Yeasts

Antisense Nucleic Acid Drug Dev, 1997 Feb, 7(1), 39 - 42
Sequence-specific cleavage of yeast tRNA(Phe) with oligonucleotides conjugated to a diimidazole construct; Vlassov V et al.; Oligonucleotide derivatives conjugated to a chemical construction with two histamine residues imitating the catalytic center of ribonuclease A have been synthesized . In experiments with the conjugates complementary to the 3'-end and to the variable loop and the T loop of yeast tRNA(Phe), it was shown that the compounds can accomplish sequence-specific cleavage of the target RNA in physiologic conditions.

Yeast, 1997 Feb, 13(2), 163 - 9
The nucleotide sequence of a 39 kb segment of yeast chromosome IV: 12 new open reading frames, nine known genes and one genes for Gly-tRNA; Bahr A et al.; The complete nucleotide sequence of a 39,090 bp segment from the left arm of yeast chromosome IV was determined . Twenty-one open reading frames (ORFs) longer than 100 amino acids and a Gly-tRNA gene were discovered . Nine of the 21 ORFs (D0892, D1022, D1037, D1045, D1057, D1204, D1209, D1214, D1219) correspond to the previously sequenced Saccharomyces cerevisiae genes for the NAD-dependent glutamate dehydrogenase (GDH), the secretory component (SHR3), the GABA transport protein (UGA4), the high mobility group-like protein (NHP2), the hydroxymethylbilane synthase (HEM3), the methylated DNA protein-cysteine S-methyltransferase (MGT1), a putative sugar transport protein, the Shm1 protein (SHM1) and the anti-silencing protein (ASF2) . The inferred amino acid sequences of 11 ORFs show significant similarity with known proteins from various organisms, whereas the remaining ORF does not share any similarity with known proteins.

Biotechniques, 1997 Feb, 22(2), 350 - 2
Mammalian two-hybrid system: a complementary approach to the yeast two-hybrid system; Luo Y et al.; Here we demonstrate the use of a mammalian two-hybrid system to study protein-protein interactions . Like the yeast two-hybrid system, this is a genetic, in vivo assay based on the reconstitution of the function of a transcriptional activator . In this system, one protein of interest is expressed as a fusion to the Gal4 DNA-binding domain and another protein is expressed as a fusion to the activation domain of the VP16 protein of the herpes simplex virus . The vectors that express these fusion proteins are cotransfected with a reporter chloramphenicol acetyltransferase (CAT) vector into a mammalian cell line . The reporter plasmid contains a cat gene under the control of five consensus Gal4 binding sites . If the two fusion proteins interact, there will be a significant increase in expression of the cat reporter gene . Previously, it was reported that mouse p53 antitumor protein and simian virus 40 large T antigen interact in a yeast two-hybrid system . Using a mammalian two-hybrid system, we were able to independently confirm this interaction . The mammalian two-hybrid system can be used as a complementary approach to verify protein-protein interactions detected by a yeast two-hybrid system screening . In addition, the mammalian two-hybrid system has two main advantages: (i) Assay results can be obtained within 48 h of transfection, and (ii) protein interactions in mammalian cells may better mimic actual in vivo interactions.

RNA, 1997 Feb, 3(2), 197 - 209
Mutations within the yeast U4/U6 snRNP protein Prp4 affect a late stage of spliceosome assembly; Ayadi L et al.; We showed previously that the yeast Prp4 protein is a spliceosomal factor that is tightly associated with the U4, U5, and U6 small nuclear RNAs . Moreover, Prp4 appears to associate very transiently with the spliceosome before the U4 snRNA dissociates from the spliceosome . Prp4 belongs to the Gbeta-like protein family, which suggests that the Prp4 Gbeta motifs could mediate interactions with other components of the spliceosome . To investigate the function of the Gbeta motifs, we introduced mutations within the second WD-repeat of Prp4 . Among the 35 new alleles found, 24 were pseudo wild-type mutants, 8 failed to grow at any temperature, and 3 were conditional sensitive mutants . The biochemical defects of the three thermosensitive prp4 mutants have been examined by immunoprecipitation, native gel electrophoresis, and glycerol gradient centrifugation . First, we show that snRNP formation is not impaired in these mutants and that Prp4 is present in the U4/U6 and U4/U6-U5 snRNP particles . We also demonstrate that spliceosome assembly is largely unaffected despite the fact that the first step of splicing does not occur . However, both Prp4 and U4 snRNA remain tightly associated with the spliceosome and this blocks the transition toward an active form of the spliceosome . Our results suggest a possible role of Prp4 in mediating important conformational rearrangements of proteins within the spliceosome that involve the region containing the Gbeta-repeats.

Curr Opin Genet Dev, 1997 Feb, 7(1), 59 - 66
Pheromone signalling and polarized morphogenesis in yeast; Leberer E et al.; Yeast cells respond to mating pheromones by activating a signal transduction pathway involving a seven transmembrane receptor/G protein complex linked to a mitogen-activated protein kinase module . Regulation of the G protein signal is controlled by the receptor and Sst2p; Sst2p may function as a GTPase-activating protein for the G protein alpha subunit . The Ste20 kinase acts in the linkage between the G protein and the MAP kinase module . Experiments suggest that binding of the Rho-like GTPase Cdc42p to Ste20p is not required for the mating response, yet is needed for the pseudohyphal growth response which involves many of the same kinases.

Curr Opin Genet Dev, 1997 Feb, 7(1), 7 - 16
Phosphorylation and proteolysis: partners in the regulation of cell division in budding yeast; Deshaies RJ; The budding yeast cell cycle oscillates between states of low and high cyclin B/cyclin-dependent kinase (CLB/CDK) activity . Remarkably, the two transitions that link these states are governed by ubiquitin-mediated proteolysis . The transition from low to high CLB activity is triggered by degradation of the CLB/CDK inhibitor SIC1, and the complementary excursion is propelled by the proteolytic destruction of CLBs . The extracellular environment controls this two-state circuit by regulating G1 cyclin/CDK activity, which is directly required for SIC1 proteolysis . Thus, stable oscillations of chromosome replication and segregation in budding yeast are propagated by the interplay between protein phosphorylation and protein degradation.

Curr Genet, 1997 Feb, 31(2), 106 - 11
Deletion of flanking ARS elements does not affect meiotic recombination at the HIS4 locus in yeast; Kirkpatrick DT; The HIS4-BIK1 interval on chromosome III of Saccharomyces cerevisiae contains a hotspot for meiotic recombination . Previous reports demonstrated that the initiating lesion is a double-stranded break which is subsequently processed in an asymmetric manner . Data presented here show that the efficiency of initiation of meiotic recombination is unaffected by the deletion of flanking ARS elements, and that the distribution of recombinants is not altered in strains heterozygous for these deletions . These results suggest that the initiation of recombination is not affected by the time of replication of the hotspot at HIS4 . The data also indicate that altering the direction of replication-fork movement through the HIS4 region does not affect meiotic recombination.

Biophys J, 1997 Feb, 72(2 Pt 1), 928 - 35
A new metal-binding site for yeast phosphoglycerate kinase as determined by the use of a metal-ATP analog; Pappu KM et al.; Suicide substrate beta, gamma-bidentate Rh(III)ATP (RhATP) was used to map the metal ion-binding site in yeast phosphoglycerate kinase (PGK) . Cleavage of the RhATP-inactivated enzyme with pepsin and subsequent separation of peptides by reverse-phase high-performance liquid chromatography gave two Rh-nucleotide bound peptides . One of the peptides corresponded to the C-terminal residues of PGK, and the other to a part of helix V . Of the four glutamates present in the C-terminal peptide, Glu 398 may be a likely metal coordination site . Therefore, importance of the C-terminal residues in PGK catalysis may be attributed, in part to the coordination of metal ion of the metal-ATP substrate . Metal coordination may then align the C-terminal peptide to extend toward the N-terminal domain and form the "closed" active site . Results presented in this paper suggest that one or more side chains of the enzyme may be coordinated to the metal ion in the PGK.3-phospho-D-glycerate-RhATP complex, and that exchange-inert metal-ATP analogs could be used to determine metal coordination sites on kinases and other metal-ATP-utilizing enzymes.

Nucleic Acids Res, 1997 Feb 1, 25(3), 668 - 74
Altered structure of the DNA duplex recognized by yeast transcription factor Reb1p; Davis DR et al.; The Saccharomyces cerevisiae REB1 gene encodes a sequence-specific DNA binding protein that has been implicated in chromatin structure, transcription regulation and transcription termination . Previous work has shown that the DNA sequence recognized by Reb1p contains an adenosine residue that is unusually reactive toward chemical modification by dimethylsulfate and that methylation of this nucleoside increases the binding affinity of the Reb1p protein for its target . Prompted by these results, we determined the solution structure of the 13mer Reb1p DNA duplex recognition site d(GTCCGGGTAATGC).d(GCATTACCCGGAC) using 2D NMR, distance geometry and iterative 2D NOESY back-calculation structure refinement . The distance geometry-refined molecule demonstrated an unusual structure in the TAAT region of the sequence that was manifested in cross-strand base stacking, as indicated by unusually strong NOE interactions between H2 protons on three adjacent adenosine bases . This structure was compared to two published NMR studies of DNA duplexes containing the related sequence TAAC . The Reb1p DNA structure does not show the conformational mobility or the 'transient kink' at TpA steps characteristic of the related TAAT-containing sequences.

Arch Biochem Biophys, 1997 Feb 1, 338(1), 1 - 6
A dual affinity tag on the 64-kDa Nlt1p subunit allows the rapid characterization of mutant yeast oligosaccharyl transferase complexes; Pathak R et al.; Oligosaccharyl transferase catalyzes the glycosylation of selected asparagine residues of nascent polypeptide chains as they are translocated into the lumen of the endoplasmic reticulum . To date, this enzyme has been purified from a number of eukaryotic organisms . Purification of transferase activity has yielded polypeptide complexes of three to six subunits depending on the source organism . Here we present the purification of an affinity-tagged version of the enzyme complex from a membrane protein fraction of the yeast Saccharomyces cerevisiae . A yeast strain was created in which the essential 64-kDa glycoprotein Nlt1p subunit of the oligosaccharyl transferase was modified by the addition of a 22-residue carboxy-terminal affinity tag; the tag included both an 8-residue FLAG epitope and a 6-residue histidine motif . Facile purification of the oligosaccharyl transferase was achieved using affinity chromatography media specific for each segment of the tag . The enzyme was purified as a heteromeric complex of five subunits in agreement with previously reported characterizations of the yeast transferase . Yeast strains bearing affinity-tagged enzyme subunits allow the rapid characterization of native and mutant transferase complexes.

Mol Endocrinol, 1997 Feb, 11(2), 193 - 202
RIP 140 enhances nuclear receptor-dependent transcription in vivo in yeast; Joyeux A et al.; RIP140 has previously been cloned as a factor that interacts with the estrogen receptor (ER) in vitro . We demonstrate in this study that RIP140 is a co-factor for nuclear receptor in yeast . RIP140 enhances the ER transcriptional activity by increasing 1.5- to 4-fold the induction factor of the reporter gene response at saturating hormone concentrations, this effect being magnified at suboptimal doses of estradiol . Moreover, RIP140 decreases the ED50 of the dose-response curve . These effects are recovered with an N-terminal truncated ER, but impaired by point mutations that abolish AF2-AD activity . We did not observe any modulation of the partial agonist 4-hydroxytamoxifen activity in the presence of RIP140 . Thus, RIP140 modulates transcriptional activity of ER through the AF2-AD domain and in a agonist-dependent fashion . RIP140 is also a strong coactivator for the retinoid pathway, as its expression enhances 10-fold the transactivation of a chimeric retinoic acid-alpha receptor at saturant hormone concentration and left shifted 5-fold the ED50 of the dose-response curve . We have investigated whether RIP140 could be involved in cross-talk between estrogenic and retinoid pathways.

Curr Opin Cell Biol, 1997 Feb, 9(1), 44 - 8
Myosins in yeast; Brown SS; It has been a banner year for the study of yeast myosins . Thanks to the completion of the Saccharomyces cerevisiae genome project, it is now known that budding yeast contains a total of five myosins . Furthermore, functions have been newly ascribed to several of them: two have been implicated in endocytosis, and another has been implicated in generating asymmetry between mother and daughter cells.

Biopolymers, 1997 Feb, 41(2), 213 - 31
A new approach to secondary structure evaluation: secondary structure prediction of porcine adenylate kinase and yeast guanylate kinase by CD spectroscopy of overlapping synthetic peptide segments; Behrends HW et al.; A new approach for evaluating the secondary structure of proteins by CD spectroscopy of overlapping peptide segments is applied to porcine adenylate kinase (AK1) and yeast guanylate kinase (GK3) . One hundred seventy-six peptide segments of a length of 15 residues, overlapping by 13 residues and covering the complete sequences of AK1 and GK3, were synthesized in order to evaluate their secondary structure composition by CD spectroscopy . The peptides were prepared by solid phase multiple peptide synthesis method using the 9-fluorenylmethoxycarbonyl/tert-butyl strategy . The individual peptide secondary structures were studied with CD spectroscopy in a mixture of 30% trifluoroethanol in phosphate buffer (pH 7) and subsequently compared with x-ray data of AK1 and GK3 . Peptide segments that cover alpha-helical regions of the AK1 or GK3 sequence mainly showed CD spectra with increasing and decreasing Cotton effects that were typical for appearing and disappearing alpha-helical structures . For segments with dominating beta-sheet conformation, however, the application of this method is limited due to the stability and clustering of beta-sheet segments in solution and due to the difficult interpretation of random-coiled superimposed beta-sheet CD signals . Nevertheless, the results of this method especially for alpha-helical segments are very impressive . All alpha-helical and 71% of the beta-sheet containing regions of the AK1 and GK3 could be identified . Moreover, it was shown that CD spectra of consecutive peptide content reveal the appearance and disappearance of alpha-helical secondary structure elements and help localizing them on the sequence string.

Mol Cell Biol, 1997 Feb, 17(2), 1027 - 36
Altered replication and inverted repeats induce mismatch repair-independent recombination between highly diverged DNAs in yeast; Tran H et al.; Replication, DNA organization, and mismatch repair (MMR) can influence recombination . We examined the effects of altered replication due to a mutation in the polymerase delta gene, long inverted repeats (LIRs) in motifs similar to those in higher eukaryotes, and MMR on intrachromosomal recombination between highly diverged (28%) truncated genes in Saccharomyces cerevisiae . A combination of altered replication and an LIR increased recombination up to 700-fold, while each alone led to a 3- to 20-fold increase . Homeologous recombination was not altered by pms1, msh2, and msh3 mismatch repair mutations . Similar to our previous observations for replication slippage-mediated deletions, there were > or = 5-bp identical runs at the recombination breakpoints . We propose that the dramatic increase in recombination results from enhancement of the effects of altered replication by the LIR, leading to recombinationally active initiating structures . Such interactions predict replication-related, MMR-independent genome changes.

Mol Cell Biol, 1997 Feb, 17(2), 906 - 20
C-terminal truncations of the yeast nucleoporin Nup145p produce a rapid temperature-conditional mRNA export defect and alterations to nuclear structure; Dockendorff TC et al.; A screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective in nucleocytoplasmic trafficking of poly(A)+ RNA has identified an allele of the NUP145 gene, which encodes an essential nucleoporin . NUP145 was previously identified by using a genetic synthetic lethal screen (E . Fabre, W . C . Boelens, C . Wimmer, I . W . Mattaj, and E . C . Hurt, Cell 78:275-289, 1994) and by using a monoclonal antibody which recognizes the GLFG family of vertebrate and yeast nucleoporins (S . R . Wente and G . Blobel, J . Cell Biol . 125:955-969, 1994) . Cells carrying the new allele, nup145-10, grew at 23 and 30 degrees C but were unable to grow at 37 degrees C . Many cells displayed a modest accumulation of poly(A)+ RNA under permissive growth conditions, and all cells showed dramatic and rapid nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C . The mutant allele contains a nonsense codon which truncates the 1,317-amino-acid protein to 698 amino acids . This prompted us to examine the role of the carboxyl half of Nup145p . Several additional alleles that encode C-terminally truncated proteins or proteins containing internal deletions of portions of the carboxyl half of Nup145p were constructed . Analysis of these mutants indicates that some sequences between amino acids 698 and 1095 are essential for RNA export and for growth at 37 degrees C . In these strains, nuclear accumulation of poly(A)+ RNA and fragmentation of the nucleolus occurred rapidly following a shift to 37 degrees C . Constitutive defects in nuclear pore complex distribution and nuclear structure were also seen in these strains . Although cells lacking Nup145p grew extremely slowly at 23 degrees C and did not grow at 30 degrees C, efficient growth at 23 or 30 degrees C occurred as long as cells produced either the amino 58% or the carboxyl 53% of Nup145p . Strains carrying alleles of NUP145 lacking up to 200 amino acids from the carboxy terminus were viable at 37 degrees C but displayed nucleolar fragmentation and some nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C . Surprisingly, these strains grew efficiently at 37 degrees C in spite of a reduction in the level of synthesis of rRNAs to approximately 25% of the wild-type level.

Mol Cell Biol, 1997 Feb, 17(2), 770 - 7
Identification of a protein that binds to the Ho endonuclease recognition sequence at the yeast mating type locus; Wang R et al.; Mating type switching in Saccharomyces cerevisiae initiates when Ho endonuclease makes a site-specific double-stranded break at MAT, the yeast mating type locus . To identify other proteins involved in this process, we examined whether extracts prepared from ho- mutants contain additional factors that bind near the recognition sequence for Ho . Using an electrophoretic mobility shift assay, we isolated a chromatographic fraction that contains an activity, named YZbp, which binds to two sequences flanking the recognition sequence at MATalpha and to one sequence overlapping it at MATa . MAT plasmids carrying mutations in the YZbp recognition sequence are cleaved by purified Ho at wild-type efficiencies in an in vitro assay . These same plasmids, however, are not cleaved by Ho inside cells, demonstrating that YZbp acts as a positive activator of in vivo cleavage . YZbp is present in all cell types, even those not undergoing mating type switching, suggesting that it has additional cellular functions.

Mol Cell Biol, 1997 Feb, 17(2), 742 - 50
A fission yeast homolog of CDC20/p55CDC/Fizzy is required for recovery from DNA damage and genetically interacts with p34cdc2; Matsumoto T; Successful recovery from DNA damage requires coordination of several biological processes . Eukaryotic cell cycle progression is delayed when the cells encounter DNA-damaging agents . This cell cycle delay allows the cells to cope with DNA damage by utilizing DNA repair enzymes . Thus, at least two processes, induction of the cell cycle delay and repair of damaged DNA, are coordinately required for recovery . In this study, a fission yeast rad mutant (slp1-362) was genetically investigated . In response to radiation, slp1 stops cell division; however, it does not restart it . This defect is suppressed when slp1-362 is combined with wee1-50 or cdc2-3w; in these mutants, the onset of mitosis is advanced due to the premature activation of p34cdc2 . In contrast, slp1 is synthetically lethal with cdc25, nim1/cdr1, or cdr2, all of which are unable to activate the p34cdc2 kinase correctly . These genetic interactions of slp1 with cdc2 and its modulators imply that slp1 is not defective in either "induction of cell cycle delay" or "DNA repair." slp1+ may be involved in a critical process which restarts cell cycle progression after the completion of DNA repair . Molecular cloning of slp1+ revealed that slp1+ encodes a putative 488-amino-acid polypeptide exhibiting significant homology to WD-domain proteins, namely, CDC20 (budding yeast), p55CDC (human), and Fizzy (fly) . A possible role of slp1+ is proposed.

