,N'-Bisformyl dityrosine is an in vivo precursor of the yeast ascospore wall; Briza P et al.; The amino acid, dityrosine, is a major component of the spore wall surface of the yeast Saccharomyces cerevisiae, where it is part of a highly cross-linked macromolecular network of yet unknown chemical structure, consisting mostly of glucosamine, dityrosine and few other amino acids . Biosynthesis of the dityrosine moiety of this network consists of several steps, including the chemical modification of free L-tyrosine and the subsequent oxidative cross-linking of the modified tyrosine residues (catalyzed by a cytochrome P-450), leading to soluble dityrosine-containing spore wall precursors . We isolated, purified and characterized the dityrosine-containing precursor that appears late in spore wall synthesis and that is thought to be directly incorporated into the maturing spore wall . Chemical and spectroscopic analyses showed that this precursor is N,N'-bisformyl dityrosine . In addition, we identified a tyrosine-containing spore wall precursor as N-formyl tyrosine . The elucidation of the chemical structure of soluble spore wall precursors is crucial for the characterization of the function of the enzymes involved in maturation of the spore surface, e.g . by in vitro systems . A dityrosine-containing fragment, which was solubilized from mature spore walls by partial hydrolysis, was identified as N-formyl dityrosine . Mature spore walls contain significant amounts of N-formyl dityrosine and N,N'-bisformyl dityrosine . This supports the assumption that the dityrosine-containing macromolecular network on the spore surface has an unusual, non-peptidic structure.

J Cell Biol, 1996 Jul, 134(1), 37 - 51
The absence of Pmp47, a putative yeast peroxisomal transporter, causes a defect in transport and folding of a specific matrix enzyme; Sakai Y et al.; Candida boidinii Pmp47, an integral peroxisomal membrane protein, belongs to a family of mitochondrial solute transporters (e.g., ATP/ADP exchanger), and is the only known peroxisomal member of this family . However, its physiological and biochemical functions have been unrevealed because of the difficulties in the molecular genetics of C . boidinii . In this study, we first isolated the PMP47 gene, which was the single gene encoding for Pmp47 in a gene-engineerable strain S2 of C . boidinii . Sequence analysis revealed that it was very similar to PMP47A and PMP47B genes from a polyploidal C . Boidinii strain (ATCC32195) . Next, the PMP47 gene was disrupted and the disruption strain (pmp47delta) was analyzed . Depletion of PMP47 from strain S2 resulted in a retarded growth on oleate and a complete loss of growth on methanol . Both growth substrates require peroxisomal metabolism . EM observations revealed the presence of peroxisomes in methanol- and oleate-induced cells of pmp47delta, but in reduced numbers, and the presence of material of high electron density in the cytoplasm in both cases . Methanol-induced cells of pmp47delta were investigated in detail . The activity of one of the methanol-induced peroxisome matrix enzymes, dihydroxyacetone synthase (DHAS), was not detected in pmp47delta . Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body, while two other peroxisome matrix enzymes, alcohol oxidase (AOD) and catalase, were active and found in peroxisomes . Two peroxisome-deficient mutants, strains M6 and M13 (described in previous studies), retained DHAS activity although it was mislocalized to the cytoplasm and the nucleus . We disrupted PMP47 in these peroxisome-deficient mutants . In both strains, M6-pmp47delta and M13-pmp47delta, DHAS was enzymatically active and was located in the cytoplasm and the nucleus . We suggest that an unknown small molecule, which PMP47 transports, is necessary for the folding or the translocation machinery of DHAS within peroxisomes . Pmp47 does not catalyze folding directly because active DHAS is observed in the M6-pmp47delta and M13-pmp47delta strains . Since both AOD and DHAS have the PTS1 motif sequences at their carboxyl terminal, our results first show that depletion of Pmp47 could dissect the peroxisomal import pathway (PTS1 pathway) of these proteins.

Biochem J, 1996 Jul 1, 317 ( Pt 1), 89 - 95
The genes YNI1 and YNR1, encoding nitrite reductase and nitrate reductase respectively in the yeast Hansenula polymorpha, are clustered and co-ordinately regulated; Brito N et al.; The nitrite reductase-encoding gene (YNI1) from the yeast Hansenula polymorpha was isolated from a lambda EMBL3 H . polymorpha genomic DNA library, using as a probe a 481 bp DNA fragment from the gene of Aspergillus nidulans encoding nitrite reductase (niiA) . An open reading frame of 3132 bp, encoding a putative protein of 1044 amino acids with high similarity with nitrite reductases from fungi, was located by DNA sequencing in the phages lambdaNB5 and lambdaJA13 . Genes YNI1 and YNR1 (encoding nitrate reductase) are clustered, separated by 1700 bp . Northern blot analysis showed that expression of YNI1 and YNR1 is co-ordinately regulated; induced by nitrate and nitrite and repressed by sources of reduced nitrogen, even in the presence of nitrate . A mutant lacking nitrite reductase activity was obtained by deletion of the chromosomal copy of YNI1 . The mutant does not grow in nitrate or in nitrite; it exhibits a similar level of transcription of YNR1 to the wild type, but the nitrate reductase enzymic activity is only about 50% of the wild type . In the presence of nitrate the delta ynil::URA3 mutant extrudes approx . 24 nmol of nitrite/h per mg of yeast (wet weight), about five times more than the wild type.

Nucleic Acids Res, 1996 Jul 1, 24(13), 2519 - 24
A new efficient gene disruption cassette for repeated use in budding yeast; Guldener U et al.; The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers . We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-lox P recombination system . This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%) . Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus . This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.

Genes Dev, 1996 Jul 1, 10(13), 1699 - 708
The yeast splicing factor Mud13p is a commitment complex component and corresponds to CBP20, the small subunit of the nuclear cap-binding complex; Colot HV et al.; The mechanism by which pre-mRNAs are initially recognized by the splicing machinery is not well understood . In the yeast system, commitment complexes are the earliest identified splicing complexes . They contain pre-mRNA, U1 snRNP, and the splicing factor Mud2p and probably correspond to the mammalian E complexes, which contain pre-mRNA, U1 snRNP, and the splicing factor U2AF . To identify other yeast commitment complex components, we have characterized mutant strains that are synthetic lethal with viable U1 snRNA mutations . We report here that MUD13 is a nonessential gene that encodes the yeast homolog of CBP20, the small subunit of the vertebrate nuclear cap-binding complex (CBC) . Characterization of splicing in the delta-MUD13 strain and extract indicates that Mud13p is a yeast splicing factor and is the second identified non-snRNP commitment complex component . The observations also suggest that CBC interacts with other commitment complex components as well as with the substrate cap . Taken together with the accompanying results for a mammalian system, our data indicate that cap-binding proteins as well as the pre-mRNA cap contribute to early steps in spliceosome assembly.

Genes Dev, 1996 Jul 1, 10(13), 1595 - 607
Multiple determinants controlling activation of yeast replication origins late in S phase; Friedman KL et al.; Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase . Previous work has shown that telomeres act as determinants for late origin activation . However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres . Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time . These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set . Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.

EMBO J, 1996 Jul 1, 15(13), 3377 - 84
Iron-regulated DNA binding by the AFT1 protein controls the iron regulon in yeast; Yamaguchi-Iwai Y et al.; Iron deprivation of Saccharomyces cerevisiae induces transcription of genes required for high-affinity iron uptake . AFT1 mediates this transcriptional control . In this report, the 5'-flanking region of FET3, which encodes a copper-dependent oxidase required for iron transport, was analyzed and found to contain a DNA sequence responsible for AFT1-regulated gene expression . AFT1 was capable of interacting specifically with this DNA sequence . A core element within this DNA sequence necessary for the binding of AFT1 was also determined . In vivo footprinting demonstrated occupancy of the AFT1 binding site in cells deprived of iron and not in cells grown in the presence of iron . Thus, the environmental signal resulting from iron deprivation was transduced through the regulated binding of AFT1 to the FET3 promoter, followed by the activation of transcription . A regulon of genes under the control of AFT1 could be defined . AFT1 was able to bind to a consensus binding site (PyPuCACCCPu) in the 5' region of FRE1, FRE2, FTR1, FTH1 and CCC2.

EMBO J, 1996 Jul 1, 15(13), 3296 - 305
Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF); Haas A et al.; In Saccharomyces cerevisiae, vacuoles are inherited by the formation of tubular and vesicular structures from the mother vacuole, the directed projection of these structures into the bud and the homotypic fusion of these vesicles . We have previously exploited a cell-free inheritance assay to show that the fusion step of vacuole inheritance requires cytosol, ATP and the GTPase Ypt7p . Here we demonstrate, using affinity-purified antibodies and purified recombinant proteins, a requirement for Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF) in homotypic vacuole fusion in vitro . Thus, Sec17p and Sec18p, which are typically involved in heterotypic transport steps, can also be involved in homotypic organelle fusion . We further show that vacuole-to-vacuole fusion is stimulated by certain fatty acyl-coenzyme A compounds in a Sec18p-dependent fashion . Finally, our data suggest the presence of a cytosolic factor which activates vacuole membrane-bound Sec18p.

Mol Cell Biol, 1996 Jul, 16(7), 3833 - 43
Functional mapping of the translation-dependent instability element of yeast MATalpha1 mRNA; Hennigan AN et al.; The determinants of mRNA stability include specific cis-acting destabilizing sequences located within mRNA coding and noncoding regions . We have developed an approach for mapping coding-region instability sequences in unstable yeast mRNAs that exploits the link between mRNA translation and turnover and the dependence of nonsense-mediated mRNA decay on the activity of the UPF1 gene product . This approach, which involves the systematic insertion of in-frame translational termination codons into the coding sequence of a gene of interest in a upf1delta strain, differs significantly from conventional methods for mapping cis-acting elements in that it causes minimal perturbations to overall mRNA structure . Using the previously characterized MATalpha1 mRNA as a model, we have accurately localized its 65-nucleotide instability element (IE) within the protein coding region . Termination of translation 5' to this element stabilized the MATalpha1 mRNA two- to threefold relative to wild-type transcripts . Translation through the element was sufficient to restore an unstable decay phenotype, while internal termination resulted in different extents of mRNA stabilization dependent on the precise location of ribosome stalling . Detailed mutagenesis of the element's rare-codon/AU-rich sequence boundary revealed that the destabilizing activity of the MATalpha1 IE is observed when the terminal codon of the element's rare-codon interval is translated . This region of stability transition corresponds precisely to a MATalpha1 IE sequence previously shown to be complementary to 18S rRNA . Deletion of three nucleotides 3' to this sequence shifted the stability boundary one codon 5' to its wild-type location . Conversely, constructs containing an additional three nucleotides at this same location shifted the transition downstream by an equivalent sequence distance . Our results suggest a model in which the triggering of MATalpha1 mRNA destabilization results from establishment of an interaction between translating ribosomes and a downstream sequence element . Furthermore, our data provide direct molecular evidence for a relationship between mRNA turnover and mRNA translation.

Mol Cell Biol, 1996 Jul, 16(7), 3730 - 41
The product of HUM1, a novel yeast gene, is required for vacuolar Ca2+/H+ exchange and is related to mammalian Na+/Ca2+ exchangers; Pozos TC et al.; Calcineurin, or PP2B, plays a critical role in mediating Ca2+-dependent signaling in many cell types . In yeast cells, this highly conserved protein phosphatase regulates aspects of ion homeostasis and cell wall synthesis . We show that calcineurin mutants are sensitive to high concentrations of Mn2+ and identify two genes, CCC1 and HUM1, that, at high dosages, increase the Mn2+ tolerance of calcineurin mutants . CCC1 was previously identified by complementation of a Ca2+-sensitive (csg1) mutant . HUM1 (for "high copy number undoes manganese") is a novel gene whose predicted protein product shows similarity to mammalian Na+/Ca2+ exchangers . hum1 mutations confer Mn2+ sensitivity in some genetic backgrounds and exacerbate the Mn2+ sensitivity of calcineurin mutants . Furthermore, disruption of HUM1 in a calcineurin mutant strain results in a Ca2+-sensitive phenotype . We investigated the effect of disrupting HUM1 in other strains with defects in Ca2+ homeostasis . The Ca2+ sensitivity of pmc1 mutants, which lack a P-type ATPase presumed to transport Ca2+ into the vacuole, is exacerbated in a hum1 mutant strain background . Also, the Ca2+ content of hum1 pmc1 cells is less than that of pmc1 cells . In contrast, the Ca2+ sensitivity of vph1 mutants, which are specifically defective in vacuolar acidification, is not significantly altered by disruption of Hum1p function . These genetic interactions suggest that Hum1p may participate in vacuolar Ca2+/H+ exchange . Therefore, we prepared vacuolar membrane vesicles from wild-type and hum1 cells and compared their Ca2+ transport properties . Vacuolar membrane vesicles from hum1 mutants lack all Ca2+/H+ antiport activity, demonstrating that Hum1p catalyzes the exchange of Ca2+ for H+ across the yeast vacuolar membrane.

Mol Cell Biol, 1996 Jul, 16(7), 3651 - 7
Identifying a species-specific region of yeast TF11B in vivo; Shaw SP et al.; The general transcription factor IIB (TFIIB) is required for RNA polymerase II transcription in eukaryotes . It provides a physical link between the TATA-binding protein (TBP) and the RNA polymerase and is a component previously suggested to respond to transcriptional activators in vitro . In this report, we compare the yeast (Saccharomyces cerevisiae) and human forms of the protein in yeast cells to study their functional differences . We demonstrate that human TFIIB fails to functionally replace yeast TFIIB in yeast cells . By analyzing various human-yeast hybrid TFIIB molecules, we show that a 14-amino-acid region at the amino terminus of the first repeat of yeast TFIIB plays an important role in determining species specificity in vivo . In addition, we identify four amino acids in this region that are critical for an amphipathic helix unique to yeast TFIIB . By site-directed mutagenesis analyses we demonstrate that these four amino acids are important for yeast TFIIB's activity in vivo . Finally, we show that mutations in the species-specific region of yeast TFIIB can differentially affect the expression of genes activated by different activators in vivo . These results provide strong evidence suggesting that yeast TFIIB is involved in the process of transcriptional activation in living cells.

Mol Cell Biol, 1996 Jul, 16(7), 3429 - 36
Yeast mitochondrial RNase P RNA synthesis is altered in an RNase P protein subunit mutant: insights into the biogenesis of a mitochondrial RNA-processing enzyme; Stribinskis V et al.; Rpm2p is a protein subunit of Saccharomyces cerevisiae yeast mitochondrial RNase P, an enzyme which removes 5' leader sequences from mitochondrial tRNA precursors . Precursor tRNAs accumulate in strains carrying a disrupted allele of RPM2 . The resulting defect in mitochondrial protein synthesis causes petite mutants to form . We report here that alteration in the biogenesis of Rpm1r, the RNase P RNA subunit, is another consequence of disrupting RPM2 . High-molecular-weight transcripts accumulate, and no mature Rpm1r is produced . Transcript mapping reveals that the smallest RNA accumulated is extended on both the 5' and 3' ends relative to mature Rpm1r . This intermediate and other longer transcripts which accumulate are also found as low-abundance RNAs in wild-type cells, allowing identification of processing events necessary for conversion of the primary transcript to final products . Our data demonstrate directly that Rpm1r is transcribed with its substrates, tRNA met f and tRNAPro, from a promoter located upstream of the tRNA met f gene and suggest that a portion also originates from a second promoter, located between the tRNA met f gene and RPM1 . We tested the possibility that precursors accumulate because the RNase P deficiency prevents the removal of the downstream tRNAPro . Large RPM1 transcripts still accumulate in strains missing this tRNA . Thus, an inability to process cotranscribed tRNAs does not explain the precursor accumulation phenotype . Furthermore, strains with mutant RPM1 genes also accumulate precursor Rpm1r, suggesting that mutations in either gene can lead to similar biogenesis defects . Several models to explain precursor accumulation are presented.

Mol Cell Biol, 1996 Jul, 16(7), 3308 - 16
TFG/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9; Cairns BR et al.; The SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 gene products are all required for proper transcriptional control of many genes in the yeast Saccharomyces cerevisiae . Genetic studies indicated that these gene products might form a multiprotein SWI/SNF complex important for chromatin transitions preceding transcription from RNA polymerase II promoters . Biochemical studies identified a SWI/SNF complex containing these and at least six additional polypeptides . Here we show that the 29-kDa component of the SWI/SNF complex is identical to TFG3/TAF30/ANC1 . Thus, a component of the SWI/SNF complex is also a member of the TFIIF and TFIID transcription complexes . TFG3 interacted with the SNF5 component of the SWI/SNF complex in protein interaction blots . TFG3 is significantly similar to ENL and AF-9, two proteins implicated in human acute leukemia . These results suggest that ENL and AF-9 proteins interact with the SNF5 component of the human SWI/SNF complex and raise the possibility that the SWI/SNF complex is involved in acute leukemia.

Mol Cell Biol, 1996 Jul, 16(7), 3275 - 84
Human ARF4 expression rescues sec7 mutant yeast cells; Deitz SB et al.; Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events . The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae . To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants . We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature . The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells . Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function . Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction . The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools . These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic . In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner . This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics.

Mol Cell Biol, 1996 Jul, 16(7), 3235 - 44
Yeast pip3/mec3 mutants fail to delay entry into S phase and to slow DNA replication in response to DNA damage, and they define a functional link between Mec3 and DNA primase; Longhese MP et al.; The catalytic DNA primase subunit of the DNA polymerase alpha-primase complex is encoded by the essential PRI1 gene in Saccharomyces cerevisiae . To identify factors that functionally interact with yeast DNA primase in living cells, we developed a genetic screen for mutants that are lethal at the permissive temperature in a cold-sensitive pril-2 genetic background . Twenty-four recessive mutations belonging to seven complementation groups were identified . Some mutants showed additional phenotypes, such as increased sensitivity to UV irradiation, methyl methanesulfonate, and hydroxyurea, that were suggestive of defects in DNA repair and/or checkpoint mechanisms . We have cloned and characterized the gene of one complementation group, PIP3, whose product is necessary both for delaying entry into S phase or mitosis when cells are UV irradiated in G1 or G2 phase and for lowering the rate of ongoing DNA synthesis in the presence of methyl methanesulfonate . PIP3 turned out to be the MEC3 gene, previously identified as a component of the G2 DNA damage checkpoint . The finding that Mec3 is also required for the G1- and S-phase DNA damage checkpoints, together with the analysis of genetic interactions between a mec3 null allele and several conditional DNA replication mutations at the permissive temperature, suggests that Mec3 could be part of a mechanism coupling DNA replication with repair of DNA damage, and DNA primase might be involved in this process.