Mol Cell Biol, 1997 Feb, 17(2), 553 - 63
CDC45, a novel yeast gene that functions with the origin recognition complex and Mcm proteins in initiation of DNA replication; Zou L et al.; The CDC45 gene of Saccharomyces cerevisiae was isolated by complementation of the cold-sensitive cdc45-1 mutant and shown to be essential for cell viability . Although CDC45 genetically interacts with a group of MCM genes (CDC46, CDC47, and CDC54), the predicted sequence of its protein product reveals no significant sequence similarity to any known Mcm family member . Further genetic characterization of the cdc45-1 mutant demonstrated that it is synthetically lethal with orc2-1, mcm2-1, and mcm3-1 . These results not only reveal a functional connection between the origin recognition complex (ORC) and Cdc45p but also extend the CDC45-MCM genetic interaction to all known MCM family members that were shown to be involved in replication initiation . Initiation of DNA replication in cdc45-1 cells was defective, causing a delayed entry into S phase at the nonpermissive temperature, as well as a high plasmid loss rate which could be suppressed by tandem copies of replication origins . Furthermore, two-dimensional gels directly showed that chromosomal origins fired less frequently in cdc45-1 cells at the nonpermissive temperature . These findings suggest that Cdc45p, ORC, and Mcm proteins act in concert for replication initiation throughout the genome.

Gene, 1997 Jan 31, 185(1), 137 - 46
ENP1, an essential gene encoding a nuclear protein that is highly conserved from yeast to humans; Roos J et al.; A novel gene in Saccharomyces cerevisiae, ENP1, was found to be essential for growth . The ENP1 gene encodes a protein of 483 amino acids (aa) . Nucleotide sequence analysis revealed that the deduced aa sequence of this gene exhibited approx . 60% sequence similarity to the deduced aa sequence of proteins of unknown function in Drosophila, Caenorhabditis elegans and humans . No well defined functional motifs were evident upon analysis of the aa sequence . The protein was found to contain 20% acidic aa residues, with most of them being localized to a very negatively charged domain between aa residues 100 and 150 . A construct encoding a fusion protein consisting of the Enp1 protein fused to the c-myc epitope that was either under the control of the ENP1 promoter or the GAL1,10 promoter was prepared . The construct was used to express the protein tagged with the c-myc epitope . Despite the presence of a naturally occurring promoter region with homology to the unfolded protein response element, the level of Enp1mycp remained unchanged after growth of the cells in the presence of tunicamycin, an inhibitor of N-linked glycosylation of proteins . Immunohistochemical studies to define the cellular localization of the Enp1myc protein revealed that it was localized to the nucleus . Accession No.: U50779.

Gene, 1997 Jan 31, 185(1), 1 - 4
Protein interaction cloning in yeast of the mouse third largest RNA polymerase II subunit, mRPB31; Korobko IV et al.; The cDNA encoding a protein that interacts with the mouse homologue of the yeast RNA polymerase II (polII) subunit, RPB11, and the human polII subunit, hRPB14, has been isolated by protein interaction cloning . Its deduced amino acid sequence has 96% homology to the human third largest polII subunit, hRPB33 {Pati and Weissman (1990) J . Biol . Chem . 265, 8400 8405} . Therefore, we conclude that the cloned cDNA encodes the mouse third largest polII subunit, mRPB31 . Isolation of cDNA by protein interaction cloning provides evidence supporting the hypothesis, first proposed for human polII assembly {Pati (1994) Gene 145, 289-292}, that the mRPB31/mRPB14 heterodimer, rather than the mRPB31 homodimer, forms in the mouse polII assembly . Indeed, in the yeast two-hybrid system, mRPB31 was shown to fail to form homodimer.

J Biol Chem, 1997 Jan 31, 272(5), 3049 - 56
Identification of a putative mitochondrial telomere-binding protein of the yeast Candida parapsilosis; Tomaska L et al.; Terminal segments (telomeres) of linear mitochondrial DNA (mtDNA) molecules of the yeast Candida parapsilosis consist of large sequence units repeated in tandem . The extreme ends of mtDNA terminate with a 5' single-stranded overhang of about 110 nucleotides . We identified and purified a mitochondrial telomere-binding protein (mtTBP) that specifically recognizes a synthetic oligonucleotide derived from the extreme end of this linear mtDNA . MtTBP is highly resistant to protease and heat treatments, and it protects the telomeric probe from degradation by various DNA-modifying enzymes . Resistance of the complex to bacterial alkaline phosphatase suggests that mtTBP binds the very end of the molecule . We purified mtTBP to near homogeneity using DNA affinity chromatography based on the telomeric oligonucleotide covalently bound to Sepharose . Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified fractions revealed the presence of a protein with an apparent molecular mass of approximately 15 kDa . UV cross-linking and gel filtration chromatography experiments suggested that native mtTBP is probably a homo-oligomer . MtTBP of C . parapsilosis is the first identified protein that specifically binds to telomeres of linear mitochondrial DNA.

J Cell Biol, 1997 Jan 27, 136(2), 345 - 54
Identification of a mid-anaphase checkpoint in budding yeast; Yang SS et al.; Activation of a facultative, dicentric chromosome provides a unique opportunity to introduce a double strand DNA break into a chromosome at mitosis . Time lapse video enhanced-differential interference contrast analysis of the cellular response upon dicentric activation reveals that the majority of cells initiates anaphase B, characterized by pole-pole separation, and pauses in mid-anaphase for 30-120 min with spindles spanning the neck of the bud before completing spindle elongation and cytokinesis . The length of the spindle at the delay point (3-4 microm) is not dependent on the physical distance between the two centromeres, indicating that the arrest represents surveillance of a dicentric induced aberration . No mid-anaphase delay is observed in the absence of the RAD9 checkpoint gene, which prevents cell cycle progression in the presence of damaged DNA . These observations reveal RAD9-dependent events well past the G2/M boundary and have considerable implications in understanding how chromosome integrity and the position and state of the mitotic spindle are monitored before cytokinesis.

J Cell Biol, 1997 Jan 27, 136(2), 307 - 17
Docking of yeast vacuoles is catalyzed by the Ras-like GTPase Ypt7p after symmetric priming by Sec18p (NSF); Mayer A et al.; Vacuole inheritance in yeast involves the formation of tubular and vesicular "segregation structures" which migrate into the bud and fuse there to establish the daughter cell vacuole . Vacuole fusion has been reconstituted in vitro and may be used as a model for an NSF-dependent reaction of priming, docking, and fusion . We have developed biochemical and microscopic assays for the docking step of in vitro vacuole fusion and characterized its requirements . The vacuoles must be primed for docking by the action of Sec17p (alpha-SNAP) and Sec18p (NSF) . Priming is necessary for both fusion partners . It produces a labile state which requires rapid docking in order to lead productively to fusion . In addition to Sec17p/Sec18p, docking requires the activity of the Ras-like GTPase Ypt7p . Unlike Sec17p/Sec18p, which must act before docking, Ypt7p is directly involved in the docking process itself.

J Cell Biol, 1997 Jan 27, 136(2), 299 - 306
A heterodimer of thioredoxin and I(B)2 cooperates with Sec18p (NSF) to promote yeast vacuole inheritance; Xu Z et al.; Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole . This inheritance reaction can be studied in vitro with isolated vacuoles . Rapid and efficient homotypic fusion between salt-washed vacuoles requires the addition of only two purified soluble proteins, Sec18p (NSF) and LMA1, a novel heterodimer with a thioredoxin subunit . We now report the identity of the second subunit of LMA1 as I(B)2, a previously identified cytosolic inhibitor of vacuolar proteinase B . Both subunits are needed for efficient vacuole inheritance in vivo and for the LMA1 activity in cell extracts . Each subunit acts via a novel mechanism, as the thioredoxin subunit is not acting through redox chemistry and LMA1 is still needed for the fusion of vacuoles which do not contain proteinase B . Both Sec18p and LMA1 act at an early stage of the in vitro reaction . Though LMA1 does not stimulate Sec18p-mediated Sec17p release, LMA1 cannot fulfill its function before Sec18p . Upon Sec17p/Sec18p action, vacuoles become labile but are rapidly stabilized by LMA1 . The action of LMA1 and Sec18p is thus coupled and ordered . These data establish LMA1 as a novel factor in trafficking of yeast vacuoles.

J Cell Biol, 1997 Jan 27, 136(2), 287 - 97
Two separate signals act independently to localize a yeast late Golgi membrane protein through a combination of retrieval and retention; Bryant NJ et al.; The localization of proteins to late-Golgi membranes (TGN) of Saccharomyces cerevisiae is conferred by targeting motifs containing aromatic residues in the cytosolic domains of these proteins . These signals could act by directing retrieval from a post-Golgi compartment or by preventing exit from the TGN . To investigate the mechanism of localization of yeast TGN proteins, we used the heterologous protein A-ALP (consisting of the cytosolic domain of dipeptidyl aminopeptidase A {DPAP A} fused to the transmembrane and luminal domains of the vacuolar protein alkaline phosphatase {ALP}), which localizes to the yeast TGN . Insertion of the aromatic residue-based TGN localization motif (FXFXD) of DPAP A into the cytosolic domain of ALP results in a protein that resides in the TGN . We demonstrate that the FXFXD motif confers Golgi localization through retrieval from a post-Golgi compartment by detecting a post-Golgi processed form of this protein in the TGN . We present an assay that uncouples retrieval-mediated Golgi localization from static retention-based localization, allowing measurement of the rate at which proteins exit the yeast TGN . We also demonstrate that the cytosolic domain of DPAP A contains additional information, separate from the retrieval motif, that slows exit from the TGN . We propose a model for DPAP A localization that involves two distinct mechanisms: one in which the FXFXD motif directs retrieval from a post-Golgi compartment, and a second that slows the rate at which DPAP A exits the TGN.

Cell, 1997 Jan 24, 88(2), 243 - 51
Characterization of the yeast transcriptome; Velculescu VE et al.; We have analyzed the set of genes expressed from the yeast genome, herein called the transcriptome, using serial analysis of gene expression . Analysis of 60,633 transcripts revealed 4,665 genes, with expression levels ranging from 0.3 to over 200 transcripts per cell . Of these genes, 1981 had known functions, while 2684 were previously uncharacterized . The integration of positional information with gene expression data allowed for the generation of chromosomal expression maps identifying physical regions of transcriptional activity and identified genes that had not been predicted by sequence information alone . These studies provide insight into global patterns of gene expression in yeast and demonstrate the feasibility of genome-wide expression studies in eukaryotes.

Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 485 - 90
Probing the environment along the protein import pathways in yeast mitochondria by site-specific photocrosslinking; Kanamori T et al.; Artificially aminoacylated suppressor tRNAs were used to introduce photoreactive amino acids into model mitochondrial precursor proteins to probe the environment along the protein import pathway . Amino acids with benzophenone side chains of various lengths {DL-2-amino-3-(p-benzoylphenyl)propanoic acid (1) and DL-2-amino-5-(p-benzoylphenyl)pentanoic acid (2)} were incorporated at specific sites throughout the cytochrome b2-dihydrofolate reductase fusion proteins, pb2(220)-DHFR and pb2 delta 19(220)-DHFR, which were destined for the intermembrane space and the matrix in mitochondria, respectively . In vitro import of pb2(220)-DHFR and pb2 delta 19(220)-DHFR bearing 1 or 2 into isolated yeast mitochondria was arrested so that the N terminus reached the intermembrane space or the matrix, respectively, while the DHFR domain remained at the mitochondrial surface . The matrix-targeted pb2 delta 19(220)-DHFR was photocrosslinked to Tom40 in the outer membrane, Tim44 in the inner membrane, and Ssc1p in the matrix, suggesting that the protein has an extended conformation in the import channels . On the other hand, incorporation of 2 at various positions in the 50-residue segment of intermembrane-space-targeted pb2(220)-DHFR gave photocrosslinks only to Tom40, suggesting that the segment is not in an extended conformation, but localized near Tom40 . The N-terminal portion of pb2(220)-DHFR, but not pb2 delta 19(220)-DHFR, was photocrosslinked to an as-yet-unidentified mitochondrial component to generate a 95-kDa crosslinked product.

Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 385 - 90
Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry; Neubauer G et al.; Here we report the rapid identification of the proteins of the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) from the yeast Saccharomyces cerevisiae by searching mass spectrometric data in genomic sequence databases . The U1 snRNP, containing a histidine-tagged 70K protein, was isolated from cell extracts by anti m3G-cap immunoaffinity and subsequent nickel nitrilotriacetic acid chromatography . A U1 snRNP fraction containing 20 proteins was obtained . Further purification by glycerol gradient centrifugation identified nine U1 snRNP specific and six common proteins . The U1 snRNP proteins were partially sequenced by nanoelectrospray mass spectrometry, and their genes were identified in the data base via multiple peptide sequence tags . Apart from the already known common proteins D1, D3, F, and G, the D2 and E homologs were also identified . The same six common proteins were detected in core U2 snRNP, which was purified and analyzed separately . The biochemical association of these six proteins with yeast snRNPs is shown here for the first time . Intriguingly, the Sm B/B' homolog was not detected . In addition to the well characterized yeast U1 specific proteins {U1-70K (Snp1p), U1-A (Mud1p), Prp39p, and Prp40p} the homolog of the U1-C protein was identified together with four additional novel U1 specific proteins, which are not found in mammalian U1 . This is the first time that the components of a multiprotein complex from an organism with a sequenced genome have been characterized by mass spectrometry . The technique should be applicable to any protein complex that can be biochemically purified from an organism whose genome is known.

J Biol Chem, 1997 Jan 17, 272(3), 1688 - 93
Reactive cysteines of the yeast plasma-membrane H+-ATPase (PMA1) . Mapping the sites of inactivation by N-ethylmaleimide; Petrov VV et al.; We have taken advantage of cysteine mutants described previously (Petrov, V . V., and Slayman, C . W . (1995) J . Biol . Chem . 270, 28535-28540) to map the sites at which N-ethylmaleimide (NEM) reacts with the plasma-membrane H+ATPase (PMA)1 of Saccharomyces cerevisiae . When membrane vesicles containing the ATPase were incubated with NEM, six of nine mutants with single cysteine substitutions showed sensitivity similar to the wild-type enzyme . By contrast, C221A and C532A were inactivated more slowly than the wild-type control, and the C221, 532A double mutant was completely resistant, indicating that Cys-221 and Cys-532 are NEM-reactive residues . In the presence of 10 mM MgADP, the wild-type ATPase was partially protected against NEM; parallel experiments with the C221A and C532A mutants showed that the protection occurred at Cys-532, located in or near the nucleotide-binding site . Unexpectedly, the inactivation of the C409A ATPase was approximately 4-fold more rapid than in the case of the wild-type enzyme . Experiments with double mutants made it clear that this resulted from an acidic shift in pKa and a consequent acceleration of the reaction rate at Cys-532 . One simple interpretation is that substitution of Cys-409 leads to a local conformational change within the central hydrophilic domain . Consistent with this idea, the reaction of fluorescein 5'-isothiocyanate at Lys-474 was also stimulated approximately 3 . 5-fold by the C409A mutation . Taken together, the results of this study provide new information about the reactivity of individual Cys residues within the ATPase and pave the way to tag specific sites for structural and functional studies of the enzyme.

Eur J Biochem, 1997 Jan 15, 243(1-2), 350 - 7
The wheat poly(A)-binding protein functionally complements pab1 in yeast; Le H et al.; Poly(A)-binding protein (PAB) binds to the poly(A) tail of most eukaryotic mRNAs and influences its translational efficiency as well as its stability . Although the primary structure of PAB is well conserved in eukaryotes, its functional conservation across species has not been extensively investigated . In order to determine whether PAB from a monocot plant species could function in yeast, a protein characterized as having PAB activity was purified from wheat and a cDNA encoding for PAB was isolated from a wheat seedling expression library . Wheat PAB (72 kDa as estimated by SDS/PAGE and a theoretical mass of 70 823 Da as determined from the cDNA) was present in multiple isoforms and exhibited binding characteristics similar to that determined for yeast PAB . Comparison of the wheat PAB protein sequence with PABs from yeast and other species revealed that wheat PAB contained the characteristic features of all PABs, including four RNA binding domains each of which contained the conserved RNP1 and RNP2 sequence motifs . The wheat PAB cDNA functionally complemented a pab1 mutant in yeast suggesting that, although the amino acid sequence of wheat PAB is only 47% conserved from that of yeast PAB, this monocot protein can function in yeast.

Biochem J, 1997 Jan 15, 321 ( Pt 2), 487 - 95
Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis; van den Broek PJ et al.; Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis . The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles . Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome c oxidase as a proton-motive-force-generating system . Addition of reduced cytochrome c generated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside . Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40-50 . Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force . Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems . Efflux of accumulated glucose was observed on dissipation of the proton-motive force . Exchange and counterflow experiments confirmed the reversible character of the H+-glucose symporter . In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose . Moreover under counterflow conditions, the expected transient accumulation was small . Thus the H+-maltose symporter has some characteristics of a carrier that is not readily reversible . It is concluded that in C . utilis the transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different.

Biochem J, 1997 Jan 15, 321 ( Pt 2), 397 - 403
The YNT1 gene encoding the nitrate transporter in the yeast Hansenula polymorpha is clustered with genes YNI1 and YNR1 encoding nitrite reductase and nitrate reductase, and its disruption causes inability to grow in nitrate; Perez MD et al.; DNA sequencing in the phage lambda JA13 isolated from a lambda EMBL3 Hansenula polymorpha genomic DNA library containing the nitrate reductase-(YNR1) and nitrite reductase-(YNI1) encoding genes revealed an open reading frame (YNT1) of 1524 nucleotides encoding a putative protein of 508 amino acids with great similarity to the nitrate transporters from Aspergillus nidulans and Chlamydomonas reinhardtii . Disruption of the chromosomal YNT1 copy resulted in incapacity to grow in nitrate and a significant reduction in rate of nitrate uptake . The disrupted strain is still sensitive to chlorate, and, in the presence of 0.1 mM nitrate, the expression of YNR1 and YNI1 and the activity of nitrate reductase and nitrite reductase are significantly reduced compared with the wild-type . Northern-blot analysis showed that YNT1 is expressed when the yeast is grown in nitrate and nitrite but not in ammonium solution.

Nucleic Acids Res, 1997 Jan 15, 25(2), 451 - 2
Recombination-mediated PCR-directed plasmid construction in vivo in yeast; Oldenburg KR et al.; We have extended the technique of PCR-directed recombination in Saccharomyces cerevisiae to develop a simple method for plasmid or gene construction in the absence of suitable restriction sites . The DNA to be cloned is PCR-amplified with 30-40 bp of homology to a linearized yeast plasmid . Co-transformation into yeast results in homologous recombination at a position directed by the PCR oligonucleotides.

Nucleic Acids Res, 1997 Jan 15, 25(2), 417 - 22
Sequence requirements of the bidirectional yeast TRP4 mRNA 3'-end formation signal; Egli CM et al.; The yeast TRP4 3'-end formation signal functions in both orientations in an in vivo test system . We show here that the TRP4 3'-end formation element consists of two functionally different sequence regions . One region of approximately 70 nucleotides is located in the untranslated region between the translational stop codon and the major poly(A) site . The major poly(A) site is not part of this region and can be deleted without a decrease in TRP4 3'-end formation . 5'and 3'deletions and point mutations within this region affected 3'-end formation similarly in both orientations . In the center of this region the motif TAGT is located on the antisense strand . Point mutations within this motif resulted in a drastic reduce of 3'-end formation activity in both orientations . A second region consists of the 3'-end of the TRP4 open reading frame and is required for 3'-end formation in forward orientation . A single point mutation in a TAGT motif of the TRP4 open reading frame abolished TRP4 mRNA 3'-end formation in forward orientation and had no effect on the reverse orientation.