Genomics, 1996 Jul 1, 35(1), 196 - 206
A yeast artificial chromosome contig and NotI restriction map that spans the tumor suppressor gene(s) locus, 11q22.2-q23.3; Arai Y et al.; Human chromosome 11q22-q23 is a pathologically important region in which a high level of loss of heterozygosity has been reported for breast, ovary, cervical, colon, and lung carcinomas, malignant melanomas, and hematologic malignancies . This strongly indicates that one or more tumor suppressor genes reside within the deleted region . In this report, we report the development of a contig map that covers most of the deleted regions found in these malignancies . The map comprises a contig of 66 overlapping yeast artificial chromosomes (YACs) and spans a region of 17 Mb from the PGR gene at 11q22.2 to the MLL gene at q23.3 . In the process of screening the YACs, 50 new sequence-tagged site markers were developed from the termini of the YAC inserts . These markers were used for chromosome walking, and the data were then integrated into the contig map . NotI restriction mapping of these YACs revealed the presence of at least 26 NotI sites in the region . Using 22 of them, a NotI restriction map of the region from PGR to D11S939 was developed . This YAC contig will provide efficient tools for identification of the putative tumor suppressor gene(s).

Genomics, 1996 Jul 1, 35(1), 79 - 86
The construction of a yeast artificial chromosome (YAC) contig in the vicinity of the Usher syndrome type IIa (USH2A) gene in 1q41; Sumegi J et al.; The gene for Usher syndrome type II (USH2A), an autosomal recessive syndromic deafness, has been mapped to a region of 1q41 flanked proximally by D1S217 and distally by D1S439 . Using sequence-tagged sites (STSs) within the region, a total of 21 yeast artificial chromosome (YAC) clones were isolated and ordered into a single contig that spans approximately 11.0 Mb . The order of microsatellite and STS markers in this region was established as D1S505-D1S425-DXS217-D1S556-D1S237-D1S4 74-EB1-EB2-KB6-AFM144XF2-KB1-K B4-D1S229-D1S490-D1S227-TGFbeta2-D1S439 . Analysis of newly positioned polymorphic markers in recombinant individuals in two Usher syndrome type IIa families has enabled us to identify DXS474 and AFM144XF2 as two flanking markers for the Usher type IIa locus . The physical distance between the two markers is 1.0 Mb . This region is covered by eight YACs from the CEPH library: 945f7, 867g9, 762a6, 919h3, 794b8, 785h4, 848b9, and 841g2 . A long-range physical map of the Usher type IIa critical region, using MluI, BssHII, NotI, EagI, and SacII, has been developed.

Yeast, 1996 Jun 30, 12(8), 809 - 13
The 2 micron plasmid of laboratory yeast strains is a type-1/type-2 hybrid; Xiao W et al.; Industrial yeast strains carry one of two homeologous 2 microns plasmids designated as type-1 or type-2 . The 2 microns plasmid, Scp1, found in common laboratory strains of Saccharomyces cerevisiae is considered a type-2 plasmid, since the ori, STB, RAF and REP1 loci and intergenic sequences of the right-unique region of Scp1 are homologous to the corresponding loci in industrial strain type-2 plasmids . However, within both its 599 bp inverted repeats Scp1 has 142-bp sequences homologous to the bakers' yeast type-1 plasmid . DNA sequence analyses and oligonucleotide hybridizations indicate that the 142-bp insertion in Scp1 was probably due to homeologous recombination between type-1 and type-2 plasmids . These results suggest that some of the plasmid and chromosomal sequence polymorphisms seen in laboratory yeast strains result from homeologous recombination in their ancestral breeding stock.

Yeast, 1996 Jun 30, 12(8), 787 - 97
Sequence analysis of a 40.7 kb segment from the left arm of yeast chromosome X reveals 14 known genes and 13 new open reading frames including homologues of genes clustered on the right arm of chromosome XI; Katsoulou C et al.; The complete nucleotide sequence of a 40.7 kb segment about 130 kb from the left end of chromosome X of Saccharomyces cerevisiae was determined from two overlapping cosmids . Computer analysis of that sequence revealed the presence of the previously known genes VPS35, INO1, SnR128, SnR190, MP12, YAK1, RPB4, YUR1, TIF2, MRS3 and URA2, three previously sequenced open reading frames (ORFs) of unknown function 5' of the INO1, 5' of the MP12 and 3' of the URA2 genes and 13 newly identified ORFs . One of the new ORFs is homologous to mammalian glycogenin glycosyltransferases and another has similarities to the human phospholipase D . Some others contain potential transmembrane regions or leucine zipper motifs . The existence of yeast expressed sequence tags for some of the newly identified ORFs indicates that they are transcribed . A cluster of six genes within 10 kb (YUR1, TIF2, two new ORFs, an RSP25 homologue and MRS3) have homologues arranged similarly within 28.5 kb on the right arm of chromosome XI.

Yeast, 1996 Jun 30, 12(8), 773 - 86
Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast; Niedenthal RK et al.; The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted much attention as a tool to study a number of biological processes . This study describes the use of GFP as a vital reporter molecule for localization and expression studies in Saccharomyces cerevisiae . Construction of GFP expression vectors which allow N- or C-terminal fusion of the gfp gene to a gene of interest allowed the generation of fusion proteins whose subcellular localization was followed by fluorescence microscopy in living yeast cells . Analysis of three unknown open reading frames obtained from the budding yeast chromosome XIV resulted in distinct staining patterns, allowing prediction of the cellular localization of these unknown proteins . Furthermore, GFP was used to construct a gene replacement cassette which, after homologous integration into the genomic locus, placed the gfp gene behind a promoter of interest . The amount of GFP produced from this promoter was then quantified in living yeast cells by flow cytometry . With this novel replacement cassette a gene of interest can be deleted and at the same time its expression level studied under various growth conditions . The experiments presented here suggest that GFP represents a convenient fluorescent marker for localization studies as well as gene expression studies in budding yeast . Systematic studies of a large number of genes should benefit from such assays.

Yeast, 1996 Jun 30, 12(8), 731 - 40
Sustained oscillations in free-energy state and hexose phosphates in yeast; Richard P et al.; In a population of intact cells of the yeast Saccharomyces cerevisiae the dynamics of glycolytic metabolism were investigated under the condition of sustained oscillations . At 5-s intervals cells were quenched in -40 degrees C methanol, extracted and the intracellular concentrations of glycolytic metabolites, adenine nucleotides and phosphate were analysed . Oscillations were found for the glycolytic intermediates glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate . At variance with earlier reports on transient glycolytic oscillations, some intermediates further down the glycolytic pathway did not oscillate significantly, even though NADH did . In addition, the adenylate energy charge and the free energy of ATP hydrolysis oscillated significantly . Dynamic coupling through the latter may be responsible for this effective compartmentation of glycolytic dynamics.

J Biol Chem, 1996 Jun 28, 271(26), 15837 - 44
HDA1 and HDA3 are components of a yeast histone deacetylase (HDA) complex; Carmen AA et al.; Histone acetylation is maintained through the action of histone acetyltransferases and deacetylases and has been correlated with increased gene activity . To investigate the functional role of these enzymes in the regulation of transcription, we have purified from Saccharomyces cerevisiae two histone deacetylase activities, HDA and HDB, with molecular masses of approximately 350 and 600 kDa, respectively . In vitro, the HDA activity deacetylates all four core histones, has a preference for histone H3, and is strongly inhibited by trichostatin A (a specific inhibitor of histone deacetylases) . HDB is considerably less sensitive to trichostatin A . We report the extensive purification of the HDA activity and the identification of peptides (p75, p73, p72, and p71) whose presence correlates with deacetylase activity on native polyacrylamide gels . An antibody to p75 immunoprecipitates peptides with molecular masses similar to those in the 350-kDa complex . Additionally, antibodies to p75 and p71 specifically precipitate histone deacetylase activity and co-immunoprecipitate each other . Gene disruptions of p75 (HDA1) or p71 (HDA3) cause the loss of the 350-kDa (but not the 600-kDa) activity from our chromatography profiles . These data argue strongly that HDA1 and HDA3 are subunits of the HDA complex, which is structurally distinct from the second, HDB complex.

Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6605 - 9
Effect of deletion of 5'HS3 or 5'HS2 of the human beta-globin locus control region on the developmental regulation of globin gene expression in beta-globin locus yeast artificial chromosome transgenic mice; Peterson KR et al.; To analyze the function of the 5' DNase I hypersensitive sites (HSs) of the locus control region (LCR) on beta-like globin gene expression, a 2.3-kb deletion of 5'HS3 or a 1.9-kb deletion of 5'HS2 was recombined into a beta-globin locus yeast artificial chromosome, and transgenic mice were produced . Deletion of 5'HS3 resulted in a significant decrease of epsilon-globin gene expression and an increase of gamma-globin gene expression in embryonic cells . Deletion of 5'HS2 resulted in only a small decrease in expression of epsilon-, gamma-, and beta-globin mRNA at all stages of development . Neither deletion affected the temporal pattern of globin gene switching . These results suggest that the LCR contains functionally redundant elements and that LCR complex formation does not require the presence of all DNase I hypersensitive sites . The phenotype of the 5'HS3 deletion suggests that individual HSs may influence the interaction of the LCR with specific globin gene promoters during the course of ontogeny.

Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6589 - 93
The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells; Cunningham E et al.; The mammalian phosphatidylinositol transfer proteins (PITP) and the yeast Saccharomyces cerevisiae PITP (SEC14p) that show no sequence homology both catalyze exchange of phosphatidylinositol (PI) between membranes compartments in vitro . In HL-60 cells where the cytosolic proteins are depleted by permeabilization, exogenously added PITPalpha is required to restore G protein-mediated phospholipase Cbeta (PLCbeta) signaling . Recently, a second mammalian PITPbeta form has been described that shows 77% identity to rat PITPalpha . We have examined the ability of the two mammalian PITPs and SEC14p to restore PLC-mediated signaling in cytosol-depleted HL-60 and RBL-2H3 cells . Both PITPalpha and PITPbeta isoforms as well as SEC14p restore G protein-mediated PLCbeta signaling with a similar potency . In RBL-2H3 cells, crosslinking of the IgE receptor by antigen stimulates inositol lipid hydrolysis by tyrosine phosphorylation of PLCgamma1 . Permeabilization of RBL cells leads to loss of PLCgamma1 as well as PITP into the extracellular medium and this coincides with loss of antigen-stimulated lipid hydrolysis . Both PLCgamma1 and PITP were required to restore inositol lipid signaling . We conclude that (i) because the PI binding/transfer activities of PITP/SEC14p is the common feature shared by all three transfer proteins, it must be the relevant activity that determines their abilities to restore inositol lipid-mediated signaling and (ii) PITP is a general requirement for inositol lipid hydrolysis regardless of how and which isoform of PLC is activated by the appropriate agonist.

Mol Cell Endocrinol, 1996 Jun 18, 120(1), 31 - 9
Identification of COUP-TFII as a peroxisome proliferator response element binding factor using genetic selection in yeast: COUP-TFII activates transcription in yeast but antagonizes PPAR signaling in mammalian cells; Marcus SL et al.; Peroxisome proliferator-response elements (PPRE) are cis-acting regulatory elements that confer responsiveness to peroxisome proliferators and various fatty acids by serving as target sites for ligand-activated peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers . Other cellular factors, including additional nuclear hormone receptors, also interact with PPREs and modulate PPAR function . We have developed a positive selection strategy in yeast to identify mammalian factors that functionally interact with PPREs . Saccharomyces cerevisiae containing an integrated copy of the HIS3 gene under transcriptional control of a minimal CYC1 promoter and two copies of the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase PPRE was constructed and transformed with a rat liver cDNA yeast expression library . Plasmids were isolated from his + transformants . One plasmid contained a cDNA encoding the complete rat chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), an orphan member of the nuclear hormone receptor superfamily . COUP-TFII potently activated PPRE-linked reporter gene expression in yeast, and COUP-TFII synthesized in yeast or in vitro formed specific protein/DNA complexes with this PPRE . Significantly, COUP-TFII did not activate transcription of PPRE-linked reporter genes in mammalian cells but rather strongly inhibited induction mediated by PPAR/RXR . Our findings demonstrate the utility of using genetic screening in yeast to identify sequence-specific DNA binding transcription factors.

EMBO J, 1996 Jun 17, 15(12), 3127 - 34
Propagation of the yeast prion-like {psi+} determinant is mediated by oligomerization of the SUP35-encoded polypeptide chain release factor; Paushkin SV et al.; The Sup35p protein of yeast Saccharomyces cerevisiae is a homologue of the polypeptide chain release factor 3 (eRF3) of higher eukaryotes . It has been suggested that this protein may adopt a specific self-propagating conformation, similar to mammalian prions, giving rise to the {psi+} nonsense suppressor determinant, inherited in a non-Mendelian fashion . Here we present data confirming the prion-like nature of {psi+} . We show that Sup35p molecules interact with each other through their N-terminal domains in {psi+}, but not {psi-} cells . This interaction is critical for {psi+} propagation, since its disruption leads to a loss of {psi+} . Similarly to mammalian prions, in {psi+} cells Sup35p forms high molecular weight aggregates, accumulating most of this protein . The aggregation inhibits Sup35p activity leading to a {psi+} nonsense-suppressor phenotype . N-terminally altered Sup35p molecules are unable to interact with the {psi+} Sup35p isoform, remain soluble and improve the translation termination in {psi+} strains, thus causing an antisuppressor phenotype . The overexpression of Hsp104p chaperone protein partially solubilizes Sup35P aggregates in the {psi+} strain, also causing an antisuppressor phenotype . We propose that Hsp104p plays a role in establishing stable {psi+} inheritance by splitting up Sup35p aggregates and thus ensuring equidistribution of the prion-like Sup35p isoform to daughter cells at cell divisions.

EMBO J, 1996 Jun 17, 15(12), 3040 - 52
A pathway in the yeast cell division cycle linking protein kinase C (Pkc1) to activation of Cdc28 at START; Marini NJ et al.; In an effort to study further the mechanism of Cdc28 function and cell cycle commitment, we describe here a genetic approach to identify components of pathways downstream of the Cdc28 kinase at START by screening for mutations that decrease the effectiveness of signaling by Cdc28 . The first locus to be characterized in detail using this approach was PKC1 which encodes a homolog of the Ca(2+)-dependent isozymes of the mammalian protein kinase C (PKC) superfamily (Levin et al., 1990) . By several genetic criteria, we show a functional interaction between CDC28 and PKC1 with PKC1 apparently functioning with respect to bud emergence downstream of START . Consistent with this, activity of the MAP kinase homolog Mpk1 (a putative Pkc1 effector) is stimulated by activation of Cdc28 . Furthermore, we demonstrate a cell cycle-dependent hydrolysis of phosphatidylcholine to diacylglycerol (a PKC activator) and choline phosphate at START . Diacylglycerol production is stimulated by Cdc28 in cycling cells and is closely associated with Cdc28 activation at START . These results imply that the activation of Pkc1, which is known to be necessary during bud morphogenesis, is mediated via the CDC28-dependent stimulation of PC-PLC activity in a novel cell cycle-regulated signaling pathway.

Yeast, 1996 Jun 15, 12(7), 667 - 72
A simplified method for the repeated replacement of yeast chromosomal sequences with in vitro mutations; Szent-Gyorgyi C; A strategy for gene replacement in Saccharomyces cerevisiae has been modified to facilitate the repeated substitution of a chromosomal locus with in vitro generated variant sequences, so that the resulting locus contains only the desired mutation and is free of extraneous vector DNA . The construction of an internally deleted chromosomal target locus carrying the counterselectable CYH2S marker and a second positively selectable marker has been simplified; the design of the locus has been altered to increase the frequency of authentic gene replacements obtained upon the subsequent integration of in vitro mutated DNA . The modified chromosomal target locus is amenable to replacement using either of two transformation protocols: (i) integration of a second positively selectable plasmid carrying mutant sequences to form a tandem intermediate structure at the locus; upon counterselection on cycloheximide, all vector sequence is excised to give the desired replacement at high frequency (> 70%); (ii) single-step integration of a linear segment of mutated genomic DNA by selection for cycloheximide resistance . A subsequent screen for the loss of the positively selectable target locus marker detects the desired replacement at modest frequency (> 2%) . Polymerase chain reaction using multiple primers in a single amplification reaction is useful for monitoring these variously modified chromosomal loci.

Genomics, 1996 Jun 15, 34(3), 368 - 75
Isolation and characterization of the human and mouse homologues (SUPT4H and Supt4h) of the yeast SPT4 gene; Chiang PW et al.; To study gene regulation mediated by chromatin in mammals, we isolated the human (SUPT4H) and murine (Supt4h) counterparts of the yeast gene encoding SPT4; the product of this gene presumably interacts with the products of the mammalian homologues (which we have also cloned) of yeast SPT5 and SPT6, thereby modulating chromatin formation and activity . We isolated two different sized human SUPT4H cDNA clones (1464 and 728 nt) and one murine Supt4h (688 nt) cDNA clone; all three encode the same 117-amino-acid protein with conservation of the zinc finger motif found in SPT4 . Conservation of this zinc finger motif from yeast to mouse and human implies functional importance . Although the overall sequence homology at the DNA level between the human 728-nt transcript and the murine 688-nt transcript is only 78.4%, the DNA sequence homology is 97.7% within the coding region . At the protein level, the amino acid sequences of the translated murine Supt4h and the human SUPT4H gene products are identical . The likely functional copy of SUPT4H, which has at least two introns, maps to human chromosome 17, with candidate intronless pseudogenes on chromosomes 2, 12, and 20 . Buttressing the hypothesis that this is a gene required constitutively, both the human SUPT4H transcripts and the murine Supt4h transcript are expressed widely, although not at equal levels (e.g., such as most histones), in all fetal and adult tissues that we examined.