Genes Dev, 1997 Jan 15, 11(2), 255 - 69
Transcriptional silencing of Ty1 elements in the RDN1 locus of yeast; Bryk M et al.; We demonstrate that in Saccharomyces cerevisiae, the tandem array of ribosomal RNA genes (RDN1) is a target for integration of the Ty1 retrotransposon that results in silencing of Ty1 transcription and transposition . Ty1 elements transpose into random rDNA repeat units and are mitotically stable . In addition, we have found that mutation of several putative modifiers of RDN1 chromatin structure abolishes silencing of Ty1 elements in the rDNA array . Disruption of SIR2, which elevates recombination in RDN1, or TOP1, which increases psoralen accessibility in rDNA, or HTA1-HTB1, which reduces histone H2A-H2B levels and causes localized chromatin perturbations, abolishes transcriptional silencing of Ty1 elements in RDN1 . Furthermore, deletion of the gene for the ubiquitin conjugating enzyme Ubc2p, which ubiquitinates histones in vitro, derepresses not only Ty1 transcription but also mitotic recombination in RDN1 . On the basis of these results, we propose that a specialized chromatin structure exists in RDN1 that silences transcription of the Ty1 retrotransposon.

Biochem Biophys Res Commun, 1997 Jan 13, 230(2), 381 - 5
Cloning and functional expression in yeast of a cDNA coding for an obtusifoliol 14alpha-demethylase (CYP51) in wheat; Cabello-Hurtado F et al.; Screening of a wheat cDNA library with an heterologous CYP81B1 probe from Helianthus tuberosus led to the isolation of a partial cDNA coding a protein with all the characteristics of a typical P450 with high homology (32-39% identity) to the fungal and mammalian CYP51s . Extensive screening of several wheat cDNA libraries isolated a longer cDNA (W516) coding a peptide of 453 amino acids . Alignment of W516 with other P450 sequences revealed that it was missing a segment corresponding to the N-terminal membrane anchor of the protein . The corresponding segment from the yeast lanosterol 14alpha-demethylase was linked to the partial wheat cDNA and the chimera expressed in Saccharomyces cerevisiae . Compared to microsomes from control yeasts, membranes of yeast expressing the chimera catalysed 14alpha-demethylation of obtusifoliol with an increased efficiency relative to lanosterol demethylase activity . W516 is thus a plant member of the most ancient and conserved P450 family, CYP51.

J Cell Biol, 1997 Jan 13, 136(1), 95 - 110
Targeting of chitin synthase 3 to polarized growth sites in yeast requires Chs5p and Myo2p; Santos B et al.; Chitin is an essential structural component of the yeast cell wall whose deposition is regulated throughout the yeast life cycle . The temporal and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomyces cerevisiae by localization of the putative catalytic subunit of chitin synthase III, Chs3p, and its regulator, Chs5p . Immunolocalization of epitope-tagged Chs3p revealed a novel localization pattern that is cell cycle-dependent . Chs3p is polarized as a diffuse ring at the incipient bud site and at the neck between the mother and bud in small-budded cells; it is not found at the neck in large-budded cells containing a single nucleus . In large-budded cells undergoing cytokinesis, it reappears as a ring at the neck . In cells responding to mating pheromone, Chs3p is found throughout the projection . The appearance of Chs3p at cortical sites correlates with times that chitin synthesis is expected to occur . In addition to its localization at the incipient bud site and neck, Chs3p is also found in cytoplasmic patches in cells at different stages of the cell cycle . Epitope-tagged Chs5p also localizes to cytoplasmic patches; these patches contain Kex2p, a late Golgi-associated enzyme . Unlike Chs3p, Chs5p does not accumulate at the incipient bud site or neck . Nearly all Chs3p patches contain Chs5p, whereas some Chs5p patches lack detectable Chs3p . In the absence of Chs5p, Chs3p localizes in cytoplasmic patches, but it is no longer found at the neck or the incipient bud site, indicating that Chs5p is required for the polarization of Chs3p . Furthermore, Chs5p localization is not affected either by temperature shift or by the myo2-66 mutation, however, Chs3p polarization is affected by temperature shift and myo2-66 . We suggest a model in which Chs3p polarization to cortical sites in yeast is dependent on both Chs5p and the actin cytoskeleton/Myo2p.

FEBS Lett, 1997 Jan 13, 401(1), 65 - 7
Binding of the N-terminal 63 kDa portion of connectin/titin to alpha-actinin as revealed by the yeast two-hybrid system; Ohtsuka H et al.; Connectin/titin is a 3000 kDa protein which links the myosin filament to the Z-line in vertebrate striated muscle sarcomeres . To search for the Z-line proteins to which connectin binds, the yeast two-hybrid system was applied using cDNA coding the N-terminal 63 kDa fragment of connectin . Two clones coding the C-terminal half region of alpha-actinin (amino acids, 343-897 and 446-897) were obtained . Enzyme-linked immunosorbent assay clearly demonstrated the interactions of alpha-actinin and the N-terminal 63 kDa fragment of connectin in vitro . Thus it is concluded that the N-terminal 63 kDa portion of connectin binds to alpha-actinin in the Z-line of myofibrillar sarcomeres.

J Biol Chem, 1997 Jan 10, 272(2), 1237 - 47
Beryllium fluoride and phalloidin restore polymerizability of a mutant yeast actin (V266G,L267G) with severely decreased hydrophobicity in a subdomain 3/4 loop; Kuang B et al.; Holmes proposed that in F-actin, hydrophobic residues in a subdomain 3/4 loop interact with a hydrophobic pocket on the opposing strand resulting in helix stabilization . We have determined how a decreased hydrophobicity of this plug affects yeast actin function . Cells harboring only the V266G, V266D, V266F, L267G, L269D, or L269K actins appear normal, although V266G cells display an altered budding pattern . However, V266G,L267G (GG) double mutant cells are cold-sensitive with randomly oriented thick actin assemblies seen in rhodamine phalloidin-stained GG cells . V266D actin polymerizes slower than wild-type actin at room temperature . At 4 degrees C, not only is polymerization slowed, but there is also an effect on critical concentration . However, the polymerization defects are milder than those associated with substitution of Asp for the neighboring Leu267 . Purified GG-actin does not polymerize in vitro alone or in the presence of wild-type F-actin seeds . GG-actin polymerization can be restored by larger amounts of wild-type actin, beryllium fluoride, or phalloidin at room temperature, although at 4 degrees C only phalloidin is effective . These results suggest that the diminished hydrophobicity of the plug in GG-actin leads to filament destabilization . However, the V266D actin results require a modification of the original Holmes filament model.

J Biol Chem, 1997 Jan 10, 272(2), 1110 - 6
The human homologue of the yeast Prt1 protein is an integral part of the eukaryotic initiation factor 3 complex and interacts with p170; Methot N et al.; Eukaryotic initiation factor 3 (eIF3) is a large multisubunit complex that stabilizes the ternary complex, eIF2 x GTP x tRNA(Met)i and promotes mRNA binding to the 40 S ribosomal subunit . eIF3 also functions as a ribosome subunit anti-association factor . The molecular mechanisms by which eIF3 exerts these functions are poorly understood . We describe here the cloning of the cDNA encoding the human homologue of the yeast eIF3 subunit Prt1 . The human PRT1 cDNA encodes a protein of predicted molecular mass of 98.9 kDa that migrates at 116 kDa on SDS-polyacrylamide gels . Human and yeast Prt1 share 31% identity and 50% similarity at the amino acid level . The homology is distributed throughout the entire protein, except for the amino terminus, and is particularly high in the central portion of the protein, which contains a putative RNA recognition motif . hPrt1 is recognized by an antibody raised against eIF3, and an affinity-purified antibody to recombinant hPrt1 recognizes a protein migrating at 116 kDa in a purified eIF3 preparation . Far Western analysis shows that hPrt1 interacts directly with the p170 subunit of eIF3 . Mapping studies identify the RNA recognition motif as the region required for association with p170 . Taken together, these experiments demonstrate that hPrt1 is a component of eIF3 . Our data, combined with those of Hershey and co-workers, suggest that mammalian eIF3 is composed of at least 10 subunits: p170, p116 (hPrt1), p110, p66, p48, p47, p44, p40, p36, and p35.

Biochemistry, 1997 Jan 7, 36(1), 119 - 26
A lysine 73-->histidine variant of yeast iso-1-cytochrome c: evidence for a native-like intermediate in the unfolding pathway and implications for m value effects; Godbole S et al.; In this paper we report thermodynamic studies on a variant of yeast iso-1-cytochrome c in which a surface lysine residue at position 73 has been replaced with a histidine (H73) . Guanidine hydrochloride denaturation studies monitored by circular dichroism spectroscopy indicated decreased thermodynamic stability (a lower delta G(o)(u)H20) and a smaller m value for the H73 protein as compared to the wild type (WT) protein . Further investigations to probe the causes for the thermodynamic stability differences between the two proteins involved guanidine hydrochloride and urea denaturations monitored by tryptophan fluorescence . The stability of heme ligation in the denatured state in the presence of either guanidine hydrochloride or urea was monitored by the spin-state transition of the heme iron induced by pH . None of these studies supported the hypothesis that the decreased m value was due to heme-His73 ligation in the denatured state . Guanidine hydrochloride denaturations monitored by the change in the extinction coefficient at 695 nm, which is sensitive to the presence of heme-Met80 ligation, revealed a native-like intermediate for the H73 protein, probably caused by displacement of the Met80 heme ligand by histidine 73 at guanidine hydrochloride concentrations much lower than required for full cooperative unfolding . Presence of the native-like intermediate is most likely the cause of the smaller m value and decreased thermodynamic stability for the CD-monitored H73 protein unfolding as compared to the unfolding of the WT protein . Guanidine hydrochloride denaturations in the presence of 200 mM imidazole provide further evidence in support of the proposed mechanism.

Proc Natl Acad Sci U S A, 1997 Jan 7, 94(1), 169 - 74
Mutation of gene-proximal regulatory elements disrupts human epsilon-, gamma-, and beta-globin expression in yeast artificial chromosome transgenic mice; Liu Q et al.; Previous studies have defined transcriptional control elements, in addition to the promoters, that both lie near individual human beta-globin locus genes and have been implicated in their differential stage-specific regulation during development (i.e., are believed to directly participate in hemoglobin switching) . We have reinvestigated the activities during erythropoiesis that might be conferred by two of the more intensively analyzed of these elements, the epsilon-globin gene 5' silencer and the beta-globin gene 3' enhancer, by deleting them from a yeast artificial chromosome that spans the human beta-globin locus, and then analyzing transgenic mice for expression of all of the human genes . These studies show that sequences within the epsilon-globin "silencer" are not only required for silencing but are also required for activation of epsilon-globin transcription; furthermore, deletion of the silencer simultaneously reduced gamma-globin transcription during the yolk sac stage of erythroid development . Analysis of the adult beta-globin gene 3' enhancer deletion showed that its deletion affects only that gene.

Gene, 1997 Jan 3, 184(1), 27 - 32
Isolation of cDNAs encoding chicken homologues of the yeast SNF2 and Drosophila Brahma proteins; Goodwin GH; The SNF2/Brahma proteins are a class of DNA-dependent ATPases which activate gene expression by disrupting chromatin repression . They also cooperate with nuclear hormone receptors to activate transcription . Two cDNAs encoding chicken homologues of the SNF2/Brahma proteins have been isolated from chicken haematopoietic libraries . The encoded proteins closely resemble the human homologues, hBRM and BRG1, and the chicken homologues have therefore been termed cBRH and cBRG1 . Homology is conserved in five characteristic domains: an N-terminal domain that binds the SNF11 protein, a conserved domain A of unknown function, a central ATPase domain, a domain that binds the retinoblastoma tumor suppressor protein Rb, and a C-terminal bromodomain of unknown function.

J Biol Chem, 1997 Jan 3, 272(1), 240 - 8
The G beta gamma complex of the yeast pheromone response pathway . Subcellular fractionation and protein-protein interactions; Hirschman JE et al.; Genetic evidence suggests that the yeast STE4 and STE18 genes encode G beta and G gamma subunits, respectively, that the G betagamma complex plays a positive role in the pheromone response pathway, and that its activity is subject to negative regulation by the G alpha subunit (product of the GPA1 gene) and to positive regulation by cell-surface pheromone receptors . However, as yet there is no direct biochemical evidence for a G betagamma protein complex associated with the plasma membrane . We found that the products of the STE4 and STE18 genes are stably associated with plasma membrane as well as with internal membranes and that 30% of the protein pool is not tightly associated with either membrane fraction . A slower-migrating, presumably phosphorylated, form of Ste4p is enriched in the non-membrane fraction . The Ste4p and Ste18p proteins that had been extracted from plasma membranes with detergent were found to co-sediment as an 8 S particle under low salt conditions and as a 6 S particle in the presence of 0.25 M NaCl; the Ste18p in these fractions was precipitated with anti-Ste4p antiserum . Under the conditions of our assay, Gpa1p was not associated with either particle . The levels of Ste4p and Ste18p accumulation in mutant cells provided additional evidence for a G betagamma complex . Ste18p failed to accumulate in ste4 mutant cells, and Ste4p showed reduced levels of accumulation and an increased rate of turnover in ste18 mutant cells . The gpa1 mutant blocked stable association of Ste4p with the plasma membrane, and the ste18 mutant blocked stable association of Ste4p with both plasma membranes and internal membranes . The membrane distribution of Ste4p was unaffected by the ste2 mutation or by down-regulation of the cell-surface receptors . These results indicate that at least 40% of Ste4p and Ste18p are part of a G betagamma complex at the plasma membrane and that stable association of this complex with the plasma membrane requires the presence of G alpha.

J Biol Chem, 1997 Jan 3, 272(1), 36 - 9
Identification of a novel Ca2+-dependent, phosphatidylethanolamine-hydrolyzing phospholipase D in yeast bearing a disruption in PLD1; Waksman M et al.; We have previously reported the identification and partial characterization of a gene encoding a phospholipase D activity (PLD1) in the yeast, Saccharomyces cerevisiae . Here we report the existence of a second phospholipase D activity, designated PLD2, in yeast cells bearing disruption at the PLD1 locus . PLD2 is a Ca2+-dependent enzyme which preferentially utilizes phosphatidylethanolamine over phosphatidylcholine as a substrate . In contrast to PLD1, the activity of PLD2 is insensitive to phosphatidylinositol 4,5-bisphosphate, and the enzyme is incapable of catalyzing the transphosphatidylation reaction with short chain alcohols as acceptors . Subcellular fractionation shows that PLD2 localizes mainly to the cytosol, but could also be detected in the particulate fraction . Thus, the biochemical properties of PLD2 appear to be substantially different from those of PLD1 . PLD2 activity is significantly and transiently elevated upon exit of wild type yeast cells from stationary phase, suggesting that it may play a role in the initiation of mitotic cell division in yeast . In view of the significantly different properties of PLD1 and PLD2, and because the yeast genome contains PLD1 as the sole member of the recently defined PLD gene family, it may be concluded that PLD2 is structurally unrelated to PLD1 . Thus, the novel PLD2 activity described herein is likely to represent the first identified member of a new PLD gene family.

EMBO J, 1997 Jan 2, 16(1), 83 - 97
Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase; Leberer E et al.; Ste20p from Saccharomyces cerevisiae belongs to the Ste20p/p65PAK family of protein kinases which are highly conserved from yeast to man and regulate conserved mitogen-activated protein kinase pathways . Ste20p fulfills multiple roles in pheromone signaling, morphological switching and vegetative growth and binds Cdc42p, a Rho-like small GTP binding protein required for polarized morphogenesis . We have analyzed the functional consequences of mutations that prevent binding of Cdc42p to Ste20p . The complete amino-terminal, non-catalytic half of Ste20p, including the conserved Cdc42p binding domain, was dispensable for heterotrimeric G-protein-mediated pheromone signaling . However, the Cdc42p binding domain was necessary for filamentous growth in response to nitrogen starvation and for an essential function that Ste20p shares with its isoform Cla4p during vegetative growth . Moreover, the Cdc42p binding domain was required for cell-cell adhesion during conjugation . Subcellular localization of wild-type and mutant Ste20p fused to green fluorescent protein showed that the Cdc42p binding domain is needed to direct localization of Ste20p to regions of polarized growth . These results suggest that Ste20p is regulated in different developmental pathways by different mechanisms which involve heterotrimeric and small GTP binding proteins.

Microbiol Immunol, 1997, 41(7), 571 - 3
Partial sequences of large subunit ribosomal DNA of a new yeast species, Trichosporon domesticum and related species; Sugita T et al.; We determined the partial sequences of large subunit rDNA of a new yeast species, Trichosporon domesticum, which was isolated from the house of a summer-type hypersensitivity pneumonitis patient . Phylogenetically, T . domesticum was positioned in the taxonomic group containing T . montevideense and T . brassicae, which indicated an identical serotype . A phylogenetic relationship among all species of the genus Trichosporon which belong to the basidiomycetous yeast is clarified.

Acta Vet Hung, 1997, 45(2), 207 - 12
Reduction of stress-induced changes in meat quality with thermolysed brewer's yeast of high nucleotide content in pigs; Kovacs-Zomborszky M et al.; The effect exerted by a biogenic performance enhancer of high nucleotide content on meat quality in 20 Norwegian Landrace pigs (90 to 95 kg) was examined . The diet of the treated group was supplemented with the performance enhancer for the last 30 days of fattening . The stress effect was transport to the slaughterhouse and slaughter itself . Plasma creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST) activities, glucose and cortisol concentrations, and muscle pH were determined . Serious stress damage was found in the cardiac and skeletal muscle, as indicated by the high CK (980 U/L), LDH (> 1600 U/L) and AST (67 U/L) activities in the untreated group; values were significantly lower in the experimental pigs (458, 468 and 17 U/L, respectively) . There were no significant differences in glucose and cortisol concentrations between the two groups . In the control group the pH values were significantly lower and more muscle samples showed PSE character than in the treated group (75 and 30%, respectively).

Mol Aspects Med, 1997, 18 Suppl, S121 - 7
Sensitivity to treatment with polyunsaturated fatty acids is a general characteristic of the ubiquinone-deficient yeast coq mutants; Poon WW et al.; The biosynthesis of ubiquinone (Q) and the functional consequences of Q-deficiency was studied in the yeast Saccharomyces cerevisiae . Lipid extracts were prepared from various respiratory deficient mutants grown in the presence of p-{U-14C}hydroxybenzoic acid . Q mutant strains harboring mutations in the coq3, coq4, coq5, coq6, coq7, or coq8 genes were unable to produce Q and accumulated an early intermediated that corresponded to 3-hexaprenyl-4-hydroxybenzoic acid . Several respiratory deficient yeast including both nuclear and mitochondrial petite mutant strains, retain the ability to produce Q . Thus, the inability to produce Q is a specific phenotype manifested in the class of mutants termed 'coq' . Previous studies described the enhanced sensitivity of the Q-deficient yeast strain containing a deletion in the COQ3 gene to the products of autoxidized polyunsaturated fatty acids (Do et al., 1996, Proceeding of the National Academy of Science USA, 93, 7534-7539) . The results presented here show this to be a general phenotype resulting from Q-deficiency, as all of the coq mutant yeast strains tested exhibit hypersensitivity to polyunsaturated fatty acid treatment.

Methods Enzymol, 1997, 283, 440 - 59
Genetic and physiological analysis of DNA replication in fission yeast; MacNeill SA et al.; Studies on DNA replication in S . pombe have provided powerful insights into the way in which the genome of this model eukaryote is replicated and how the replication process is controlled . These studies have been facilitated by the simplicity and range of methods available in this organism for physiological and genetic analysis of DNA replication mutants . In the future, continued focus on the analysis of such mutants, coupled with increasingly sophisticated biochemical investigation of the processes of DNA replication in both wild-type and mutant cells, will ensure continued rapid progress in this area.

Braz J Med Biol Res, 1997 Jan, 30(1), 9 - 13
Anhydrobiosis in yeast: activation effect; Rapoport AI et al.; Intracellular substances released into the medium during rehydration of dry yeast cells can significantly improve the quality of a synthetic medium . Acceleration of yeast growth in this medium and increased yield of biomass are observed simultaneously . The change in the molecular arrangement of intracellular membranes as a result of the strong dehydration of live organisms is a negative phenomenon that reduces the level of cell viability . However, this phenomenon also represents an adaptive mechanisms which facilitates the maintenance of population viability as a whole under extreme environmental conditions.