Nucleic Acids Res, 1996 Jun 15, 24(12), 2395 - 403
Interactions of the yeast centromere and promoter factor, Cpf1p, with the cytochrome c1 upstream region and functional implications on regulated gene expression; Oechsner U et al.; The upstream activation site (UAS) of the cytochrome c1 gene, CYT1, contains sequences for DNA-binding of several transcription factors . Among them are the heme-dependent protein, Hap1p, and the multiprotein complex, Hap2/3/4/5, which mediate transcriptional induction under aerobic conditions and after exhaustion of glucose, respectively . The multiple interactions of nuclear proteins with the UAS region of CYT1 observed in electrophoretic mobility shift experiments are influenced by carbon source and oxygen tension, but are independent of both regulators, Hap1p and Hap2/3/4/5 . All protein-DNA complexes obtained are solely due to the association of the centromere and promoter factor 1 (Cpf1p) with the centromere determining element (CDE I)-like motif at the 5' boundary of the UAS(CYT1) . This motif overlaps with a consensus sequence for the binding of the general factor Abf1p . Functional analyses after the separate introduction of point mutations into both elements reveal no role for the latter protein and only a minor role for Cpf1p in the regulated expression of CYT1/lacZ chimaeric proteins . However, in cpf1-mutants, induction of CYT1 reaches higher steady state levels and adaptation to aerobic conditions occurs faster than in wild-type . Thus, Cpf1p seems to reduce CYT1 promoter activity under partly inducing conditions, e.g . when only one of the activators, Hap1p or the Hap2 complex, exerts its function.

Nucleic Acids Res, 1996 Jun 15, 24(12), 2338 - 46
Yeast retrotransposon Ty4: the majority of the rare transcripts lack a U3-R sequence; Hug AM et al.; The retrotransposon Ty4 is found in different yeast strains at only one to three copies per haploid genome . In the present study, we aimed at relating the apparent low transpositional activity of Ty4 to transcriptional features of this element . RT-PCR revealed that Ty4 is transcribed at a very low level, being comparable with that of GAL4 . Contrary to other Ty elements, the transcriptional rate of Ty4 is not affected in a sin4 background nor by treatment of cells with alpha factor . From experiments measuring the expression levels in 1acZ fusion constructs, we conclude that Ty4 transcription is repressed by a negative regulating element residing within the LTR, whereas positive cis-acting elements, like those that have been found to mediate expression of Ty1/2 and Ty3, are absent from Ty4 . Analysing Ty4 transcript termini by the RACE-PCR method, we found several distinct transcriptional initiation sites . But surprisingly, the majority of the polyadenylated Ty4 transcripts terminate shortly upstream from the 3' LTR boundary, so that these transcripts do not contain a U3-R sequence, which is normally required for obligate strand transfer during DNA synthesis . Thus, the extremely low transcription rate of Ty4 and imperfect Ty4 transcripts are the reason for the low transpositional activity of this element.

Nucleic Acids Res, 1996 Jun 15, 24(12), 2331 - 7
Dual influence of the yeast Cat1p (Snf1p) protein kinase on carbon source-dependent transcriptional activation of gluconeogenic genes by the regulatory gene CAT8; Rahner A et al.; The CSRE (carbon source-responsive element) is a sequence motif responsible for the transcriptional activation of gluconeogenic structural genes in Saccharomyces cerevisiae . We have isolated a regulatory gene, DIL1 (derepression of isocitrate lyase, = CAT8), which is specifically required for derepression of CSRE-dependent genes . Expression of CAT8 is carbon source regulated and requires a functional Cat1p (Snf1p) protein kinase . The derepression defect of CAT8 in a cat1 mutant could be suppressed by a mutant Mig1p repressor protein . Derepression of CAT8 also requires a functional HAP2 gene, suggesting a regulatory connection between respiratory and gluconeogenic genes . Carbon source-dependent protein-CSRE complexes detected in a gel retardation analysis with wild-type extracts were absent in cat8 mutant extracts . However, similar experiments with an epitope-tagged CAT8 gene product in the presence of tag-specific antibodies gave evidence against a direct binding of Cat8p to the CSRE . A constitutively expressed GAL4-CAT8 fusion gene revealed a carbon source-dependent transcriptional activation of a UAS(GAL)-containing reporter gene . Activation mediated by Cat8p was no longer detectable in a cat1 mutant . Thus, biosynthetic control of CAT8 as well as transcriptional activation by Cat8p requires a functional Cat1p protein kinase . A model proposing CAT8 as a specific activator of a transcription factor(s) binding to the CSRE is discussed.

Nucleic Acids Res, 1996 Jun 15, 24(12), 2271 - 80
Replication of yeast DNA and novel chromosome formation in mouse cells; McGuigan A et al.; To determine whether yeast DNA can replicate or segregate in mammalian cells, we have transferred genomic DNA from the yeast Saccharomyces cerevisiae into mouse cells . Most of the lines contained stably integrated yeast DNA . However, in two of the lines, the yeast DNA was maintained as numerous small extrachromosomal elements which were still present after 26 cell divisions in selection but which were lost rapidly out of selection . This indicates that, although yeast DNA can replicate in mouse cells, the yeast centromere does not function to give segregation . In one cell line we observed a large novel chromosome consisting almost entirely of yeast DNA . This chromosome segregates well and contains mouse centromeric minor satellite DNA and variable amounts of major satellite DNA which probably comprise the functional centromere . The yeast DNA in the novel chromosome has a compacted chromatin structure which may be responsible for the efficient formation of anaphase bridges . Furthermore, yeast DNA integrated into mouse chromosomes forms constrictions at the point of integration . These features have previously been presumed to be hallmarks of centromeric function in transfection assays aimed at identifying putative centromeric DNA . Hence our results suggest caution be exercised in the interpretation of such assays.

Biochem J, 1996 Jun 15, 316 ( Pt 3), 833 - 9
Leishmania mexicana p12cks1, a homologue of fission yeast p13suc1, associates with a stage-regulated histone H1 kinase; Mottram JC et al.; We have isolated a Leishmania mexicana homologue of the fission yeast suc1 gene using PCR with oligonucleotides designed to conserved regions of cdc2 kinase subunits (cks) . The product of cks1 is a 12 kDa polypeptide, which has 70% identity with human p9cks1 and 44% identity with fission yeast p13suc1.p12cks1 was detected in the three life-cycle stages of L . mexicana by immunoblotting . Recombinant p12cks1 (p12cks1his) bound to agarose beads was used as a matrix to affinity-select histone H1 kinase complexes from Leishmania, yeast and bovine extracts . Immunoblotting showed that yeast and bovine cdc2 kinase bound to p12cks1his, thus demonstrating functional homology between L . mexicana p12cks1 and yeast p13suc1 . Histone H1 kinase activity was found at a high level in the proliferative promastigote and amastigote forms of L . mexicana, but at a low level in the non-dividing metacyclic form . These activities are likely to be the same as the leishmanial p13suc1 binding kinase (SBCRK) described previously {Mottram, Kinnaird, Shiels, Tait and Barry (1993) J . Biol . Chem . 268, 21044-21051} . A distinct cdc2-related kinase, L . mexicana CRK1, was also found to associate with p12cks1his but affinity-depletion experiments showed that CRK1 was not responsible for the histone H1 kinase activity associating with p12cks1his in promastigote cell extracts . The finding that p12cks1 associates with at least two cdc2-related kinases, SBCRK and CRK1, is consistent with the presence of a large gene family of cdc2-related kinases in trypanosomatids, a situation thought to be more similar to higher eukaryotes than yeast.

Genes Dev, 1996 Jun 15, 10(12), 1516 - 31
Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast; Piatti S et al.; In eukaryotic cells, DNA replication is confined to a discrete period of the cell cycle and does not usually recur until after anaphase . In the budding yeast Saccharomyces cerevisiae, assembly of pre-replication complexes (pre-RCs) at future origins as cells exit mitosis (or later during G1 is necessary for subsequent initiation of DNA replication triggered by activation in late G1 of Cdc28/Cdk1 kinases associated with B-type cyclins Clb1-Clb6 . The absence of pre-RCs during G2 and M phases could explain why origins of DNA replication fire only once during the cell cycle, even though S-phase-promoting Cdks remain active from the beginning of S phase through the end of M phase . Formation of pre-RCs and their maintenance during G1 depend on the synthesis and activity of an unstable protein encoded by CDC6 . We find that Cdc6 synthesis can only promote DNA replication in a restricted window of the cell cycle: between destruction of Clbs after anaphase and activation of Clb5/ and Clb6/Cdk1 in late G1 . The latter corresponds to a "point of no return," after which Cdc6 synthesis can no longer promote DNA replication . Cdc6 protein can be made throughout the cell cycle and, in certain circumstances, can accumulate within the nuclei of G2 and M phase cells without inducing re-replication . Thus, control over Cdc6 degradation and/or nuclear localization is not crucial for preventing origin re-firing . Our data are consistent with the notion that cells can no longer incorporate de novo synthesized Cdc6 into pre-RCs once C1b/Cdk1 kinases have been activated . We show that Cdc6p associates with Clb/Cdk1 kinases from late G1 until late anaphase, which might be important for inhibiting pre-RC assembly during S, G2, and M phases . Inhibition of pre-RC assembly by the same kinases that trigger initiation explains how origins are prevented from re-firing until Clb kinases are destroyed after anaphase.