Planta, 1997, 202(1), 126 - 36
Solute accumulation and decreased photosynthesis in leaves of potato plants expressing yeast-derived invertase either in the apoplast, vacuole or cytosol; Bussis D et al.; Potato (Solanum tuberosum cv . Desiree) plants expressing yeast invertase directed either to the apoplast, vacuole or cytosol were biochemically and physiologically characterised . All lines of transgenic plants showed similarities to plants growing under water stress . Transformants were retarded in growth, and accumulated hexoses and amino acids, especially proline, to levels up to 40-fold higher than those of the wild types . In all transformants rates of CO2 assimilation and leaf conductance were reduced . From the unchanged intercellular partial pressure of CO2 and apoplastic cis-abscisic acid (ABA) content of transformed leaves it was concluded that the reduced rate of CO2 assimilation was not caused by a limitation in the availability of CO2 for the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) . In the transformants the amount of Rubisco protein was not reduced, but both activation state and carboxylation efficiency of photosynthesis were lowered . In vacuolar and cytosolic transformants this inhibition of Rubisco might be caused by a changed ratio of organic bound and inorganic phosphate, as indicated by a doubling of phosphorylated intermediates . But in apoplastic transformants the pattern of phosphorylated intermediates resembled that of leaves of water-stressed potato plants, although the cause of inhibition of photosynthesis was not identical . Whereas in water-stressed plants increased contents of the phytohormone ABA are supposed to mediate the adaptation to water stress, no contribution of ABA to reduction of photosynthesis could be detected in invertase transformants.

Yi Chuan Xue Bao, 1997, 24(1), 87 - 93
{Fine physical mapping of yeast chromosome V}; Yang Z et al.; Electrophoretic karyotype of yeast strain A364a was obtained by pulsed field gel electrophoresis and the position of chromosome V on such karyotype was determined by means of dot hybridization with chromosome V-specific probe URA3 . By cloning partially digested BamHI fragments of this chromosome DNA into integrative vector Yip5, a gene library specific to this chromosome was constructed . The number of the recombinants was much more than theoretically required . After screening probe-homologous fragments from this library and analysing such fragments with restriction enzymes BamHI, EcoRI, HindIII, PstI, and SalI, a fine physical map covering about 9.4% of A364a chromosome V (which was estimated as 620kb) was constructed . Further colony hybridization with boundary clones will enable us to "walk" throughout the whole chromosome.

Cytogenet Cell Genet, 1997, 76(1-2), 87 - 93
Isolation and mapping of karyopherin alpha 3 (KPNA3), a human gene that is highly homologous to genes encoding Xenopus importin, yeast SRP1 and human RCH1; Takeda S et al.; From a human fetal-brain cDNA library, we isolated and characterized a novel gene (KPNA3) encoding a protein highly homologous to certain nuclear transport proteins of Xenopus and human . The complete cDNA clone, designated karyopherin alpha 3, contained an open reading frame of 1,563 nucleotides encoding 521 amino acids . The predicted amino acid sequence showed 48%, 45% and 48% identity with Xenopus importin, yeast SRP1 and human RCH1, respectively . The similarities among these proteins suggest that karyopherin alpha 3 may be involved in the nuclear transport system . Eight repeats of the arm motif were well conserved among these proteins . The N-terminal region of the predicted karyopherin alpha 3 product was highly basic and the C-terminal region was strongly acidic . A 4.3-kb transcript was expressed in all adult human tissues examined by Northern blotting . The cDNA clone was assigned to chromosome band 13q14.3 by fluorescence in situ hybridization.

Indian J Pathol Microbiol, 1997 Jan, 40(1), 55 - 8
A simple synthetic liquid medium for development of yeast and mycelial form of pathogenic species of Candida; Prakash P et al.; Thirty two known strains of Candida species were used for evaluation of glucose, serine, ornithine, methionine, GSOM medium and its comparison with Lee's medium for the production of yeast and mycelial phase at different temperatures and on prolonged incubation . No mycelial form was observed when various Candida species in GSOM and Lee's medium were incubated at 25 degrees C up to 72 hours . Percentage of mycelial forming cells of Candida species were more in GSOM medium than Lee's medium in 48 hours at 37 degrees C . Among various species of Candida, albicans and C . parapsilosis showed maximum mycelium formation . GSOM medium can be used for growing Candida species particularly C . albicans in mycelial phase.

Genes Cells, 1997 Jan, 2(1), 65 - 79
Involvement of the MRE2 gene of yeast in formation of meiosis-specific double-strand breaks and crossover recombination through RNA splicing; Nakagawa T et al.; BACKGROUND: The mre2 mutant of Saccharomyces cerevisiae is defective in meiotic recombination and produces inviable spores, but the sensitivities to DNA damaging agents, methyl methanesulphonate and ultraviolet light are not altered by the mutation . Mre2 has two copies of RNA recognition motif (RRM), suggesting its participation in RNA metabolism in meiosis . RESULTS: An amino acid substitution in the N-terminal RRM of Mre2 confers a meiotic recombination defect . Using this mre2N strain, the MER2 gene was isolated as a multi-copy suppressor of the recombination defect . Meiosis-specific splicing of MER2 pre-mRNA was impaired in the mre2 deletion (mre2delta) mutant . The mre2delta mutant was defective in the formation of meiosis-specific double-strand breaks (DSBs) and crossover and noncrossover recombinants . When the chromosomal MER2 gene was replaced with the intronless derivative of MER2 gene, cMER2, the formation of DSBs and of noncrossover recombinants were restored in the mre2delta mutant . However, the amount of crossover recombinants produced in the mre2delta cMER2 strain was approximately 30% that in the wild-type . In addition, the mre2delta cMER2 mutant was defective in chromosome segregation and in viable spore formation . CONCLUSIONS: Mre2 participates in the formation of DSBs through meiosis-specific splicing of MER2 pre-mRNA . Besides, Mre2 is also involved in crossover recombination, possibly through splicing of RNA from another gene(s).

Vopr Virusol, 1997 Jan-Feb, 42(1), 17 - 9
{Study of anti-HIV activity of the yeast RNA-tilorone molecular complex}; Karpov AV et al.; Anti-HIV activity of the molecular complex forming during interaction between yeast RNA and tilorone was studied in vitro on the models of acute and chronic infection . Addition of this agent to the cells infected with HIV-1/IIIB and HIV-1/BRU decreased the virus reproduction controlled by assessing the viability of cells, syncytium production, and accumulation of p24 antigen in culture medium . The authors hypothesize that the detected anti-HIV effect is due to its capacity to produce type I interferon and direct antiviral action.

J Basic Microbiol, 1997, 37(1), 23 - 8
Control of the Myc-Max mediated transactivation in yeast by natural promoter elements; Hanel F et al.; Transcriptional activation studies involving the human oncoprotein and transcription factor Myc and its helix-loop-helix partner protein Max in mammalian cells are critical due to the presence of endogenous Myc and Max proteins . Here we show that co-expression of the human c-myc and max genes from 2micro circle derived high copy number vectors in yeast cells stimulate the transcriptional activation of a LacZ reporter gene fused to the yeast cytochrome-c1 oxidase minimal promoter containing the adenovirus major late promoter element (AMLPE) . The exchange of the single Myc binding site in the AMLPE by the two E-box DNA motifs (CACGTG) present in the Myc responsive element of a human Myc target gene (ornithine decarboxylase) in front of a promoter-reporter gene cassette results in a two-fold enhanced beta-galactosidase expression . Low expression of max and high level expression of c-myc at the same time led to a further enhancement of transcriptional activation from this promoter-reporter gene cassette.

Chem Res Toxicol, 1997 Jan, 10(1), 27 - 33
Comparative inhibition of yeast glutathione reductase by arsenicals and arsenothiols; Styblo M et al.; Tri(gamma-glutamylcysteinylglycinyl)trithioarsenite (AsIII(GS)3) is formed in cells and is a more potent mixed-type inhibitor of the reduction of glutathione disulfide (GSSG) by yeast glutathione (GSH) reductase than either arsenite (AsIII) or GSH . The present work examines the effects of valence and complexation of arsenicals with GSH or L-cysteine (Cys) upon potency as competitive inhibitors of the reduction of GSH disulfide (GSSG) by yeast GSH reductase . Trivalent arsenicals were more potent inhibitors than their pentavalent analogs, and methylated trivalent arsenicals were more potent inhibitors than was inorganic trivalent As . Complexation of either inorganic trivalent As or methylarsonous diiodide (CH3As(III)I2) with Cys or GSH produced inhibitors of GSH reductase that were severalfold more potent than the parent arsenicals . In contrast, dimethylarsinous iodide ((CH3)2As(III)I) was a more potent inhibitor than its complexes with either GSH or Cys . Complexes of CH3AsIII with GSH (CH3-AsIII(GS)2) or with Cys (CH3AsIII(Cys)2) were the most potent inhibitors, with Ki's of 0.009 and 0.018 mM, respectively . Inhibition of GSH reductase by arsenicals or arsenothiols was prevented by addition of meso-2,3-dimercaptosuccinic acid (DMSA) to a mixture of enzyme, GSSG, and inhibitor before addition of NADPH . DMSA added to the reaction mixture after NADPH reversed inhibition by (CH3)2As(III)I but had little effect on inhibition by CH3As(III)I2, Ch3AsIII(GS)2, CH3AsIII(Cys)2, or AsIII(GS)3 . Partial redox inactivation of the enzyme with NADPH increased the inhibitory potency of CH3As(III)I2 and (CH3)2As(III)I and changed the mode of inhibition for CH3As(III)I2 from competitive to noncompetitive . The greater potency of methylated trivalent arsenicals and arsenothiols than of inorganic trivalent As suggests that biomethylation of As could yield species that inhibit reduction of GSSG and alter the redox status of cells.

Curr Biol, 1997 Jan 1, 7(1), R24 - 7
Genetic recombination: sex-change operations in yeast; Shore D; The 'directionality' of mating-type switching in building yeast is determined by mechanisms that regulate genetic recombination along the whole left arm of chromosome III . In MATa cells, a cis-acting 'recombinational enhancer' activates this entire region, while in MATalpha cells the enhancer is turned off by the alpha2 repressor.

Gynecol Obstet Invest, 1997, 43(2), 120 - 4
Effects of a yeast-based dietary supplementation on premenstrual syndrome . A double-blind placebo-controlled study; Facchinetti F et al.; A dietary approach has proven to be effective in alleviating symptoms of premenstrual syndrome . In our previous studies, magnesium improved premenstrual irritability and mood scoings . In this double-blind, placebo-controlled study, we evaluated the effects of a new dietetic preparation (Sillix Donna, Giuliani) in 40 patients affected by mild to moderate premenstrual syndrome . Premenstrual symptoms were scored in both follicular and luteal phases, at baseline, at 2nd, 4th and 6th month of treatment by using the Menstrual Distress Questionnaire (MDQ) . Twenty patients were randomised to receive the active preparation and 20 placebo . MDQ scores at baseline were similar in the two groups . Five patients of the placebo group dropped out because of treatment failure . No side effects were observed . Both treatments reduced symptoms already in the 2nd month, but the active preparation was more effective at all time controls (p < 0.05); at the 6th month it significantly reduced premenstrual MDQ scores to 18% of baseline values, placebo only to 73% . These data demonstrate that Sillix Donna is effective in reducing premenstrual distress.

DNA Seq, 1997, 7(2), 123 - 5
A cDNA encodes the Drosophila homolog of yeast 60S ribosomal protein YL43; Fox MG et al.; We describe the nucleotide sequence of a cDNA clone isolated from Drosophila Kc cells which encodes an amino acid sequence homologous to a 60S ribosomal protein from yeast (YL43) and rat (p23) . The DL43 cDNA is 320 nucleotides in length and predicts a protein of 76 amino acids and a calculated molecular mass of 8.9 kiloDaltons . Northern blot analysis demonstrates the presence of the DL43 transcript under both control (25 degrees C) and heat shock (37 degrees C) conditions . The Drosophila protein shares an 86% identity over the first 22 amino acids with the yeast YL43 protein and a 60% identity over the entire length of the partial sequence available for this protein.

J Biochem (Tokyo), 1997 Jan, 121(1), 8 - 14
Identification and functional characterization of yeast zeta-COP; Yamazaki S et al.; Coatomer, the cytosolic protein complex, consists of seven subunits (alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP) and is involved in vesicle trafficking early in the secretory pathway in collaboration with a 20 kDa GTP-binding protein (ARF) . In the present study, we have identified a yeast gene which encodes a protein having 39% amino acid sequence identity with bovine zeta-COP . This gene (YZC1 for Yeast Zeta COP) is essential for vegetative growth and the growth defect of delta yzc1 cells was restored by bovine zeta-COP cDNA . We isolated a temperature-sensitive mutant of YZC1 (yzc1ts) and examined its capacity for both the ER-to-Golgi transport and the double lysine motif (KKXX)-mediated retrograde transport from Golgi to ER . At non-permissive temperature, the yzc1ts cells exhibited a weak defect in the anterograde transport, but a strong defect in the retrograde vesicle transport . We conclude that Yzc1p is a yeast homologue of mammalian zeta-COP and participates mainly in the Golgi-to-ER retrograde transport.

J Biochem (Tokyo), 1997 Jan, 121(1), 1 - 4
Crystallization of eukaryotic E3, lipoamide dehydrogenase, from yeast, for exhibiting X-ray diffraction beyond 2.5 A resolution, and preliminary structure analysis; Toyoda T et al.; Lipoamide dehydrogenase, which is a common component of alpha-keto acid dehydrogenase complexes, has been highly purified from yeast (Saccharomyces cerevisiae) to reveal its structure at higher resolution . New crystals obtained by a desalting method exhibited diffraction beyond 2.5 A resolution . The cell dimensions are a = 97.1, b = 158.7, and c = 67.9 A, and the space group is P2(1)2(1)2(1) . There is a dimeric enzyme in the asymmetric unit . The crystal structure was solved by means of the molecular-replacement technique and refined in a preliminary manner.

Can J Microbiol, 1997 Jan, 43(1), 70 - 7
Membrane fatty acid composition and membrane fluidity as parameters of stress tolerance in yeast; Swan TM et al.; The relationship among membrane fatty acid composition, membrane fluidity, and stress tolerance was investigated in yeast cells . Several strains were examined for their ability to survive heat, ethanol, and hydrogen peroxide stresses . Membrane fluidity was determined by measuring fluorescence anisotropy using diphenylhexatriene as a probe . There was no obvious relationship among membrane fatty acyl composition, membrane fluidity, and stress tolerance in the strains examined . A consistent trend in the present study was an observed decrease in membrane fluidity following thermal treatment, which coincided with a reduction in cell viability . We suggest that protein denaturation may be responsible for the observed effect of elevated temperature on membrane fluidity and viability . This was implied by observations on the irreversible nature of thermal transitions, as measured by breaks in Arrhenius plots, in which stationary phase cells were shown to exhibit higher transition temperatures (53.9-55.5 degrees C) than exponential phase cells (49.5-51 degrees C) . Furthermore, the thermal transition temperature was shown to increase in exponential phase cells following heat shock, which was associated with an increase in thermotolerance . We suggest that the thermotolerant state of heat-shocked cells and cells entering stationary phase may be associated with increased protein stability . However, despite the relatively good correlation between thermal transition temperature and stress tolerance, the thermal transition temperature did not predict the stress tolerance of a given strain, as stress-sensitive strains had similar transition temperatures to those of stress-resistant strains.

Yeast, 1997 Jan, 13(1), 1 - 8
Yeast PIG genes: PIG1 encodes a putative type 1 phosphatase subunit that interacts with the yeast glycogen synthase Gsy2p; Cheng C et al.; The biosynthesis of glycogen involves multiple proteins that associate with each other and the glycogen macromolecule . In efforts to understand the nature of these proteins, a two-hybrid screen was undertaken to detect proteins able to interact with Gsy2p, a major form of glycogen synthase in Saccharomyces cerevisiae . Two positives expressed proteins derived from genes designated PIG1 and PIG2, on chromosomes XIIR and IXL respectively . PIG1 codes for a protein with 38% identity over a 230 residue segment to Gac1p, a protein thought to be a type 1 protein phosphatase targeting subunit whose loss impairs glycogen synthesis . Pig2p has 30% identify to the protein corresponding to an open reading frame, YER054, on chromosome V . Deletion of PIG1 on its own had little effect on glycogen storage but, in combination with loss of GAC1, caused a more severe glycogen-deficient phenotype than seen in gac1 mutants . This result is consistent with Pig1p being functionally related to Gac1p and we propose that Pig1p may be a type 1 phosphatase regulatory subunit . Delection of PIG2, YER054, or both genes together caused no detectable change in glycogen metabolism under the conditions tested . Gac1p, Pig1p, Pig2p and the YER054p are the only four proteins coded by the yeast genome that share a conserved segment of approximately 25 residues, designated the GVNK motif, that is identifiable also in RGI, the mammalian type 1 phosphatase targeting subunit.

Genome Res, 1997 Jan, 7(1), 1 - 9
Mapping expressed sequence tag sites on yeast artificial chromosome clones of Arabidopsis thaliana DNA; Agyare FD et al.; We describe a method for efficient parallel mapping of expressed sequence tag (EST) sites onto yeast artificial chromosome (YAC) clones . The strategy involves an initial YAC clone pooling scheme that minimizes the number of required PCR amplifications . This is followed by parallel analysis of PCR amplicons of EST sequences . Using this method, we have screened 600 EST sites in combinatorial pools of 3449 YAC clones that contain Arabidopsis thaliana DNA inserts . The presence of these genes on YACs was detected by amplifying EST sequences with PCR and analyzing the reaction products by agarose gel electrophoresis . Of the 600 ESTs, 271 were found to map to individual YACs . Software tools are presented that allow for the automated analysis of this electrophoresis data . Suggestions for the scale-up of this method to map large genomes are discussed.

Genomics, 1997 Jan 1, 39(1), 55 - 65
A yeast artificial chromosome (YAC) contig encompassing the critical region of the X-linked lymphoproliferative disease (XLP) locus; Lanyi A et al.; X-linked lymphoproliferative disease (XLP) is characterized by a marked vulnerability to Epstein-Barr virus (EBV) infection . Infection of XLP patients with EBV invariably results in fatal mononucleosis, agammaglobulinemia, or malignant lymphoma . Initially the XLP gene was assigned to a 10-cM region in Xq25 between DXS42 and DXS37 . Subsequently, an interstitial, cytogenetically visible deletion in Xq25 was identified in one XLP family, 43 . In this study we estimated the deletion in XLP patient 43-004 by dual-laser flow karyotyping to involve 2% of the X chromosome, or approximately 3 Mb of DNA sequence . From a human chromosome Xq25-specific yeast artificial chromosome (YAC) sublibrary, five YACs containing DNA sequences deleted in patient 43-004 have been isolated . Sequence-tagged sites (STSs) from these YACs have been used to identify interstitial deletions in unrelated XLP patients . Three more families with interstitial deletions were found . Two of the patients (63-003 and 73-032) carried an interstitial deletion of 3.0 Mb overlapping the 43-004 deletion . In one XLP patient (30-011) who exhibited the characteristic postinfectious mononucleosis phenotype of XLP with hypogammaglobulinemia and malignant lymphoma, a deletion of approximately 250 kb was detected overlapping the deletion detected in patients 43-004, 63-003, and 73-032 . A YAC contig of 2.2 Mb spanning the XLP critical region, whose orientation on chromosome X was determined by double-color fluorescence in situ hybridization and which consists of 15 overlapping YAC clones, has been constructed . A detailed restriction enzyme map of the region has been constructed . YAC insert sizes were determined by counter-clamped homogenous electric field gel electrophoresis . Chimerism of YACs was determined by FISH and restriction mapping . On the basis of lambda subclones, YAC end-derived plasmids, and STSs with an average spacing of 100 kb, a long-range physical map was constructed using 5 rare-cutter restriction enzymes . The STSs and lambda subclones were used in Southern hybridization and PCR analyses . The work presented here substantially refines the critical region for XLP . The YAC contig with the overlapping interstitial deletions constitutes the basis for the construction of a transcriptional map of the critical region and facilitates the identification of the XLP gene.