Virology, 1996 Jun 15, 220(2), 516 - 21
Molecular interactions between the HSV-1 capsid proteins as measured by the yeast two-hybrid system; Desai P et al.; HSV-1 B capsids are composed of seven major proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26 . VP indicates that the capsid protein is also a component of the infectious virion . Capsid proteins 21, 22a, and VP24 are specified by a single open reading frame (UL26) that encodes 635 amino acids . An objective of the work in our laboratory is to identify and map interactions among and between capsid proteins . In the present studies we employed the yeast GAL4 two-hybrid system developed by Fields and his colleagues (Nature 240, 245-246 (1989)) for this purpose . DNA corresponding to the capsid open reading frames was derived as a PCR product and fused to sequences of the GAL4 activation and DNA binding domains . Using this system each of the capsid proteins has been tested for interactions with all of the other capsid proteins . Three interactions have been identified: a relatively strong self-interaction between 22a molecules (residues 307-635 of UL26), bimolecular interactions between 22a and VP5, and another between VP19C and VP23 . The interactions were detected by the expression of beta-galactosidase enzyme activity, and yielded 289, 86, and 63 units of enzyme activity, respectively . For the 22a self-interaction, elimination of residues 611-635 resulted in an approximately twofold decrease in enzyme activity . The C-terminal 25 amino acids of 22a were also essential for the bimolecular interaction between 22a and VP5.

Anal Biochem, 1996 Jun 15, 238(1), 26 - 8
Assay for in vivo yeast invertase activity using NaF; Silveira MC et al.; The methods used for invertase activity determination are based on the measurement of glucose or reducing sugars produced by the enzymatic hydrolysis of sucrose into glucose and fructose . When whole yeast cells are used in these assays, the monosaccharides formed by the action of the periplasmic enzyme can be taken up and metabolized, leading to errors on the enzyme activity determination . This study reports a method for a more accurate invertase activity measurement by blocking the glycolytic pathway . In this method the cells were preincubated with 50 mM sodium fluoride, and inhibitor of enolase . This in vivo measurement of the enzyme activity, under initial rate conditions, was performed using cell concentrations up to 64 mg cell/ml . The results obtained showed that this method is particularly useful for cells with low invertase activity.

Anal Biochem, 1996 Jun 15, 238(1), 14 - 8
Entrapment by immobilized metal ion affinity chromatography of assembled yeast mitochondrial ATP synthase containing individual subunits tagged with hexahistidine; Bateson M et al.; We demonstrate the use of immobilized metal ion affinity chromatography for isolating the constituent subunits of assembled mitochondrial ATP synthase (mtATPase) wherein a single subunit of the complex has been modified to contain hexahistidine . Genes encoding subunit d or OSCP of mtATPase from Saccharomyces cerevisiae were modified each to encode a polypeptide having a C-terminal addition of six consecutive histidines . Expression of plasmid-borne modified genes, in host yeast cells lacking a functional copy of the relevant endogenous gene, generated functional mtATPase complexes as judged by growth of rescued cells on the nonfermentable substrate ethanol . Significantly, the oligomycin-sensitive ATP hydrolase activity in mitochondria from cells expressing tagged subunits was similar to that of cells expressing unmodified subunits, indicating that there had been no impairment of the functional integrity of mtATPase . Mitochondrial lysates were prepared from each strain and subjected to chromatography under nondenaturing conditions on a resin containing immobilized Ni2+ . It is likely that the mtATPase complexes adsorbed by immobilized metal ion affinity chromatography are fully assembled because their subunit composition closely matches that of a preparation of assembled mtATPase conventionally isolated from mitochondrial lysates by ammonium sulfate precipitation and purification by sucrose gradient centrifugation . Furthermore, assembled mtATPase containing a tagged subunit could be adsorbed, albeit at lower yield, when the relevant modified gene was expressed in wild-type host cells . The general application of this novel isolation procedure greatly simplifies and reduces the number of steps required for the isolation of assembled multi-subunit complexes . Moreover, the approach may be used for studying subunit-subunit interactions within the mtATPase complex.

J Biol Chem, 1996 Jun 14, 271(24), 13931 - 4
Interaction of transforming growth factor-beta receptor I with farnesyl-protein transferase-alpha in yeast and mammalian cells; Ventura F et al.; Transforming growth factor beta (TGF-beta) signals through two transmembrane serine/threonine kinases, known as TbetaR-I and TbetaR-II . Several lines of evidence suggest that TbetaR-II acts as a primary receptor, binding TGF-beta and phosphorylating TbetaR-I whose kinase activity then propagates the signal to unknown substrates . We report an interaction between TbetaR-I and the farnesyl-protein transferase-alpha subunit (FT-alpha) both in a yeast two-hybrid system and in mammalian cells . These findings raise the possibility that TGF-beta might regulate cellular functions by altering the ability of FT-alpha to catalyze isoprenylation of targets such as G proteins, lamins, or cytoskeletal components . However, we provide evidence that TGF-beta action does not alter the overall protein isoprenyl transferase activity in Mv1Lu mink lung epithelial cells . In fact, the beta subunits of farnesyl transferase and geranylgeranyl transferase, which are necessary for the activity of FT-alpha, prevent the association of FT-alpha with TbetaR-I . Furthermore, farnesyl transferase activity is shown to be dispensable for TGF-beta signaling of growth inhibitory and transcriptional responses in these cells . These results suggest that the interaction between TbetaR-I and FT-alpha does not affect the known functions of these two proteins.

J Biol Chem, 1996 Jun 14, 271(24), 14041 - 4
Characterization of interactions between the neurofilament triplet proteins by the yeast two-hybrid system; Leung CL et al.; In the adult axon, the neurofilaments (NFs) are heteropolymers formed from the low (NFL), middle (NFM), and high (NFH) molecular weight neurofilament triplet proteins (NFTPs) . All three proteins have the basic intermediate filament protein tripartite structure, which consists of a short amino-terminal head region, an alpha-helical rod region of approximately310 amino acids, and a carboxyl-terminal tail region of variable length . In vitro polymerization studies have shown that only NFL can assemble into homopolymeric 10-nm filaments . The assembly of intermediate filaments, including the NFs, begins with the formation of a coiled-coil dimer involving the alpha-helical rod domains of two molecules . In order to determine whether homodimers or heterodimers of NFTPs are the preferred intermediates in the assembly of NFs, we have used the yeast two-hybrid system to study the interactions between the different NFTPs . By monitoring the activity of the lacZ reporter gene product, we are able to show that the interactions of NFL with NFL, NFM, or NFH are stronger than the interactions of NFM with NFM or NFH and the interaction of NFH with NFH . These results imply that NFM and NFH are more likely to form heterodimers with NFL than homodimers and are consistent with the inability of NFM and NFH to self-polymerize in vitro and in vivo.

Science, 1996 Jun 14, 272(5268), 1646 - 9
Thymine-thymine dimer bypass by yeast DNA polymerase zeta; Nelson JR et al.; The REV3 and REV7 genes of the yeast Saccharomyces cerevisiae are required for DNA damage-induced mutagenesis . The Rev3 and Rev7 proteins were shown to form a complex with DNA polymerase activity . This polymerase replicated past a thymine-thymine cis-syn cyclobutane dimer, a lesion that normally severely inhibits replication, with an efficiency of approximately 10 percent . In contrast, bypass replication efficiency with yeast DNA polymerase alpha was no more than 1 percent . The Rev3-Rev7 complex is the sixth eukaryotic DNA polymerase to be described, and is therefore called DNA polymerase zeta.

Proc Natl Acad Sci U S A, 1996 Jun 11, 93(12), 5905 - 9
Complementation of the beige mutation in cultured cells by episomally replicating murine yeast artificial chromosomes; Perou CM et al.; Chediak-Higashi syndrome in man and the beige mutation of mice are phenotypically similar disorders that have profound effects upon lysosome and melanosome morphology and function . We isolated two murine yeast artificial chromosomes (YACs) that, when introduced into beige mouse fibroblasts, complement the beige mutation . The complementing YACs exist as extrachromosomal elements that are amplified in high concentrations of G418 . When YAC-complemented beige cells were fused to human Chediak-Higashi syndrome or Aleutian mink fibroblasts, complementation of the mutant phenotype also occurred . These results localize the beige gene to a 500-kb interval and demonstrate that the same or homologous genes are defective in mice, minks, and humans.