Plant J, 1997 Jan, 11(1), 1 - 14
AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression; Klimyuk VI et al.; Based on homologies between the yeast DMC1 and the lily LIM15 meiosis-specific genes, degenerate PCR primers were designed that amplified the Arabidopsis DMC1 gene (AtDMC1) . AtDMC1 genomic DNA (8 kb) was sequenced, and the transcript was characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) and by 5' and 3' RACE (rapid amplification of cDNA ends) . The AtDMC1 gene contains 15 exons and 14 introns . RNA in situ hybridization analysis showed that expression of the AtDMC1 is restricted to pollen mother cells in anthers and to megaspore mother cells in ovules . The AtDMC1 promoter was fused to the GUS reporter gene, and conferred meiosis-associated expression in both male and female floral lineages . Comparison of AtDMC1 isolated from Landsberg erecta ecotype to its Columbia allele ArLIM15, revealed the presence of a 1874 bp transposon-like element within the promoter region of ArLIM15 . RT-PCR analysis showed that the expression levels of AtDMC1 and ArLIM15 are similar . Possible uses for the AtDMC1 promoter are discussed.

Trends Biochem Sci, 1997 Jan, 22(1), 33 - 4
False positives from the yeast two-hybrid system; Hengen PN; Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methods-reagents, available on the Internet . This month's column discusses false positives found when using the yeast two-hybrid system to isolate interactive proteins . For details on how to partake in the newsgroup, see the accompanying box.

Trends Biochem Sci, 1997 Jan, 22(1), 18 - 22
The protein kinases of budding yeast: six score and more; Hunter T et al.; The completion of the budding yeast genome sequencing project has made it possible to determine not only the total number of genes, but also the exact number of genes of a particular type 1-3 . As a consequence, we now know exactly how many protein kinases are encoded by the yeast genome, a number of considerable interest because of the importance of protein phosphorylation in the control of so many cellular processes.

Mol Biol Cell, 1997 Jan, 8(1), 171 - 87
Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1; Kominami K et al.; Nin1p, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries . By exploiting the temperature-sensitive phenotype of the nin1-1 mutant, we have screened for genes encoding proteins with related functions to Nin1p and have cloned and characterized two new multicopy suppressors, SUN1 and SUN2, of the nin1-1 mutation . SUN1 can suppress a null nin1 mutation, whereas SUN2, an essential gene, does not . Sun1p is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome . Sun1p binds ubiquitin-lysozyme conjugates as do S5a and MBP1 . Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome . cDNA encoding the p58 component was cloned . Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2 . During glycerol density gradient centrifugation, both Sun1p and Sun2p comigrated with the known proteasome components . These results, as well as other structural and functional studies, indicate that both Sun1p and Sun2p are components of the regulatory module of the yeast 26S proteasome.

Mol Biol Cell, 1997 Jan, 8(1), 13 - 31
Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein; Hicke L et al.; Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates . Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes . The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate . We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome . Treatment of sensitive cells with brefeldin A also blocks transport from this compartment . We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway . Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome . As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes.

Genetics, 1997 Jan, 145(1), 63 - 73
Proteolytic activation of Rim1p, a positive regulator of yeast sporulation and invasive growth; Li W et al.; In the yeast Saccharomyces cerevisiae, rim1, 8, 9, or 13 mutations cause four phenotypes: poor growth at low temperature, altered colony morphology, inefficient sporulation due to reduced expression of the meiotic activator IME1, and, as shown here, defective invasive growth . In this report, we have determined the relationship between RIM1 and the other genes, RIM8, 9, and 13, in this group . We have analyzed production of epitope-tagged Rim1p derivatives with HA epitopes at the N-terminus or in the middle of the protein . These Rim1p derivatives exist primarily as a small form (90 kD for Rim1-HA2p) in wild-type cells and as a large form (98 kD for Rim1-HA2p) in rim8, 9, and 13 mutants . We have also analyzed production of beta-galactosidase in strains that express a RIM1-lacZ fusion gene . beta-galactosidase exists primarily as a approximately 130 kD form in wild-type cells and as a approximately 190 kD form in rim9 mutants . These results indicate that Rim1p undergoes C-terminal proteolytic cleavage, and that rim8, 9, and 13 mutations block cleavage . Expression of a Rim1p C-terminal deletion derivative suppresses rim8, 9, and 13 mutations . Thus the phenotypes of rim8, 9, and 13 mutants arise from the defect in Rim1p C-terminal cleavage . Cleavage of Rim1p, like that of its Aspergillus nidulans homologue PacC, is stimulated under alkaline growth conditions . Therefore, Rim1p, PacC and their respective processing pathways may represent a conserved signal transduction pathway.

Nucleic Acids Res, 1997 Jan 1, 25(1), 28 - 30
MIPS: a database for protein sequences, homology data and yeast genome information; Mewes HW et al.; The MIPS group (Martinsried Institute for Protein Sequences) at the Max-Planck-Institute for Biochemistry, Martinsried near Munich, Germany, collects, processes and distributes protein sequence data within the framework of the tripartite association of the PIR-International Protein Sequence Database (,) . MIPS contributes nearly 50% of the data input to the PIR-International Protein Sequence Database . The database is distributed on CD-ROM together with PATCHX, an exhaustive supplement of unique, unverified protein sequences from external sources compiled by MIPS . Through its WWW server MIPS permits internet access to sequence databases, homology data and to yeast genome information . (i) Sequence similarity results from the FASTA program () are stored in the FASTA database for all proteins from PIR-International and PATCHX . The database is dynamically maintained and permits instant access to FASTA results . (ii) Starting with FASTA database queries, proteins have been classified into families and superfamilies (PROT-FAM) . (iii) The HPT (hashed position tree) data structure () developed at MIPS is a new approach for rapid sequence and pattern searching . (iv) MIPS provides access to the sequence and annotation of the complete yeast genome (), the functional classification of yeast genes (FunCat) and its graphical display, the 'Genome Browser' () . A CD-ROM based on the JAVA programming language providing dynamic interactive access to the yeast genome and the related protein sequences has been compiled and is available on request.

Protein Sci, 1997 Jan, 6(1), 197 - 210
Analysis of the structure and stability of omega loop A replacements in yeast iso-1-cytochrome c; Fetrow JS et al.; Omega (omega)-loop A, residues 18-32 in wild-type yeast iso-1-cytochrome c, has been deleted and replaced with loop sequences from three other cytochromes c and one from esterase . Yeast expressing a partial loop deletion do not contain perceptible amounts of holoprotein as measured by low-temperature spectroscopy and cannot grow on nonfermentable media . Strains expressing loop replacement mutations accumulate holoprotein in vivo, but the protein function varies depending on the sequence and length of the replacement loop; in vivo expression levels do not correlate with their thermal denaturation temperatures . In vitro spectroscopic studies of the loop replacement proteins indicate that all fold into a native-like cytochrome c conformation, but are less stable than the wild-type protein . Decreases in thermal stability are caused by perturbation of loop C backbone in one case and a slight reorganization of the protein hydrophobic core in another case, rather than rearrangement of the loop A backbone . A single-site mutation in one of the replacement mutants designed to relieve inefficient hydrophobic core packing caused by the new loop recovers some, but not all, of the lost stability.

Protein Sci, 1997 Jan, 6(1), 109 - 18
Recombinant hirustasin: production in yeast, crystallization, and interaction with serine proteases; Di Marco S et al.; A synthetic gene coding for the 55-amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast alpha-factor leader sequence and expressed in Saccharomyces cerevisiae . Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease . The processed hirustasin was purified to better than 97% purity . N-terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N-terminus and the expected amino acid sequence and molecular mass . The biological activity of recombinant hirustasin was identical to that of the authentic leech protein . Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 A and 2.4 A, respectively . In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor . During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor . In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly . Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31 . Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.

Hum Mol Genet, 1997 Jan, 6(1), 59 - 68
Functional human CFTR produced by stable Chinese hamster ovary cell lines derived using yeast artificial chromosomes; Mogayzel PJ Jr et al.; The cystic fibrosis transmembrane conductance regulator gene (CFTR) encodes a transmembrane protein (CFTR) which functions in part as a cyclic adenosine monophosphate (cAMP)-regulated chloride channel . CFTR expression is controlled temporally and cell specifically by mechanisms that are poorly understood . Insight into CFTR regulation could be facilitated by the successful introduction of the entire 230 kb human CFTR and adjacent sequences into mammalian cells . To this end, we have introduced two different CFTR-containing yeast artificial chromosomes (YACs) (320 and 620 kb) into Chinese hamster ovary-K1 (CHO) cells . Clonal cell lines containing human CFTR were identified by PCR, and the genetic and functional analyses of one clone containing each YAC are described . Integration of the human CFTR-containing YACs into the CHO genome at a unique site in each cell line was demonstrated by fluorescence in situ hybridization (FISH) . Southern blot analysis suggested that on the order of one copy of human CFTR was integrated per CHO cell genome . Fiber-FISH and restriction analysis suggested that CFTR remained grossly intact . Northern analysis showed full-length, human CFTR mRNA . Immunoprecipitation followed by phosphorylation with protein kinase demonstrated mature, glycosylated CFTR . Finally, chloride secretion in response to cAMP indicated the functional nature of the human CFTR . This study provides several novel results including: (i) functional human CFTR can be expressed from these YACs; (ii) CHO cells are a permissive environment for expression of human CFTR; (iii) the level of human CFTR expression in CHO cells is unexpectedly high given the lack of endogenous CFTR production; and (iv) the suggestion by Fiber-FISH of CFTR integrity correlates with functional gene expression . These YACs and the cell lines derived from them should be useful tools for the study of CFTR expression.

Parasitol Res, 1997, 83(1), 87 - 9
Construction and rapid screening of a representative yeast artificial chromosome library from the Plasmodium falciparum strain Dd2; Camargo AA et al.; Large genomic DNA fragments from the Plasmodium falciparum clone Dd2 have been cloned as artificial chromosomes in yeast (YAC) . The resulting library has a 10-fold redundancy for single-copy genes and consists of 1440 individual clones, including 240 telomeric clones, with an average insert size of 150 kb . A novel hybridization method was developed for the rapid and cost-effective screening of protozoan YAC libraries . The Dd2 YAC clones will facilitate a positional approach to the parasite's genes and aid in the dissection of genetic loci associated with the virulence and pathogenicity of P . falciparum.

Genes Dev, 1997 Jan 1, 11(1), 139 - 51
A human protein required for the second step of pre-mRNA splicing is functionally related to a yeast splicing factor; Horowitz DS et al.; We have identified a human splicing factor required for the second step of pre-mRNA splicing . This new protein, hPrp18, is 30% identical to the yeast splicing factor Prp18 . In HeLa cell extracts immunodepleted of hPrp18, the second step of pre-mRNA splicing is abolished . Splicing activity is restored by the addition of recombinant hPrp18, demonstrating that hPrp18 is required for the second step . The hPrp18 protein is bound tightly to the spliceosome only during the second step of splicing . hPrp18 is required for the splicing of several pre-mRNAs, making it the first general second-step splicing factor found in humans . Splicing activity can be restored to hPrp18-depleted HeLa cell extracts by yeast Prp18, showing that important functional regions of the proteins have been conserved . A 90-amino-acid region near the carboxyl terminus of hPrp18 is strongly homologous to yeast Prp18 and is also conserved in rice and nematodes . The homology identifies one region important for the function of both proteins and may define a new protein motif . In contrast to yeast Prp18, hPrp18 is not stably associated with any of the snRNPs . A 55-kD protein that cross-reacts with antibodies against hPrp18 is a constituent of the U4/U6 and U4/U6 x U5 snRNP particles.

Genes Dev, 1997 Jan 1, 11(1), 83 - 93
SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast; Strahl-Bolsinger S et al.; Yeast core telomeric heterochromatin can silence adjacent genes and requires RAP1, SIR2, SIR3, and SIR4 and histones H3 and H4 for this telomere position effect . SIR3 overproduction can extend the silenced domain . We examine here the nature of these multiprotein complexes . SIR2 and SIR4 were immunoprecipitated from whole-cell extracts . In addition, using formaldehyde cross-linking we have mapped SIR2, SIR4, and RAP1 along telomeric chromatin before and after SIR3 overexpression . Our data demonstrate that SIR2 and SIR4 interact in a protein complex and that SIR2, SIR3, SIR4, and RAP1 map to the same sites along telomeric heterochromatin in wild-type cells . However, when overexpressed, SIR3 spreads along the chromosome and its interactions are dominant to those of SIR4 and especially SIR2, whose detection is decreased in extended heterochromatin . RAP1 binding at the core region is unaffected by SIR3 overproduction and RAP1 shows no evidence of spreading . Thus, we propose that the structure of core telomeric heterochromatin differs from that extended by SIR3.

Immunogenetics, 1997, 45(3), 180 - 7
A yeast artificial chromosome contig spanning the mouse immunoglobulin kappa light chain locus; Schupp IW et al.; A single contig spanning the entire mouse immunoglobulin kappa light chain (Igk) locus on chromosome 6 has been established using yeast and bacterial artificial chromosome clones . Detailed mapping of the Igk locus indicates that a member of the Igk-V2 gene family, located about 3.5 megabases upstream of the Igk-J-C complex, is the most distal functional Igk-V gene . Sequence analyses of Igk-V genes and anonymous DNA segments provide indications for internal duplications at the 5' end of the Igk-V locus and identify the likely origin of Igk-V orphon gene clusters located elsewhere in the mouse genome.

RNA, 1997 Jan, 3(1), 27 - 36
RNA-dependent RNA polymerase activity associated with the yeast viral p91/20S RNA ribonucleoprotein complex; Garcia-Cuellar MP et al.; 20S RNA is a noninfectious viral single-stranded RNA found in most laboratory strains of the yeast Saccharomyces cerevisiae . 20S RNA encodes a protein of 91 kDa (p91) that contains the common motifs found among RNA-dependent RNA polymerases from RNA viruses . p91 and 20S RNA are noncovalently associated in vivo, forming a ribonucleoprotein complex . We detected an RNA polymerase activity in p91/20S RNA complexes isolated by high-speed centrifugation . The activity was not inhibited by actinomycin D nor alpha-amanitin . The majority of the in vitro products was 20S RNA and the rest was the complementary strands of 20S RNA . Because the extracts were prepared from cells accumulating 20S RNA over its complementary strands, these in vitro products reflect the corresponding activities in vivo . When the p91/20S RNA complexes were subjected to sucrose gradient centrifugation, the polymerase activity cosedimented with the complexes . Furthermore, an RNA polymerase activity was detected in the complex by an antibody-linked polymerase assay using anti-p91 antiserum, suggesting that p91 is present in the active RNA polymerase machinery . These results together indicate that p91 is the RNA-dependent RNA polymerase or a subunit thereof responsible for 20S RNA replication.

J Virol, 1997 Jan, 71(1), 812 - 7
Mapping the multimerization domains of the Gag protein of yeast retrotransposon Ty1; Brachmann CB et al.; The two-hybrid system was used to define regions of the Ty1 Gag protein responsible for multimerization . Gag truncations lacking the first 146 or the last 97 amino acids (Gag is 440 amino acids in length) interact . A severely C-terminally truncated molecule (lacking the last 207 amino acids) was the smallest truncation to interact, suggesting that some protein-protein interactions between Gag molecules are mediated through the first 233 amino acids . However, an internal deletion of amino acids 147 to 233 does not abolish Gag-Gag interaction, indicating that more than one region can mediate Gag interaction . Surprisingly, we found that a truncation lacking the last 97 amino acids interacts with itself but not with full-length Gag . This is apparently due to an artifact of the two-hybrid assay, since these same molecules coassemble with wild-type Gag into Ty1 virus-like particles.

Mol Cell Biol, 1997 Jan, 17(1), 519 - 27
Histone acetyltransferase activity is conserved between yeast and human GCN5 and is required for complementation of growth and transcriptional activation; Wang L et al.; Yeast and human ADA2 and GCN5 (y- and hADA2 and y- and hGCN5, respectively) have been shown to potentiate transcription in vivo and may function as adaptors to bridge physical interactions between DNA-bound activators and the basal transcriptional machinery . Recently it was shown that yGCN5 is a histone acetyltransferase (HAT), suggesting a link between enzymatic modification of nucleosomes and transcriptional activation . In this report, we demonstrate that hGCN5 is also an HAT and has the same substrate specificity as yGCN5 . Since hGCN5 does not complement functional defects caused by deletion of yGCN5, we constructed a series of hGCN5-yGCN5 chimeras to identify human regions capable of activity in yeast . Interestingly, only the putative HAT domain of hGCN5, when fused to the remainder of yGCN5, complemented gcn5- cells for growth and transcriptional activation . Moreover, an amino acid substitution mutation within the HAT domain reduced both HAT activity in vitro and transcription in vivo . These findings directly link enzymatic histone acetylation and transcriptional activation and show evolutionary conservation of this potentially crucial pathway in gene regulation.

Mol Cell Biol, 1997 Jan, 17(1), 378 - 88
Nop2p is required for pre-rRNA processing and 60S ribosome subunit synthesis in yeast; Hong B et al.; To investigate the function of the nucleolar protein Nop2p in Saccharomyces cerevisiae, we constructed a strain in which NOP2 is under the control of a repressible promoter . Repression of NOP2 expression lengthens the doubling time of this strain about fivefold and reduces steady-state levels of 60S ribosomal subunits, 80S ribosomes, and polysomes . Levels of 40S subunits increase as the free pool of 60S subunits is reduced . Nop2p depletion impairs processing of the 35S pre-rRNA and inhibits processing of 27S pre-rRNA, which results in lower steady-state levels of 25S rRNA and 5.8S rRNA . Processing of 20S pre-rRNA to 18S rRNA is not significantly affected . Processing at sites A2, A3, B1L, and B1S and the generation of 5' termini of different pre-rRNA intermediates appear to be normal after Nop2p depletion . Sequence comparisons suggest that Nop2p may function as a methyltransferase . 2'-O-ribose methylation of the conserved site UmGm psi UC2922 is known to take place during processing of 27S pre-rRNA . Although Nop2p depletion lengthens the half-life of 27S pre-RNA, methylation of UmGm psi UC2922 in 27S pre-rRNA is low during Nop2p depletion . However, methylation of UmGm psi UC2922 in mature 25S rRNA appears normal . These findings provide evidence for a close interconnection between methylation at this conserved site and the processing step that yields the 25S rRNA.

Mol Cell Biol, 1997 Jan, 17(1), 256 - 66
The mammalian homolog of yeast Sec13p is enriched in the intermediate compartment and is essential for protein transport from the endoplasmic reticulum to the Golgi apparatus; Tang BL et al.; The role of COPII components in endoplasmic reticulum (ER)-Golgi transport, first identified in the yeast Saccharomyces cerevisiae, has yet to be fully characterized in higher eukaryotes . A human cDNA whose predicted amino acid sequence showed 70% similarity to the yeast Sec13p has previously been cloned . Antibodies raised against the human SEC13 protein (mSEC13) recognized a cellular protein of 35 kDa in both the soluble and membrane fractions . Like the yeast Sec13p, mSEC13 exist in the cytosol in both monomeric and higher-molecular-weight forms . Immunofluorescence microscopy localized mSEC13 to the characteristic spotty ER-Golgi intermediate compartment (ERGIC) in cells of all species examined, where it colocalized well with the KDEL receptor, an ERGIC marker, at 15 degrees C . Immunoelectron microscopy also localized mSEC13 to membrane structures close to the Golgi apparatus . mSEC13 is essential for ER-to-Golgi transport, since both the His6-tagged mSEC13 recombinant protein and the affinity-purified mSEC13 antibody inhibited the transport of restrictive temperature-arrested vesicular stomatitis virus G protein from the ER to the Golgi apparatus in a semi-intact cell assay . Moreover, cytosol immunodepleted of mSEC13 could no longer support ER-Golgi transport . Transport could be restored in a dose-dependent manner by a cytosol fraction enriched in the high-molecular-weight mSEC13 complex but not by a fraction enriched in either monomeric mSEC13 or recombinant mSEC13 . As a putative component of the mammalian COPII complex, mSEC13 showed partially overlapping but mostly different properties in terms of localization, membrane recruitment, and dynamics compared to that of beta-COP, a component of the COPI complex.