FEBS Lett, 1996 Jun 10, 388(1), 5 - 10
Association of yeast SAP1, a novel member of the 'AAA' ATPase family of proteins, with the chromatin protein SIN1; Liberzon A et al.; The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes . In addition, sin1 mutants are defective in proper mitotic chromosome segregation . In an effort to understand the basis for these phenotypes, we employed the yeast two-hybrid system to identify proteins that interact with SIN1 in vivo . Here, we demonstrate that SAP1, a novel protein belonging to the 'AAA' family of ATPases, is able to directly interact with SIN1 . Furthermore, we show, using recombinant molecules in vitro, that a short 27 amino acid sequence near the N-terminal of SIN1 is sufficient to bind SAP1 . Previous experiments defined different domains of SIN that interact with other proteins and with DNA . The C-terminal domain of SIN1 was shown to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SIN1 . The central 'HMG1-like region' of SIN1 binds DNA, while the N-terminal of SIN1 can bind CDC23, a protein that regulates chromosome segregation . These data, taken together with the results presented here, suggest that SIN1 is a multifunctional chromatin protein that can interact with a number of different proteins that are involved in several different cellular functions.

J Biol Chem, 1996 Jun 7, 271(23), 13706 - 15
Isolation and characterization of TAF25, an essential yeast gene that encodes an RNA polymerase II-specific TATA-binding protein-associated factor; Klebanow ER et al.; We describe the cloning and analysis of TAF25, a previously uncharacterized yeast gene that encodes a yeast TATA-binding protein-associated factor or yTAF of Mr = 25,000 . The gene encoding yTAF25 is a single copy essential gene, and the protein sequence deduced from TAF25 exhibits sequence similarity to a metazoan hTAFII . The results from immunological studies confirm that yTAF25 is a subunit of a large multiprotein TATA-binding protein-yeast TATA-binding protein-associated factor complex that contains a subset of the total number of the yTAFs present in yeast cell extracts . Both genetic and biochemical analyses demonstrate that yTAF25 can interact directly with itself . Transcriptional data show that the activity of the multiprotein complex containing yTAF25 is RNA polymerase II-specific, thus indicating that TAF25 encodes a bona fide yeast RNA polymerase II TAF . Hence the protein encoded by TAF25 has been termed yTAFII25.

Biochemistry, 1996 Jun 4, 35(22), 7204 - 13
Expression in yeast and purification of functional macrophage nitric oxide synthase . Evidence for cysteine-194 as iron proximal ligand; Sari MA et al.; Mouse macrophage NO-synthase (mNOS) was expressed in a unique yeast-based system by using a three-step procedure which allows yeast growth and NOS expression to be uncoupled . Despite cytotoxic effects related to mNOS expression, levels of catalytically active enzyme up to 0.5 mg of protein per 5 L of culture was obtained after purification . Its electrophoretic, spectroscopic {lambda max = 446 nm for its Fe(II)-CO complex}, and catalytic properties were similar to those previously reported for mNOS purified from macrophages . Recombinant mNOS catalyzed the NADPH-dependent oxidation of L-arginine to citrulline (Km = 7 +/- 3 microM) as well as the reduction of cytochrome C by NADPH {Km = 34 +/- 8 microM and Vm = 25 +/- 5 mumol min-1 (mg of protein-1)} . Two mutants of mNOS in which Cys 194 was replaced with either serine or histidine were constructed and expressed in the same yeast strain at a level higher than that of the wild type protein, as they appear less toxic for the host . Both mutants exhibited electrophoretic properties and activities toward cytochrome C reduction identical to those of wild type NOS . However, they were unable to catalyze the oxidation of L-arginine to citrulline and did not appear to bind heme (no appearance of peaks around 400 and 446 nm for the resting enzyme and its CO complex, respectively, in visible spectroscopy) . These data provide the first experimental evidence in favor of previous suggestions that Cys 194 was the proximal iron ligand of mouse mNOS.

EMBO J, 1996 Jun 3, 15(11), 2640 - 50
A novel Hsp70 of the yeast ER lumen is required for the efficient translocation of a number of protein precursors; Craven RA et al.; The yeast genome sequencing project predicts an open reading frame (YKL073) that would encode a novel member of the Hsp70 family of molecular chaperones . We report that this 881 codon reading frame represents a functional gene expressing a 113-119 kDa glycoprotein localized within the lumen of the endoplasmic reticulum (ER) . We therefore propose to designate this gene LHS1 (Lumenal Hsp Seventy) . Our studies indicate that LHS1 is regulated by the unfolded protein response pathway, as evidenced by its transcriptional induction in cells treated with tunicamycin, and in various mutants defective in precursor processing (sec11-7, sec53-6 and sec59-1) . LHS1 is not essential for viability, but an Lhs1 null mutant strain exhibits a coordinated induction of genes regulated by the unfolded protein response indicating a role for Lhs1p in protein folding in the ER . Furthermore, the null mutation is synthetically lethal in combination with (delta)ire1, thus activation of the unfolded protein response pathway is essential for cells to tolerate loss of Lhs1p . Synthetically lethality is also seen with mutations in KAR2, strongly suggesting that Kar2p and Lhs1p have overlapping functions . The Lhs1 null mutant exhibits a severe constitutive defect in the translocation of several secretory preproteins . We therefore propose that Lhs1p is a molecular chaperone of the ER lumen involved in both polypeptide translocation and subsequent protein folding.

Bioseparation, 1996 Jun, 6(3), 159 - 63
An investigation into the possible use of hydrocyclones for the removal of yeast from beer; Yuan H et al.; This investigation has focused on the importance of hydrocyclone design and running conditions for the removal of yeast from suspensions . Experiments have been carried out using both a model system of yeast grown in suspension cultures and rough beer . It appeared that the most important factor was the design of the hydrocyclone with a positive correlation between higher residence times and acceleration with the efficiency of separation . The other factor that affected the separation efficiency was the concentration of the yeast suspension and a new phenomenon of a lower efficiency of hydrocyclones at very low feed concentrations has been observed . The separation efficiency was also affected by temperature with lower temperatures giving poorer separations.

Cell Struct Funct, 1996 Jun, 21(3), 175 - 81
Topogenic effect of positively charged N-terminal amino acid in ER translocation of yeast alpha-factor precursor; Lee MA et al.; Expression of fusion proteins between prepro-alpha-factor and somatostatin (SRIF) in yeast, resulted in the correct processing and secretion of the heterologous 14-amino acid SRIF peptide (1) . When the chimeric genes were placed under the control of yeast acid phosphatase (PHO5) promoter, significant amount of an unglycosylated form of the fusion precursor molecule accumulated intracellularly, suggesting disruption of an endoplasmic reticulum-mediated function . We report here that the appearance of the precursor is due to an alteration in the three amino terminal residues of the chimera, i.e., Met-Arg-Phe in native prepro-alpha-factor is changed to Met-Phe-Lys in the hybrids . The unglycosylated precursor represents a population of molecules that are disrupted at an early stage of targeting to or translocation across the endoplasmic reticulum membrane . Our data demonstrate that the N-terminus plays an important role in topogenesis . Furthermore, these results show that translocation and glycosylation can be uncoupled from protein synthesis in vivo, and therefore can be posttranslational events in yeast.

Int J Pept Protein Res, 1996 Jun, 47(6), 484 - 90
Alkaline unfolding and salt-induced folding of yeast alcohol dehydrogenase under high pH conditions; Le WP et al.; The conformational changes of yeast alcohol dehydrogenase during unfolding at alkaline pH have been followed by fluorescence emission and circular dichroism spectra . A result of comparison of inactivation and conformational changes shows that much lower values of alkaline pH are required to bring about inactivation than significant conformational change of the enzyme molecule . At pH 9.5, although the enzyme has been completely inactivated, no marked conformational changes can be observed . Even at pH 12, the apparently fully unfolded enzyme retains some ordered secondary structure . After removal of Zn2+ from the enzyme molecule, the conformational stability decreased . At pH 12 by adding the salt, the relatively unfolded state of denatured enzyme changes into a compact conformational state by hydrophobic collapsing . Folded states induced by salt bound ANS strongly, indicating the existence of increased hydrophobic surface . More extensive studies showed that although apo-YADH and holo-YADH exhibited similar behavior, the folding cooperative ability of apo-enzyme was lower than that of holo-enzyme . The above results suggest that the zinc ion plays an important role in helping the folding of YADH and in stabilizing its native conformation.

Biochem Mol Biol Int, 1996 Jun, 39(3), 439 - 45
Purification and partial characterization of a fatty acid-binding protein from the yeast, Yarrowia lipolytica; Dell'Angelica EC et al.; A low-molecular-mass fatty acid-binding protein was isolated from the cytosol of the yeast Yarrowia lipolytica . Purification was achieved by a two-step procedure involving size-exclusion and cation-exchange chromatography . The isolated protein exists as a monomer of 15 kDa, is basic and has a blocked N-terminus . Internal amino acid sequencing suggests that this protein may belong to a novel class of fatty acid-binding proteins.

Indian J Biochem Biophys, 1996 Jun, 33(3), 177 - 83
Use of pH studies to determine the kinetic and chemical mechanism of yeast alcohol dehydrogenase with primary aliphatic alcohols and aldehydes; Leskovac V et al.; A complete list of all steady-state kinetic constants for the yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzed oxidation of ethanol, propan-1-ol and butan-1-ol, and for the reduction of acetaldehyde and propionaldehyde was collected in the pH range 6-10, and an appropriate pH profile for each constant was constructed . A common minimal mechanism with all these substrates has been postulated and pKa values and the pH independent limiting values have been assigned for the rate constants.

Chromosome Res, 1996 Jun, 4(4), 277 - 81
In situ localization of yeast artificial chromosome sequences on tomato and potato metaphase chromosomes; Fuchs J et al.; In situ localization of short low- or single-copy sequences is still difficult in plants . One solution to this problem could be the use of large yeast artificial chromosomes (YACs) for fluorescence in situ hybridization . Two YACs specific for a single copy marker on the long arm of the NOR-chromosome 2 of tomato (Lycopersicon esculentum) were selected . Both probes hybridized exclusively to this chromosome, although one produced a slightly dispersed hybridization signal . Hybridization of these YACs onto potato chromosome showed a clear single locus on the homoeologous potato chromosome in both cases.

Mol Biol Cell, 1996 Jun, 7(6), 985 - 99
Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant; Rieder SE et al.; In the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y transit from the Golgi to the lysosome-like vacuole via an endosome-like intermediate compartment . The vacuolar protein sorting (vps) mutant vps28, a member of the "class E" vps mutants, accumulates vacuolar, endocytic, and late Golgi markers in an aberrant endosome-like class E compartment . Sequence analysis of VPS28 revealed an open reading frame predicted to encode a hydrophilic protein of 242 amino acids . Consistent with this, polyclonal antiserum raised against Vps28p recognized a cytoplasmic protein of 28 kDa . Disruption of VPS28 resulted in moderate defects in both biosynthetic traffic and endocytic traffic destined for the vacuole . The transport of soluble vacuolar hydrolases to the vacuole was impaired in vps28 null mutant cells (approximately 40-50% carboxypeptidase Y missorted) . Internalization of the endocytic marker FM 4-64, a vital lipophilic dye, resulted in intense staining of a small intracellular compartment adjacent to an enlarged vacuole in delta vps28 cells . Furthermore, the vacuolar H+-ATPase accumulated in the perivacuolar class E compartment in delta vps28 cells, as did a-factor receptor Ste3p that was internalized from the plasma membrane . Electron microscopic analysis revealed the presence of a novel compartment consisting of stacks of curved membrane cisternae . Immunolocalization studies demonstrated that the vacuolar H+-ATPase is associated with this cupped cisternal structure, indicating that it corresponds to the class E compartment observed by fluorescence microscopy . Our data indicate that kinetic defects in both anterograde and retrograde transport out of the prevacuolar compartment in vps28 mutants result in the accumulation of protein and membrane in an exaggerated multilamellar endosomal compartment . We propose that Vps28p, as well as other class E Vps proteins, may facilitate (possibly as coat proteins) the formation of transport intermediates required for efficient transport out of the prevacuolar endosome.

Mol Biol Cell, 1996 Jun, 7(6), 853 - 70
CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p; Yokota K et al.; The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112 . Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865 . The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2 . Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease . The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1 . NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase . Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth . The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p . Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation . The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome.

Fungal Genet Biol, 1996 Jun, 20(2), 133 - 42
Structure and chemical composition of the cell walls from the haploid yeast and mycelial forms of Ustilago maydis; Ruiz-Herrera J et al.; Isolated or in vivo cell walls from the yeast and mycelial forms of haploid Ustilago maydis were not stained by the normal osmium procedure for electron microscopy . KMnO4 stained mycelial walls, revealing a layered structure with a loose electron-dense layer at the cell surface, but stained only the outer surface layer of yeast walls . Walls were purified from extracts obtained by ballistic and ultrasonic disruption . Chemical analysis showed that composition of yeast and mycelial walls was similar . Yeast walls contained higher amounts of neutral sugars and protein, whereas mycelial walls contained more chitin and phosphate . No chitosan or uronic acids were detected . Higher proportions of xylose and mannose were present in yeast walls, whereas the amounts of glucose and galactose were higher in mycelial walls . Fucose, arabinose, and ribose were detected in yeast walls only . Electrophoretic patterns of proteins extracted with SDS, beta 1, 3-glucanase, or chitinase were similar in walls of both morphologies, although some differential bands were identified . Most antigenic proteins appeared in the covalently bound fraction of the wall . Some were common to both morphologies, but others were stage specific.

Mol Microbiol, 1996 Jun, 20(6), 1135 - 43
Depletion in the levels of the release factor eRF1 causes a reduction in the efficiency of translation termination in yeast; Stansfield I et al.; In Saccharomyces cerevisiae, translation termination is mediated by a complex of two proteins, eRF1 and eRF3, encoded by the SUP45 and SUP35 genes, respectively . Mutations in the SUP45 gene were selected which enhanced suppression by the weak ochre (UAA) suppressor tRNA(Ser) SUQ5 . In each of four such allosuppressor alleles examined, an in-frame ochre (TAA) mutation was present in the SUP45 coding region; therefore each allele encoded both a truncated eRF1 protein and a full-length eRF1 polypeptide containing a serine missense substitution at the premature UAA codon . The full-length eRF1 generated by UAA read-through was present at sub-wild-type levels . In an suq5+ (i.e . non-suppressor) background none of the truncated eRF1 polypeptides were able to support cell viability, with the loss of only 27 amino acids from the C-terminus being lethal . The reduced eRF1 levels in these sup45 mutants did not lead to a proportional reduction in the levels of ribosome-bound eRF3, indicating that eRF3 can bind the ribosome independently of eRF1 . A serine codon inserted in place of the premature stop codon at codon 46 in the sup45-22 allele did not generate an allosuppressor phenotype, thereby ruling out this "missense' mutation as the cause of the allosuppressor phenotype . These data indicate that the cellular levels of eRF1 are important for ensuring efficient translation termination in yeast.

J Cell Sci, 1996 Jun, 109 ( Pt 6), 1347 - 57
A nitrogen starvation-induced dormant G0 state in fission yeast: the establishment from uncommitted G1 state and its delay for return to proliferation; Su SS et al.; Fission yeast cells either remain in the mitotic cell cycle or exit to meiotic sporulation from an uncommitted G1 state dependent on the presence or absence of nitrogen source in the medium (Nurse and Bissett, 1981) . We examined how heterothallic haploid cells, which cannot sporulate, behave under nitrogen-starvation for longer than 25 days at 26 degrees C . These cells were shown to enter a stable state (designated the dormant G0) with nearly full viability . Maintaining the dormant cells required glucose, suggesting that the cells remained metabolically active although cell division had ceased . They differed dramatically from mitotic and uncommitted G1 cells in heat resistance, and also in cytoplasmic and nuclear morphologies . After nitrogen replenishment, the initial responses of dormant G0 cells were investigated . The kinetics for reentry into the proliferative state were delayed considerably, and the changes in cell shape were enhanced particularly for those recovering from extended nitrogen starvation . A part of the delay could be accounted for by the duration of nuclear decondensation and cell elongation for the first cell division.

Protein Sci, 1996 Jun, 5(6), 1144 - 9
Substrate-induced conformational changes in yeast 3-phosphoglycerate kinase monitored by fluorescence of single tryptophan probes; Cheung CW et al.; 3-Phosphoglycerate kinase (PGK) catalyzes the reversible conversion of 3-phosphoglycerate (3-PG) and ATP to 1,3-diphosphoglycerate (1,3-diPG) and ADP in the presence of magnesium ions . PGK is a single polypeptide chain arranged in two domains, with an active site located in the interdomain cleft . The large distance between the binding sites for 3-PG and ATP, deduced from the crystallographic structures of the binary complexes, gave rise to the hypothesis that this enzyme undergoes a hinge-bending domain motion from open to closed conformation during catalysis . However, no direct experimental evidence exists for the "closed" conformation in the presence of both substrates . In this study, several PGK mutants with single tryptophans placed in various location were used as intrinsic fluorescent probes to examine the extent and delocalization of conformational changes induced by the binding of 3-PG, 1,3-diPG, ADP, ATP, and PNP-AMP (nonhydrolyzable analogue of ATP), and by 3-PG and PNP-AMP together . The results showed that only the probes situated in the hinge and in parts of each domain close to the hinge reflect substrate-induced conformational changes . Binding of substrates to one domain was found to induce spectral perturbation of the probes in the opposite domain, indicating a transmission of conformational changes between the domains . A combination of both substrates generated much larger fluorescence changes than the individual substrates . The binding constants were determined for each substrate using probes situated in different locations.

Sci China C Life Sci, 1996 Jun, 39(3), 225 - 33
Secretory expression of a single-chain insulin precursor in yeast and its conversion into human insulin; Zhang Y et al.; A synthetic single-chain porcine insulin precursor (PIP) gene and an alpha-mating factor leader sequence (alpha MFL) gene obtained by the PCR method are inserted between the promoter and 3'-terminating sequence of the alcohol dehydrogenase gene ADH1 in plasmid pVT102-U to form plasmid pVT102-U/alpha MFL-PIP . The single-chain insulin precursor is expressed and secreted to the culture medium by Saccharomyces cerevisiae transformed by pVT102-U/alpha MFL-PIP . The precursor is purified and converted into human insulin by tryptic transpeptidation . The purified human insulin is fully active and can be crystallized . The overall yield of human insulin is 25 mg per liter of culture medium.

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