Clin Chim Acta, 1996 Dec 30, 256(2), 175 - 88
Glutamic acid decarboxylase autoantibody assay using 125I-labelled recombinant GAD65 produced in yeast; Powell M et al.; We describe a new method for measuring autoantibodies (Ab) to the 65 kDa isoform of glutamic acid carboxylase (GAD65) . In particular, GAD65 without the hydrophobic N-terminal region has been produced in yeast, purified, labelled with 125I and reacted with GAD65 Ab . Antibody bound 125I-GAD65 is then precipitated by the addition of solid phase protein A . With the assay, GAD65 Ab were detected in 59 of 71 (83%) islet cell antibody (ICA) positive IDDM patients and in 8 of 23 (35%) ICA negative IDDM patients (overall 67 of 94 (71%) of IDDM patients) . Low concentrations of GAD65 Ab were also detected in 2/98 (2%) healthy blood donors and 1/27 (4%) Graves' disease patients had a high level of antibody . GAD65 Ab were not detected in any of 10 Hashimoto's thyroiditis, 20 Addison's disease or 19 myasthenia gravis sera . There was good agreement between the 125I assay and the current reference method based on 35S-labelled full-length GAD65 (produced by in vitro transcription/translation reaction) and solid phase protein A (r = 0.91, n = 108) . Overall, our 125I assay showed sensitivity, precision and disease group specificity at least as good as any assay so far described . These features, combined with a simple assay protocol and the convenience of 125I counting and handling indicate that the method is suitable for routine GAD65 Ab measurements.

J Biol Chem, 1996 Dec 27, 271(52), 33632 - 8
A family of putative chloride channels from Arabidopsis and functional complementation of a yeast strain with a CLC gene disruption; Hechenberger M et al.; We have cloned four novel members of the CLC family of chloride channels from Arabidopsis thaliana . The four plant genes are homologous to a recently isolated chloride channel gene from tobacco (CLC-Nt1; Lurin, C., Geelen, D., Barbier-Brygoo, H., Guern, J., and Maurel, C . (1996) Plant Cell 8, 701-711) and are about 30% identical in sequence to the most closely related CLC-6 and CLC-7 putative chloride channels from mammalia . AtCLC transcripts are broadly expressed in the plant . Similarly, antibodies against the AtCLC-d protein detected the protein in all tissues, but predominantly in the silique . AtCLC-a and AtCLC-b are highly homologous to each other ( approximately 87% identity), while being approximately 50% identical to either AtCLC-c or AtCLC-d . None of the four cDNAs elicited chloride currents when expressed in Xenopus oocytes, either singly or in combination . Among these genes, only AtCLC-d could functionally substitute for the single yeast CLC protein, restoring iron-limited growth of a strain disrupted for this gene . Introduction of disease causing mutations, identified in human CLC genes, abolished this capacity . Consistent with a similar function of both proteins, the green fluorescent protein-tagged AtCLC-d protein showed the identical localization pattern as the yeast ScCLC protein . This suggests that in Arabidopsis AtCLC-d functions as an intracellular chloride channel.

J Biol Chem, 1996 Dec 27, 271(52), 33181 - 6
Direct association between the yeast Rad51 and Rad54 recombination proteins; Jiang H et al.; The RAD54 and RAD51 genes are involved in genetic recombination and double-strand break repair in the yeast Saccharomyces cerevisiae . The Rad51 protein is thought to be a yeast analogue of the Eschericia coli recA gene product and catalyzes strand exchange between homologous single- and double-stranded DNAs in vitro . RAD54 exhibits homologies to several known ATPases and is a member of the SWI2/MOT1 family . We show here that the Rad54 protein interacts with the Rad51 protein in vivo and in vitro and that the NH2-terminal 115 residues of the Rad54 protein are necessary for this interaction . Combined with previously reported results, these data imply that the Rad54 protein is part of a multiprotein yeast recombination complex.

Biochemistry, 1996 Dec 24, 35(51), 16871 - 8
Yeast sterol C8-C7 isomerase: identification and characterization of a high-affinity binding site for enzyme inhibitors; Moebius FF et al.; The yeast gene ERG2 encodes a sterol C8-C7 isomerase and is essential for ergosterol synthesis and cell proliferation . Its striking homology with the so-called sigma1 receptor of guinea pig brain, a polyvalent steroid and drug binding protein, suggested that the yeast sterol C8-C7 isomerase (ERG2) carries a similar high affinity drug binding domain . Indeed the sigma ligands {3H}haloperidol (Kd = 0.3 nM) and {3H}ifenprodil (Kd = 1.4 nM) bound to a single population of sites in ERG2 wild type yeast microsomes (Bmax values of 77 and 61 pmol/mg of protein, respectively), whereas binding activity was absent in strains carrying ERG2 gene mutations or disruptions . {3H}Ifenprodil binding was inhibited by sterol isomerase inhibitors such as fenpropimorph (Ki = 0.05 nM), tridemorph (Ki = 0.09 nM), MDL28,815 (Ki = 0.44 nM), triparanol (Ki = 1.5 nM), and AY-9944 (Ki = 5.8 nM) . {3H}Haloperidol specifically photoaffinity-labeled a protein with an apparent molecular weight of 27400, in agreement with the molecular mass of the sterol C8-C7 isomerase (24900 Da) . 9E10 c-myc antibodies specifically immunoprecipitated the c-myc tagged protein after {3H}haloperidol photolabeling, unequivocally proving that the drug binding site is localized on the ERG2 gene product . Haloperidol, trifluperidol, and ifenprodil inhibited the growth of Saccharomyces cerevisiae and reduced the ergosterol content of cells grown in their presence . Our results demonstrate that the yeast sterol C8-C7 isomerase has a polyvalent high-affinity drug binding site similar to mammalian sigma receptors and that in yeast sigma ligands inhibit sterol biosynthesis.

Biochemistry, 1996 Dec 24, 35(51), 16566 - 72
Polymerization and in vitro motility properties of yeast actin: a comparison with rabbit skeletal alpha-actin; Kim E et al.; Actin purified from the yeast (Saccharomyces cerevisae) was polymerized faster than rabbit skeletal alpha-actin by MgCl2 . The two actins polymerized at similar rates in the presence of CaCl2 . Yeast actin, up to 25 microM, was not polymerized by KCl (100-300 mM); the monovalent salt also inhibited the MgCl2-induced polymerization of actin . The local structure of the subdomain-2 region in yeast actin filaments was probed by subtilisin and trypsin digestions . Loop 38-52 appeared more flexible and accessible to subtilisin in yeast than in rabbit actin . In contrast, tryptic digestions at Lys-61 and -68 occurred at the same rate for yeast and alpha-actin filaments . Modification of yeast actin by a sulfhydryl reagent CPM {7-(diethylamino)-3-(4'-maleimidophenyl)-4-methylcoumain} was specific to the Cys-374 residue; no labeling of a yeast actin mutant containing an alanine substitution for cysteine 374 was observed . The rates of Cys-374 labeling by CPM were similar for yeast and muscle actin, suggesting a similar environment for the C terminus in both polymers . In the in vitro motility assays, yeast actin required higher concentrations of heavy meromyosin (HMM) for its sliding than did the rabbit actin . At saturating concentrations of HMM, the sliding velocities of both actins were the same (3.0 microns/s) . Relative forces generated by HMM with yeast and muscle actin were assessed by monitoring their in vitro motility in the presence of NEM-HMM load . The sliding of yeast actin was stopped at a level of external load (molar ratio NEM-HMM/HMM = 0.25) lower than that of muscle actin (NEM-HMM/HMM = 0.43), suggesting lower force production with yeast actin . These results are discussed in terms of the myosin cross-bridge cycle and actomyosin interactions.

Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15075 - 80
The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast; Brush GS et al.; Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination . In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation . Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well . We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested . Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response . MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T) . A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation . Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T.

J Biol Chem, 1996 Dec 20, 271(51), 33039 - 46
Mapping the contacts of yeast TFIIIB and RNA polymerase III at various distances from the major groove of DNA by DNA photoaffinity labeling; Persinger J et al.; The structure of the Saccharomyces cerevisiae RNA polymerase III transcription complex on the SUP4 tRNATyr gene was probed at distances of approximately 10 to approximately 23 A from the C-5 methyl of thymidine in the major groove of DNA using photoreactive aryl azides attached to deoxyuridine by variable chain lengths . The nucleotide analogs contained an azidobenzoyl group attached with chain lengths that were incrementally increased by approximately 4 . 3 A by inserting 1-3 glycine residues into the chain . Another photoreactive deoxyuridine analog was made that contained a butyl chain (ABU-dUMP) to assess the effect of the chain's hydrophobicity on its ability to photoaffinity label the transcription complex . These nucleotide analogs were incorporated at base pairs (bp) -26/-21, -17, or -3/-2 on the nontranscribed strand of the SUP4 tRNATyr gene along with an {alpha-32P}dNMP by primer extension using an immobilized single-stranded DNA template annealed to specific oligonucleotides . The 27-kDa subunit of TFIIIB or the TATA box binding protein was photoaffinity labeled at bp -26/-21 with nucleotide analogs containing a approximately 19- or approximately 23-A chain and not with shorter chains of approximately 10 to approximately 15 A in length . The B" subunit of TFIIIB (Mr = 90 kDa) was photoaffinity labeled at bps -26/-21 with DNA containing a approximately 14-A chain and not with shorter or longer chains . Cross-linking of the B" subunit was inhibited by binding of RNA polymerase III (Pol III) to the TFIIIB-DNA complex and suggested that Pol III binding causes a conformational change in the TFIIIB-DNA complex resulting in the displacement of the 90-kDa subunit at bps -26/-21 . Next, the chain length dependence of photoaffinity labeling the 34-kDa subunit of Pol III at bps -17 and -3/-2 indicated that the 34-kDa subunit of Pol III is slightly removed from the major groove at bp -17 in the initiation complex and makes closer contact at bps -3/-2 in a stalled elongation complex.

J Biol Chem, 1996 Dec 20, 271(51), 32881 - 5
Mouse RNA polymerase I 16-kDa subunit able to associate with 40-kDa subunit is a homolog of yeast AC19 subunit of RNA polymerases I and III; Yao Y et al.; We have previously isolated a mouse RPA40 (mRPA40) cDNA encoding the 40-kDa subunit of mouse RNA polymerase I and demonstrated that mRPA40 is a mouse homolog of the yeast subunit AC40, which is a subunit of RNA polymerases I and III, having a limited homology to bacterial RNA polymerase subunit alpha (Song, C . Z., Hanada, K., Yano, K., Maeda, Y., Yamamoto, K., and Muramatsu, M . (1994) J . Biol . Chem . 269, 26976-26981) . In an extension of the study we have now cloned mouse RPA16 (mRPA16) cDNA encoding the 16-kDa subunit of mouse RNA polymerase I by a yeast two-hybrid system using mRPA40 as a bait . The deduced amino acid sequence shows 45% identity to the yeast subunit AC19 of RNA polymerases I and III, known to associate with AC40, and a local similarity to bacterial alpha subunit . We have shown that mRPA40 mutants failed to interact with mRPA16 and that neither mRPA16 nor mRPA40 can interact by itself in the yeast two-hybrid system . These results suggest that higher eukaryotic RNA polymerase I conserves two distinct alpha-related subunits that function to associate with each other in an early stage of RNA polymerase I assembly.

J Biol Chem, 1996 Dec 20, 271(51), 32810 - 7
Isolation and characterization of SUG2 . A novel ATPase family component of the yeast 26 S proteasome; Russell SJ et al.; Using a genetic strategy designed to find proteins involved in the function of the Saccharomyces cerevisiae transcriptional activator GAL4, we isolated mutants in two genes which rescue a class of gal4 activation domain mutants . One of these genes, SUG1, encodes a member of a large family of putative ATPases, the Conserved ATPase containing Domain (CAD) proteins (also known as AAA proteins) that are involved in a wide variety of cellular functions . Subsequently, SUG1 was identified as a subunit of the 26 S proteasome . We have now cloned the gene defined by the second complementation group . SUG2 encodes an essential 49-kDa protein that is also a member of the CAD family and is 43% identical to SUG1 . The mutation in sug2-1, like that in sug1-1, is found in the CAD near the highly conserved ATPase motif . We present biochemical and genetic evidence that SUG2 is associated in vivo with SUG1 and is a novel CAD protein subunit of the 26 S proteasome . With its highly conserved mammalian homologs, human p42 and ground squirrel CADp44, SUG2 defines a new class of proteasomal CAD proteins.

J Biol Chem, 1996 Dec 20, 271(51), 32503 - 6
Disruption of re-replication control by overexpression of human ORC1 in fission yeast; Wolf DA et al.; Initiation of DNA replication in Saccharomyces cerevisiae requires the binding of the origin recognition complex (ORC) to autonomously replicating sequences . HsORC1, a recently identified human protein related to yeast Orc1p and Cdc6p/Cdc18p, may be a component of the replication initiation complex in human cells . We have independently isolated the gene for HsORC1 and begun to address its function in eukaryotic DNA replication and its relationship to Cdc18p . Although HsORC1 failed to rescue the temperature-sensitive S . pombe cdc18-K46 strain, overexpression in a wild-type strain led to continuous DNA synthesis in the absence of mitosis . Deletion mutagenesis identified a short N-terminal region of HsORC1 that contains potential phosphorylation sites for cyclin-dependent kinase (CDK) as being sufficient to induce re-replication . In addition, we found that HsORC1 is an efficient substrate for CDKs in vitro . We propose that perturbation of the re-replication control by overexpression of HsORC1 is due to a titration of components involved in inactivating Cdc18p upon initiation of replication.

Science, 1996 Dec 20, 274(5295), 2069 - 74
Functional analysis of the genes of yeast chromosome V by genetic footprinting; Smith V et al.; Genetic footprinting was used to assess the phenotypic effects of Ty1 transposon insertions in 268 predicted genes of chromosome V of Saccharomyces cerevisiae . When seven selection protocols were used, Ty1 insertions in more than half the genes tested (157 of 268) were found to result in a detectable reduction in fitness . Results could not be obtained for fewer than 3 percent of the genes tested (7 of 268) . Previously known mutant phenotypes were confirmed, and, for about 30 percent of the genes, new mutant phenotypes were identified.

Biochemistry, 1996 Dec 17, 35(50), 16144 - 52
Probing the role of positive residues in the ADP/ATP carrier from yeast . The effect of six arginine mutations on transport and the four ATP versus ADP exchange modes; Heidkamper D et al.; Mutagenesis of three intrahelical arginines, R96, R204, or R294, and of each member of the arginine triplet R252, R253, R254 into neutral residues had resulted in a strong suppression of oxidative phosphorylation in cells and isolated mitochondria {Muller, V., Basset, G., Nelson, D . R., & Klingenberg, M . (1996) Biochemistry 35, 16132-16143} . Here we determine the transport activity of wild-type and mutant AAC in reconstituted proteoliposomes using a new rapid removal-stop method without relying on the inhibitor stop which can be compromised by mutations . The basic electroneutral ADP/ADP exchange activity is strongly or totally suppressed in six out of seven of these mutations, with the exception of R294A, which retains nearly wild-type activity . Carboxyatractylate (CAT) inhibits the ADP/ATP exchange rate only to 3-10% in wild type and R294A and up to 40% in other mutants, whereas bongkrekic acid (BKA) inhibits 50% (wild type and R294A) and 90% (other mutants) . Consequently, AAC is preferentially reconstituted with the matrix surface outside . All these mutations drastically change activity distribution among the four exchange modes ADP/ADP, ADP/ATP, ATP/ADP, and ATP/ATP . Whereas in wild-type AAC the homo ATP/ATP exchange is twice as high as the ADP/ADP exchange, in mutants it is 10 to 15 times lower . Similarly, the hetero ATP/ADP exchange in wild-type AAC is higher than the ADP/ ATP exchange, but in mutants it is several times lower . Thus, these mutations afflict the ATP-linked modes, in particular those linked to external ATP . The inhibition of oxidative phosphorylation is thus explained by the suppression of ATP export versus ADP import mode . The "extra"-inhibition of oxidative phosphorylation in mutant cells is explained by the extreme shift in mutants in favour of ATP import versus ADP export . Besides structural changes, the mutant effects indicate electrostatic interactions of these arginines with the anionic substrates . The loss of one positive charge raises the translocation barrier the more negative the substrate, i.e . more for ATP4- than for ADP3- . In none of these arginine mutants was the binding of CAT or BKA abolished.

Biochemistry, 1996 Dec 17, 35(50), 16132 - 43
Probing the role of positive residues in the ADP/ATP carrier from yeast . The effect of six arginine mutations of oxidative phosphorylation and AAC expression; Muller V et al.; ADP/ATP transport is the terminal step of oxidative phosphorylation in mitochondria . In this paper seven mutants of AAC2 from Saccharomyces cerevisiae are studied on the cellular and mitochondrial level . Six conspicuously located arginines were mutated into mostly neutral residues {Nelson, D . R., Lawson, J . E., Klingenberg, M., & Douglas, M . G . (1993) J . Mol . Biol . 230, 1159-1170} . R96A, R96H, R204L, and R294A are located in the second transmembrane helix of each repeat while R252I, R253I, and R254I are in the arginine triplet of the last domain . All six arginine residues are conserved in all known ADP/ATP carrier sequences . At the cellular level, oxidative phosphorylation in R96H and R294A retains 8% of the wild-type rate, but it is virtually zero in the other mutants . However, cytochrome c, a parameter of oxidative capacity, remains at 4-42% of wt . The weak coordination of respiratory chain and AAC expression indicates that respiration is needed also for other purposes . In mitochondria the AAC-linked ATP synthesis is measured and segregated by using the AAC inhibitor bongkrekate (BKA) . Only the R96H and R294A mutants express a significant rate of AAC-dependent ATP synthesis amounting to 2-18% of the plasmid-borne wild-type AAC2 mitochondria . In all other mutants it is virtually zero . However, respiratory capacity and cytochrome c content are reduced only by 20-70% . Whereas in immunoblots the presence of AAC is detected in all mutant mitochondria, by quantitative ELISA no AAC can be measured down to 0.05 mumol of AAC dimer/g of protein in R96A and R204L, whereas in R96H, R2521, R2531, and R254I the content is around 0.2 and in R294A the content is 0.46 as compared to 0.6 in the plasmid wild type . Also the {3H}CAT and {3H}BKA binding is virtually zero in some mutants and closely parallels the ELISA-determined AAC content, indicating that the mutations did not affect the inhibitor binding site . The turnover of AAC {V(ATP)/AAC content} in oxidative phosphorylation is reduced to 10% or 20% except for the two intrahelical mutants R96H and R294A . In the three Arg triplet mutants, it is nearly zero . In conclusion, the first two intrahelical arginines R96 and R204, are essential for expression but probably also for the activity of AAC . R294A still retains good transport activity and remarkably high expression of AAC . All arginines in the triplet 252, 253, 254 are essential . Extrapolation of the in vitro phosphorylation rates to the cellular level by the cytochrome c factor reveals a large discrepancy to the in vivo rates in particular for R294A . This indicates that these mutations render the AAC more sensitive to the regulatory intracellular ATP/ADP ratio than the wt AAC.

Biochemistry, 1996 Dec 17, 35(50), 16125 - 31
Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence . 2 . Assignment of tryptophanyl residues of the carrier to the responses to specific ligands; Roux P et al.; Tryptophanyl substitution of the Saccharomyces cerevisiae adenine nucleotide carrier (Anc2p isoform) was not deleterious for the transport activity or the folding of the carrier {preceding paper by Le Saux et al . (1996) Biochemistry 35, 16116-16124} . Conformational changes of the isolated wild-type and Trp-substituted Anc2p variants, induced upon binding of specific substrates {adenosine triphosphate (ATP) or diphosphate (ADP)} or inhibitors {carboxyatractyloside (CATR) or bongkrekic acid (BA)}, were studied by measurement of intrinsic fluorescence . Titration of CATR and BA binding sites ended in the same number of sites, namely, 6-7 nmol/mg of wild-type and variant Anc2p . Isolated Anc2p in detergent presented similar emission spectra, suggesting that all tryptophanyl residues were in environments of similar hydrophobicity . Trp87 and Trp126 contributed largely and to a similar extent to the fluorescence enhancement observed in response to ATP binding, while Trp235 contributed negatively and to a small extent to the fluorescence change . Both Trp126 and Trp235, and to a lower extent Trp87, participate in the CATR-induced fluorescence decrease of Anc2p . Responses to BA binding were observed only in the presence of ATP; they consisted of a further fluorescence increase of the Anc2p.ATP complex, which was mainly due to Trp87 and Trp126, Trp235 being much less responsive . The different fluorescence responses of the three Trp residues of Anc2 variants to ATP, CATR, and BA are in agreement with distinct binding sites for these ligands and distinct conformations of the carrier protein recognizing specifically CATR or BA . A mechanistic model is proposed to interpret the transitions between the different conformational states of Anc2p.

EMBO J, 1996 Dec 16, 15(24), 7168 - 77
Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G; Tarun SZ Jr et al.; Although the cap structure and the poly(A) tail are on opposite ends of the mRNA molecule, previous work has suggested that they interact to enhance translation and inhibit mRNA degradation . Here we present biochemical data that show that the proteins bound to the mRNA cap (eIF-4F) and poly(A) tail (Pab1p) are physically associated in extracts from the yeast Saccharomyces cerevisiae . Specifically, we find that Pab1p co-purifies and co-immunoprecipitates with the eIF-4G subunit of eIF-4F . The Pab1p binding site on the recombinant yeast eIF-4G protein Tif4632p was mapped to a 114-amino-acid region just proximal to its eIF-4E binding site . Pab1p only bound to this region when complexed to poly(A) . These data support the model that the Pablp-poly(A) tail complex on mRNA can interact with the cap structure via eIF-4G.

EMBO J, 1996 Dec 16, 15(24), 7046 - 59
Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast; Peter M et al.; STE20 encodes a protein kinase related to mammalian p65Pak which functions in several signal transduction pathways in yeast, including those involved in pseudohyphal and invasive growth, as well as mating . In addition, Ste20 plays an essential role in cells lacking Cla4, a kinase with significant homology to Ste20 . It is not clear how the activity of Ste20 is regulated in response to these different signals in vivo, but it has been demonstrated recently that binding of the small GTP binding protein Cdc42 is able to activate Ste20 in vitro . Here we show that Ste20 functionally interacts with Cdc42 in a GTP-dependent manner in vivo: Ste20 mutants that can no longer bind Cdc42 were unable to restore growth of ste20 cla4 mutant cells . They were also defective for pseudohyphal growth and agar invasion, and displayed reduced mating efficiency when mated with themselves . Surprisingly, however, the kinase activity of such Ste20 mutants was normal when assayed in vitro . Furthermore, these alleles were able to fully activate the MAP kinase pathway triggered by mating pheromones in vivo, suggesting that binding of Cdc42 and Ste20 was not required to activate Ste20 . Wild-type Ste20 protein was visualized as a crescent at emerging buds during vegetative growth and at shmoo tips in cells arrested with alpha-factor . In contrast, a Ste20 mutant protein unable to bind Cdc42 was found diffusely throughout the cytoplasm, suggesting that Cdc42 is required to localize Ste20 properly in vivo.

Eur J Biochem, 1996 Dec 15, 242(3), 641 - 7
Pregnenolone-7 beta-hydroxylating activities of yeast-expressed mouse cytochrome P450-1A1 and mouse-tissue microsomes; Doostzadeh J et al.; In many tissues from different species, pregnenolone and dehydroepiandrosterone (DHEA) are hydroxylated mainly at the 7 alpha position by a cytochrome P450 (P450)-containing microsomal enzyme complex . In addition, 7-hydroxysteroids have been shown to activate immune processes in mice . The reported production of 7 beta-hydroxypregnenolone and 7 beta-hydroxy-DHEA was not supported by formal identification, and the P450 responsible for 7 alpha-hydroxylation and 7 beta-hydroxylation of pregnenolone and DHEA have not been identified . Based on results of analyses by crystallization to constant specific activity and gas chromatography/mass spectrometry, we report that mouse-liver and mouse-brain microsomes carried out 7 beta-hydroxylation of pregnenolone and DHEA, and that yeast-expressed mouse cytochrome P450-1A1 (P450 1A1) transformed pregnenolone into 7 beta-hydroxypregnenolone (Km = 25.1 +/- 0.4 microM, turnover number = 979 +/- 30 pmol.min-1.nmol-1 mouse P450 1A1) . Neither 7-hydroxy derivatives of DHEA nor 7 alpha-hydroxypregnenolone was produced by P450 1A1 . The presence of P450 1A1 in liver and brain microsomes was shown by Western blot analysis, and induction of mouse P450 1A1 by beta-naphthoflavone resulted in increased 7 beta-hydroxylation of pregnenolone in liver microsomes . Studies of the brain-microsome 7 beta-hydroxylating enzyme with pregnenolone or DHEA gave Km of 5.0 microM and 4.9 microM, respectively, and Vmax of 4.5 pmol.min-1.mg-1 and 6.1 pmol.min-1.mg-1, respectively, and showed the absence of cross-inhibitions between the two steroids . These findings indicate that, in addition to unidentified P450, P450 1A1 is involved in 7 beta-hydroxylation of pregnenolone and may contribute in part to the production of the 7-hydroxylated steroids necessary for activation of immune defenses.

Biochem J, 1996 Dec 15, 320 ( Pt 3), 769 - 75
Subunit VII of ubiquinol:cytochrome-c oxidoreductase from Neurospora crassa is functional in yeast and has an N-terminal extension that is not essential for mitochondrial targeting; Lobo-Hajdu G et al.; cDNA clones encoding subunit VII of the Neurospora crassa bc1 complex (ubiquinol:cytochrome-c oxidoreductase), which is homologous with subunit VIII of the complex from yeast (encoded by QCR8), were identified on the basis of functional complementation of a yeast QCR8 deletion strain . The clones contain an open reading frame encoding a protein with a calculated molecular mass of 11.8 kDa . The N-terminal eight residues of the amino acid sequence deduced from the cDNA clones are absent from the mature protein, as revealed by direct sequencing of the isolated protein . To investigate the potential role of the N-terminal octapeptide in mitochondrial targeting, constructs were made encoding the precursor and the mature form of subunit VII from Neurospora . Incubation of isolated mitochondria with the two proteins revealed that the N-terminal extension of the precursor is removed on import . However, the presequence does not encode information for targeting, as the proteins encoded by both constructs can be imported into isolated mitochondria with equal efficiency . In contrast, the octapeptide seems to have functional importance: the defect in the yeast qcr8-null mutant is not complemented on transformation with the construct encoding mature subunit VII from N . crassa in a single-copy plasmid . We therefore speculate that the N-terminal extension plays a role in intramitochondrial sorting of N . crassa subunit VII . This is supported by the fact that the subunit VII precursor is processed by a protease other than the general mitochondrial processing peptidase . Interestingly, the presequence of N . crassa subunit VII has an amino acid composition similar to the octapeptides cleaved off by the mitochondrial intermediate peptidase.

Experientia, 1996 Dec 15, 52(12), 1148 - 57
Molecular genetics of the peptidyl transferase center and the unusual Var1 protein in yeast mitochondrial ribosomes; Mason TL et al.; Mitochondria possess their own ribosomes responsible for the synthesis of a small number of proteins encoded by the mitochondrial genome . In yeast, Saccharomyces cerevisiae, the two ribosomal RNAs and a single ribosomal protein, Var1, are products of mitochondrial genes, and the remaining approximately 80 ribosomal proteins are encoded in the nucleus . The mitochondrial translation system is dispensable in yeast, providing an excellent experimental model for the molecular genetic analysis of the fundamental properties of ribosomes in general as well as adaptations required for the specialized role of ribosomes in mitochondria . Recent studies of the peptidyl transferase center, one of the most highly conserved functional centers of the ribosome, and the Var1 protein, an unusual yet essential protein in the small ribosomal subunit, have provided new insight into conserved and divergent features of the mitochondrial ribosome.

Experientia, 1996 Dec 15, 52(12), 1136 - 47
Nuclear organization and transcriptional silencing in yeast; Gotta M et al.; Transcriptional repression at the yeast silent mating type loci requires the formation of a nucleoprotein complex at specific cis-acting elements called silencers, which in turn promotes the binding of a histone-associated Sir-protein complex to adjacent chromatin . A similar mechanism of long-range transcriptional repression appears to function near telomeric repeat sequences, where it has been demonstrated that Sir3p is a limiting factor for the propagation of silencing . A combined immunofluorescence/in situ hybridization method for budding yeast was developed that maintains the three-dimensional structure of the nucleus . In wild-type cells the immunostaining of Sir3p, Sir4p and Rap1 colocalizes with Y' subtelomeric sequences detected by in situ hybridization . All three antigens and the subtelomeric in situ hybridization signals are clustered in foci, which are often adjacent to, but not coincident with, nuclear pores . This colocalization of Rap1, Sir3p and Sir4p with telomeres is lost in sir mutants, and also when Sir4p is overexpressed . To test whether the natural positioning of the two HM loci, located roughly 10 and 25 kb from the ends of chromosome III, is important for silencer function, a reporter gene flanked by wild-type silencer elements was integrated at various internal sites on other yeast chromosomes . We find that integration at internal loci situated far from telomeres abrogates the ability of silencers to repress the reporter gene . Silencing can be restored by creation of a telomere at 13 kb from the reporter construct, or by insertion of 340 bp of yeast telomeric repeat sequence at this site without chromosomal truncation . Elevation of the internal nuclear pools of Sir1p, Sir3p and Sir4p can relieve the lack of repression at the LYS2 locus in an additive manner, suggesting that in wild-type cells silencer function is facilitated by its juxtaposition to a pool of highly concentrated Sir proteins, such as those created by telomere clustering.

Experientia, 1996 Dec 15, 52(12), 1130 - 5
Translational control of endogenous and recoded nuclear genes in yeast mitochondria: regulation and membrane targeting; Fox TD; Mitochondrial gene expression in yeast, Saccharomyces cerevisiae, depends on translational activation of individual mRNAs by distinct proteins encoded in the nucleus . These unclearly coded mRNA-specific translational activators are bound to the inner membrane and function to mediate the interaction between mRNAs and mitochondrial ribosomes . This complex system, found to date only in organelles, appears to be an adaptation for targeting the synthesis of mitochondrially coded integral membrane proteins to the membrane . In addition, mRNA-specific translational activation is a rate-limiting step used to modulate expression of at least one mitochondrial gene in response to environmental conditions . Direct study of mitochondrial gene regulation and the targeting of mitochondrially coded proteins in vivo will now be possible using synthetic genes inserted into mtDNA that encode soluble reporter/passenger proteins.

Experientia, 1996 Dec 15, 52(12), 1101 - 10
Vacuolar H(+)-ATPase: from mammals to yeast and back; Nelson N et al.; Vacuolar H(+)-adenosine triphosphatase (V-ATPase) is composed of distinct catalytic (V1) and membrane (V0) sectors containing several subunits . The biochemistry of the enzyme was mainly studied in organelles from mammalian cells such as chromaffin granules and clathrin-coated vesicles . Subsequently, mammalian cDNAs and yeast genes encoding subunits of V-ATPase were cloned and sequenced . The sequence information revealed the relation between V- and F-ATPase that evolved from a common ancestor . The isolation of yeast genes encoding subunits of V-ATPase opened an avenue for molecular biology studies of the enzyme . Because V-ATPase is present in every known eukaryotic cell and provides energy for vital transport systems, it was anticipated that disruption of genes encoding V-ATPase subunits would be lethal . Fortunately, yeast cells can survive the absence of V-ATPase by 'drinking' the acidic medium . So far only yeast cells have been shown to be viable without an active V-ATPase . In contrast to yeast, mammalian cells may have more than one gene encoding each of the subunits of the enzyme . Some of these genes encode tissue- and/or organelle-specific subunits . Expression of these specific cDNAs in yeast cells may reveal their unique functions in mammalian cells . Following the route from mammals to yeast and back may prove useful in the study of many other complicated processes.

Exp Cell Res, 1996 Dec 15, 229(2), 181 - 8
Yeast pre-mRNA is composed of two populations with distinct kinetic properties; Elliott DJ et al.; As an approach to the study of yeast pre-mRNA splicing in vivo, we have examined properties of transcripts derived from a gal-UAS intron-containing fusion gene encoding RP51A and a series of its derivatives . RNA half-life measurements were carried out after transcription initiation was blocked by the addition of glucose . Pre-mRNA encoded by GalRP51A decayed with a half-life of approximately 6 min and was substantially polyadenylated, and transcripts derived from a nonspliced version of the same gene decayed with a similar half-life ( approximately 4 min) . A comparison of the steady-state levels of these two transcripts suggests that the bulk of GalRP51A pre-mRNA is processed much more rapidly, with an average lifetime of about 2 s . We propose that this inferred population of rapidly processed molecules is spliced cotranscriptionally and that it is the principal precursor to GalRP51A mRNA . Although the pre-mRNA molecules detected are therefore unlikely to be the major splicing precursors, an in vivo assay suggests that they are likely to have bound splicing factors . They must then be spliced much more slowly than most primary transcripts, or not spliced at all and then degraded through a different cellular pathway . As a result of its comparatively long lifetime, this minor fraction of the pre-mRNA population makes up the majority of the steady-state level of GalRP51A pre-mRNA.

Genes Dev, 1996 Dec 15, 10(24), 3105 - 15
Glucose regulates protein interactions within the yeast SNF1 protein kinase complex; Jiang R et al.; The SNF1 protein kinase is broadly conserved in eukaryotes and has been implicated in responses to environmental and nutritional stress . In yeast, the SNF1 kinase has a central role in the response to glucose starvation . SNF1 is associated with its activating subunit, SNF4, and other proteins in complexes . Using the two-hybrid system, we show that interaction between SNF1 and SNF4 is strongly regulated by the glucose signal . Moreover, this interaction is appropriately affected by mutations in regulators, including protein phosphatase 1 . We show that SNF4 binds to the SNF1 regulatory domain in low glucose, whereas in high glucose the regulatory domain binds to the kinase domain of SNF1 itself . Genetic analysis further suggests that the SNF1 regulatory domain autoinhibits the kinase activity and that in low glucose SNF4 antagonizes this inhibition . Finally, these interactions have been conserved from yeast to plants, indicating that homologs of the SNF1 kinase complex respond to regulatory signals by analogous mechanisms.

Genomics, 1996 Dec 15, 38(3), 421 - 4
Isolation, sequencing, and mapping of the human homologue of the yeast transcription factor, SPT5; Chiang PW et al.; We isolated the human homologue, SUPT5H, of the yeast transcription factor, SPT5 . The human homologue is 1088 aa long compared to 1063 aa for the yeast gene . SUPT5H maps to 19q13, near the ryanodine receptor . Like its family member, SUPT6H, and like yeast SPT5, SUPT5H has a very acidic 5' domain . Like its family member, SUPT6H, but unlike yeast SPT5 or SPT6, SUPT5H has seven MAP kinase sites at its 5' end . In addition, SUPT5H lacks the novel 6-amino-acid repeat (consensus is S-T/A-W-G-G-A/Q) at the C-terminus of yeast SPT5 . This argues that while there is functional similarity between SPT5 and SUPT5H, the molecules differ in the signals to which they respond.

Anal Biochem, 1996 Dec 15, 243(2), 270 - 6
An in vitro transcription assay for yeast mitochondria using organellar lysates; Iqbal J et al.; The nuclear gene NUC1 encodes the major mitochondrial (mt) ribonuclease in the yeast Saccharomyces cerevisiae . We describe an in vitro mt transcription assay system based on lysates of purified mitochondria from a petite (rho-, mt deletion mutant) yeast strain in which NUC1 has been insertionally inactivated . Control in vitro run-on transcription assays using intact mitochondria demonstrate that the rate of incorporation of labeled precursor into mt RNA is identical in organelles from the nuc1 rho- mutant and its otherwise isochromosomal NUC1 parent strain . Brij-35 lysates of mitochondria from the nuc1 strain incorporate precursor into mt RNA at nearly the same rate as do intact organelles from that strain, while similar mt lysates from NUC1 cells show no such incorporation . Other control studies show that mt lysates from the nuc1 strain retain functional mt cAMP-dependent protein kinase and other critical activities . When the cloned template DNA encoding the yeast mt 21S rRNA gene, which is not retained in the nuc1 rho- strain, is added to mt lysates from that strain, transcripts are produced from the template under standard assay conditions.

Mol Gen Genet, 1996 Dec 13, 253(3), 393 - 6
Measuring the toxic effects of high gene dosage on yeast cells; Daniel J; A novel method, which is rapid, reliable and quantitative, is presented for measuring the toxic effects on yeast cells of high dosage of any given gene . It is based on the possibility of monitoring the presence in cells of a plasmid carrying the ADE2 gene from Saccharomyces cerevisiae by direct observation of colonies, the construction of this particular plasmid being easily made by marked homologous recombination in yeast . Four yeast regulatory genes tested were found to result in various degrees of toxicity at high dosage . Possible implications of the measurement of gene toxicity for eukaryotic cell regulatory mechanisms and for the use of novel general approaches to gene selection, such as the gene-gene interference method, are discussed.

Mol Gen Genet, 1996 Dec 13, 253(3), 278 - 88
A meiosis-specific protein kinase, Ime2, is required for the correct timing of DNA replication and for spore formation in yeast meiosis; Foiani M et al.; In this report we study the regulation of premeiotic DNA synthesis in Saccharomyces cerevisiae . DNA replication was monitored by fluorescence-activated cell sorting analysis and by analyzing the pattern of expression of the DNA polymerase alpha-primase complex . Wild-type cells and cells lacking one of the two principal regulators of meiosis, Ime1 and Ime2, were compared . We show that premeiotic DNA synthesis does not occur in ime1 delta diploids, but does occur in ime2 delta diploids with an 8-9 h delay . At late meiotic times, ime2 delta diploids exhibit an additional round of DNA synthesis . Furthermore, we show that in wild-type cells the B-subunit of DNA polymerase alpha is phosphorylated during premeiotic DNA synthesis, a phenomenon that has previously been reported for the mitotic cell cycle . Moreover, the catalytic subunit and the B-subunit of DNA polymerase alpha are specifically degraded during spore formation . Phosphorylation of the B-subunit does not occur in ime1 delta diploids, but does occur in ime2 delta diploids with an 8-9 h delay . In addition, we show that Ime2 is not absolutely required for commitment to meiotic recombination, spindle formation and nuclear division, although it is required for spore formation.

Cell, 1996 Dec 13, 87(6), 1115 - 22
Induction of large DNA palindrome formation in yeast: implications for gene amplification and genome stability in eukaryotes; Butler DK et al.; Many amplified genes, including some oncogenes, are organized as large inverted repeats . How such giant palindromes are generated remains largely unknown . Recent studies of a palindrome in the ciliate Tetrahymena suggest a novel mechanism that requires chromosome breakage next to short inverted repeats . The prevalence of short inverted repeats in eukaryotic genomes raises the interesting possibility that this process may occur widely as a response to chromosome damage . Here we demonstrate that in Saccharomyces cerevisiae, large DNA palindromes are formed efficiently, probably by intramolecular recombination, when a double-strand break is introduced next to short inverted repeats . These results suggest a general mechanism for large palindromic DNA formation and reveal an important new source of genome instability resulting from chromosome breakage at selective sites.

Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 518 - 23
Inhibition of progesterone receptor activity in yeast by synthetic chemicals; Tran DQ et al.; Numerous synthetic chemicals have estrogenic activity by interacting with the estrogen receptor . In this report, we test the hypothesis that some estrogenic chemicals may also modulate the human progesterone receptor (hPR) signaling pathway . This was evaluated by examining synthetic chemicals for their ability to modulate the activity of hPR expressed in yeast . The transcriptional activity of hPR was not increased in the presence of several synthetic chemicals . However, the estrogenic chemicals p-nonylphenol and 4-tert-octyphenol, and pentachlorophenol, effectively inhibited the activity of the hPR in yeast . Competition binding studies indicated these chemicals effectively competed with radiolabeled R5020, a synthetic progestin, for binding to the hPR in yeast . These results indicate that some synthetic chemicals directly inhibit the activity of hPR in yeast . The observations that some estrogenic chemicals can also inhibit hPR activity suggest a potential mechanism for the reported potent estrogenic activities of these chemicals.

J Biol Chem, 1996 Dec 13, 271(50), 32064 - 72
Phosphorylation of yeast plasma membrane H+-ATPase by casein kinase I; Estrada E et al.; The plasma membrane H+-ATPase of Saccharomyces cerevisiae is subject to phosphorylation by a casein kinase I activity in vitro . We show this casein kinase I activity to result from the combined function of YCK1 and YCK2, two highly similar and plasma membrane-associated casein kinase I homologues . First, H+-ATPase phosphorylation is severely impaired in the plasma membrane of YCK-deficient yeast strains . Furthermore, the wild-type level of the phosphoprotein is restored by the addition of purified mammalian casein kinase I to the mutant membranes . We used the H+-ATPase as well as a synthetic peptide substrate that contains a phosphorylation site for casein kinase I to compare kinase activity in membranes prepared from yeast cells grown in the presence or absence of glucose . The addition of glucose results in increased H+-ATPase activity which is associated with a decline in the phosphorylation level of the enzyme . Mutations in both YCK1 and YCK2 affect this regulation, suggesting that H+-ATPase activity is modulated by glucose via a combination of a "down-regulating" casein kinase I activity and another, yet uncharacterized, "up-regulating" kinase activity . Biochemical mapping of phosphorylated H+-ATPase identifies a major phosphopeptide that contains a consensus phosphorylation site (Ser-507) for casein kinase I . Site-directed mutagenesis of this consensus sequence indicates that Glu-504 is important for glucose-induced decrease in the apparent Km for ATP.

Gene, 1996 Dec 12, 183(1-2), 7 - 14
Mammalian homologues of yeast vacuolar protein sorting (vps) genes implicated in Golgi-to-lysosome trafficking; Pevsner J et al.; Sec1p, Vps33p, Vps45p and Sly1p constitute a family of proteins implicated in vesicle trafficking at distinct stages of the yeast secretory pathway . Several mammalian homologues of Sec1p have been described, including n-sec1 which has been implicated in the regulation of synaptic vesicle docking at the nerve terminal . We have characterized cDNA clones encoding three additional mammalian homologues belonging to this family: r-vps33a and r-vps33b from rat, which are 30 and 26% identical to yeast Vps33p, respectively, and h-vps45 from human which is 38% identical to yeast Vps45p at the amino acid (aa) level . Phylogenetic analysis of 16 Sec1p-related proteins from several species is consistent with the hypothesis that the evolution of this gene family parallels the specialization of vesicle trafficking to distinct intracellular compartments . By Northern analysis, each of these genes is expressed in all tissues examined (brain, spleen, lung, liver, skeletal muscle, kidney, testis) . While n-sec1 binds syntaxin 1a, 2, and 3, r-vps33a, r-vps33b and h-vps45 do not bind any of the known syntaxins . We propose that the three proteins bind as yet unidentified syntaxin homologues involved in vesicle trafficking between the Golgi apparatus, prelysosomal compartment(s), and the lysosome.

Nature, 1996 Dec 12, 384(6609), 589 - 91
The histone deacetylase RPD3 counteracts genomic silencing in Drosophila and yeast; De Rubertis F et al.; Both position-effect variegation (PEV) in Drosophila and telomeric position-effect in yeast (TPE) result from the mosaic inactivation of genes relocated next to a block of centromeric heterochromatin or next to telomeres . In many aspects, these phenomena are analogous to other epigenetic silencing mechanisms, such as the control of homeotic gene clusters, X-chromosome inactivation and imprinting in mammals, and mating-type control in yeast . Dominant mutations that suppress or enhance PEV are thought to encode either chromatin proteins or factors that directly affect chromatin structure . We have identified an insertional mutation in Drosophila that enhances PEV and reduces transcription of the gene in the eye-antenna imaginal disc . The gene corresponds to that encoding the transcriptional regulator RPD3 in yeast, and to a human histone deacetylase . In yeast, RRD3-deletion strains show enhanced TPE, suggesting a conserved role of the histone deacetylase RPD3 in counteracting genomic silencing . This function of RPD3, which is in contrast to the general correlation between histone acetylation and increased transcription, might be due to a specialized chromatin structure at silenced loci.

Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14654 - 8
Yeast homologues of higher eukaryotic TFIID subunits; Moqtaderi Z et al.; In eukaryotic cells the TATA-binding protein (TBP) associates with other proteins known as TBP-associated factors (TAFs) to form multisubunit transcription factors important for gene expression by all three nuclear RNA polymerases . Computer searching of the complete Saccharomyces cerevisiae genome revealed five previously unidentified yeast genes with significant sequence similarity to known human and Drosophila RNA polymerase II TAFs . Each of these genes is essential for viability . A sixth essential gene (FUN81) has previously been noted to be similar to human TAFII18 . Coimmunoprecipitation experiments show that all six proteins are associated with TBP, demonstrating that they are true TAFs . Furthermore, these proteins are present in complexes containing the TAFII130 subunit, indicating that they are components of TFIID . Based on their predicted molecular weights, these genes have been designated TAF67, TAF61(68), TAF40, TAF23(25), TAF19(FUN81), and TAF17 . Yeast TAF61 is significantly larger than its higher eukaryotic homologues, and deletion analysis demonstrates that the evolutionarily conserved, histone-like domain is sufficient and necessary to support viability.

Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14503 - 8
HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription; Rundlett SE et al.; Increased histone acetylation has been correlated with increased transcription, and regions of heterochromatin are generally hypoacetylated . In investigating the cause-and-effect relationship between histone acetylation and gene activity, we have characterized two yeast histone deacetylase complexes . Histone deacetylase-A (HDA) is an approximately 350-kDa complex that is highly sensitive to the deacetylase inhibitor trichostatin A . Histone deacetylase-B (HDB) is an approximately 600-kDa complex that is much less sensitive to trichostatin A . The HDA1 protein (a subunit of the HDA activity) shares sequence similarity to RPD3, a factor required for optimal transcription of certain yeast genes . RPD3 is associated with the HDB activity . HDA1 also shares similarity to three new open reading frames in yeast, designated HOS1, HOS2, and HOS3 . We find that both hda1 and rpd3 deletions increase acetylation levels in vivo at all sites examined in both core histones H3 and H4, with rpd3 deletions having a greater impact on histone H4 lysine positions 5 and 12 . Surprisingly, both hda1 and rpd3 deletions increase repression at telomeric loci, which resemble heterochromatin with rpd3 having a greater effect . In addition, rpd3 deletions retard full induction of the PHO5 promoter fused to the reporter lacZ . These data demonstrate that histone acetylation state has a role in regulating both heterochromatic silencing and regulated gene expression.

Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14440 - 5
Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels; Shevchenko A et al.; The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome . However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology . Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome . As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein . Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy . The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags . In blind trials, the method led to unambiguous identification in all cases . In the largest individual protein identification project to date, a total of 150 gel spots-many of them at subpicomole amounts-were successfully analyzed, greatly enlarging a yeast two-dimensional gel data base . More than 32 proteins were novel and matched to previously uncharacterized open reading frames in the yeast genome . This study establishes that mass spectrometry provides the required throughput, the certainty of identification, and the general applicability to serve as the method of choice to connect genome and proteome.

Biochemistry, 1996 Dec 10, 35(49), 15791 - 9
A complete mechanism for steady-state oxidation of yeast cytochrome c by yeast cytochrome c peroxidase; Miller MA; Steady-state oxidation of yeast cytochrome c (yCc) was monitored as a function of ionic strength (mu) for mutants of a cloned cytochrome c peroxidase {CcP(MI)} . The data are best interpreted in the context of a two binding site model, where the affinity of the two sites for yCc differs by approximately 1000-fold and rapid intracomplex electron transfer (ET) occurs only at the high-affinity site identified in the crystal structure . At low mu, catalysis is apparently limited by the rate of yCc dissociation from the reactive high-affinity site (koff) . Binding of yCc at the low-affinity site increases koff and therefore increases the rate of catalysis . Mutations at the high-affinity site also increase the rate of catalysis by the 1:1 CcP(MI):yCc complex by increasing koff . Mutations at residues that interact strongly with yCc at the high-affinity site (Asp 34, Glu 290, and Ala 193) cause the greatest increase in koff (25-38-fold at mu = 20 mM) . Mutations at residues that interact less strongly with yCc (Glu 32 and Glu 291) cause smaller increases in koff (10- and 3-fold, respectively, at mu = 20 mM) . The results provide additional evidence that the high-affinity site formed in solution is similar to the one identified in the crystal structure and that yCc dissociation from this site limits enzyme turnover at low ionic strength . Numerical integration simulations show that the model accurately predicts enzyme turnover rates at the high-affinity site, using published rate constants for the elementary reaction steps.

J Biol Chem, 1996 Dec 6, 271(49), 31021 - 4
Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase; Finegold AA et al.; A plasma membrane iron reductase, required for cellular iron acquisition by Saccharomyces cerevisiae, and the human phagocytic NADPH oxidase, implicated in cellular defense, contain low potential plasma membrane b cytochromes that share elements of structure and function . Four critical histidine residues in the FRE1 protein of the iron reductase were identified by site-directed mutagenesis . Individual mutation of each histidine to alanine eliminated the entire heme spectrum without affecting expression of the apoprotein, documenting the specificity of the requirement for the histidine residues . These critical residues are predicted to coordinate a bis-heme structure between transmembrane domains of the FRE1 protein . The histidine residues are conserved in the related gp91(phox) protein of the NADPH oxidase of human granulocytes, predicting the sites of heme coordination in that protein complex . Similarly spaced histidine residues have also been implicated in heme binding by organelle b cytochromes with little overall sequence similarity to the plasma membrane b cytochromes . This bis-heme motif may play a role in transmembrane electron transport by distinct families of polytopic b cytochromes.

Gene, 1996 Dec 5, 182(1-2), 89 - 96
Analysis of yeast trimethylguanosine-capped RNAs by midwestern blotting; Rasmussen TP et al.; Immuno-detection by 'Midwestern' blotting provides a simple way to identify trimethylguanosine (TMG) capped RNAs . With this technique, over 20 bands are observed when total cellular RNA from Saccharomyces cerevisiae is transferred to a nylon membrane and probed with anti-TMG antibodies . Most, if not all, species known to contain a TMG cap are detected by this method . Only TMG-capped RNAs are detected on Midwestern blots unlike anti-TMG immunoprecipitates . Midwestern blotting is a useful alternative to immunoprecipitation and Northern analysis and may prove to be a better method for determining the relative abundance of capped RNAs . The blots can be reprobed multiple times with labeled antisense oligonucleotides to determine the identity of any TMG-capped species for which the primary sequence or a clone is available . This dual detection capability provides a powerful tool for the analysis of TMG-capped snRNAs and snoRNAs.

Nature, 1996 Dec 5, 384(6608), 479 - 81
Nucleotide exchange on ARF mediated by yeast Gea1 protein; Peyroche A et al.; The ADP-ribosylation factor ARF is a small GTP-binding protein that is involved in the transport of vesicles between the endoplasmic reticulum (ER) and Golgi complex and within the Golgi complex itself . ARF cycles between inactive and membrane-associated active forms as a result of exchange of bound GDP for GTP; the GTP-bound form is an essential participant in the formation of transport vesicles . This nucleotide exchange is inhibited by the fungal metabolite brefeldin A (BFA) . Here we identify a protein (Gea1) from Saccharomyces cerevisiae that is a component of a complex possessing guanine-nucleotide-exchange activity for ARF . We show that the activity of the complex is sensitive to brefeldin A and that Gea1 function is necessary for ER-Golgi transport in vivo . Gea1 contains a domain that is similar to a domain of Sec7, a protein necessary for intra-Golgi transport . We propose that Gea1 and ARNO, a human protein with a homologous Sec7 domain, are members of a new family of ARF guanine-nucleotide exchange factors.

Biochim Biophys Acta, 1996 Dec 4, 1285(2), 175 - 82
H+/K+ exchange in reconstituted yeast plasma membrane vesicles; Ramirez J et al.; The activity of a putative H+/K+ exchange system in the plasma membrane of yeast was studied following the alkalinization of the interior of vesicles prepared with lecithin and yeast plasma membrane containing pyranine entrapped inside . The fluorescence of pyranine was used as an indicator of the internal pH of the vesicles . The addition of monovalent cations produced an increase of the internal pH, probably due to the activity of an exchange system, allowing H+ to leave the vesicle in an exchange for the cation added . The system showed partial selectivity towards K+ against other monovalent cations, and it was inhibited by amiloride . The activity of this system required the presence of the yeast plasma membrane in the vesicles, and it did not produce important changes of the membrane potential of the vesicles . The exchange depended partially on the relative values of the internal and the external pH of the vesicles . The system shows low affinity for the cations, and appears to be different from the mitochondrial H+/K+ exchange system, which is non-selective toward the different monovalent cations . This system could be involved in the regulation of the internal pH of the cells when they accumulate high concentrations of K+.

Biochem Biophys Res Commun, 1996 Dec 4, 229(1), 102 - 8
The Anti-estrogenic Activity of Selected Polynuclear Aromatic Hydrocarbons in Yeast Expressing Human Estrogen Receptor
Tran DQ, Ide CF, Mclachlan JA, Arnold SF.
Polynuclear aromatic hydrocarbons (PAH) represent a large class of chemicals present in the environment . We have used yeast strain ER(wt) expressing human estrogen receptor (hER) and an estrogen-sensitive reporter to characterize the estrogenic or anti-estrogenic activities of 21 PAHs . The PAHs did not exhibit estrogenic activity in yeast strain ER(wt) . Four of the PAHs, dibenz{a,h}anthracene, 6-hydroxy-chrysene, 2,3-benzofluorene, and benzo(a)pyrene, inhibited estradiol-dependent reporter activity in strain ER(wt) . A mutant hER lacking the amino terminus expressed in yeast was inhibited by the four PAHs to a lesser extent than the full-length hER . 6-hydroxy-chrysene, 2,3-benzofluorene, and benzo(a)pyrene, but not dibenz{a,h}anthracene, weakly displaced {3H}estradiol from the hER in a competition binding assay . A strong correlation between the inhibition of {3H}estradiol-binding from the hER and the reduction of hER-mediated transactivation in yeast was not observed . These observations suggest that the PAHs dibenz{a,h}anthracene, 6-hydroxy-chrysene, 2,3-benzofluorene, and benzo(a)pyrene may antagonize estradiol activity in yeast by binding to an anti-estrogen binding site on the hER or by mechanisms independent of the hER.

Biochem Biophys Res Commun, 1996 Dec 4, 229(1), 134 - 8
Molecular cloning of a novel rat salt-tolerant protein by functional complementation in yeast; Tsuji E et al.; To elucidate the genetic basis of salt-sensitivity in mammalian hypertension, we isolated six rat complementary DNAs by functional complementation in yeast . These genes were able to substitute for the salt-tolerant activity of HALI which confers salt tolerance by modulating the cation transport system in yeast . We identified these genes as beta-globin, lambda-crystallin, androgen-regulated protein, mitochondrial cytochrome b, a homologue of infant brain cDNA, and a novel gene, called salt-tolerant protein (STP) . STP contains 1964 bp nucleotides and an open reading frame which encodes 496 amino acid residues . Northern blot analysis showed that STP mRNA is expressed in various rat tissues.

Biochem Biophys Res Commun, 1996 Dec 4, 229(1), 101 - 8
The anti-estrogenic activity of selected polynuclear aromatic hydrocarbons in yeast expressing human estrogen receptor; Tran DQ et al.; Polynuclear aromatic hydrocarbons (PAH) represent a large class of chemicals present in environment . We have used yeast strain ER(wt) expressing human estrogen receptor (hER) and an estrogen-sensitive reporter to characterize the estrogenic or anti-estrogenic activities of 21 PAHs . The PAHs did not exhibit estrogenic activity in yeast strain ER(wt) . Four of the PAHs, dibenz{a,h}anthracene, 6-hydroxy-chrysene, 2,3-benzofluorene, and benzo(a)pyrene, inhibited estradiol-dependent reporter activity in strain ER(wt) . A mutant hER lacking the amino terminus expressed in yeast was inhibited by the four PAHs to a lesser extent than the full-length hER . 6-hydroxy-chrysene, 2,3-benzofluorene, and benzo(a)pyrene, but not dibenz{a,h}anthracene, weakly displaced {3H}estradiol from the hER in a competition binding assay . A strong correlation between the inhibition of {3H}estradiol-binding from the hER and the reduction of hER-mediated transactivation in yeast was not observed . These observations suggest that the PAHs dibenz{a,h}-anthracene, 6-hydroxy-chrysene, 2,3-benzofluorene, and benzo(a)pyrene may antagonize activity in yeast by binding to an anti-estrogen binding site on the hER or by mechanisms independent of the hER.

Biochemistry, 1996 Dec 3, 35(48), 15322 - 31
Functional replacement of hamster lysyl-tRNA synthetase by the yeast enzyme requires cognate amino acid sequences for proper tRNA recognition; Agou F et al.; We cloned the cDNA encoding a 597-aa hamster lysyl-tRNA synthetase . This enzyme is a close homologue of the 591-aa Saccharomyces cerevisiae enzyme, with the noticeable exception of their 60-aa N-terminal regions, which differ significantly . Several particular features of this polypeptide fragment from the hamster lysyl-tRNA synthetase suggest that it is implicated in the assembly of that enzyme within the multisynthetase complex . However, we show that this protein domain is dispensable in vivo to sustain growth of CHO cells . The cross-species complementation was investigated in the lysine system . The mammalian enzyme functionally replaces a null-allele of the yeast KRS1 gene . Conversely, the yeast enzyme cannot rescue Lys-101 cells, a CHO cell line with a temperature-sensitive lysyl-tRNA synthetase . The yeast and mammalian enzymes, overexpressed in yeast, were purified to homogeneity . The hamster lysyl-tRNA synthetase efficiently aminoacylates both mammalian and yeast tRNA(Lys), whereas the yeast enzyme aminoacylates mammalian tRNA(Lys) with a catalytic efficiency 20-fold lower, as compared to its cognate tRNA . The 152-aa C-terminus extremity of the hamster enzyme provides the yeast enzyme with the capacity to complement Lys-101 cells . This hybrid protein is fairly stable and aminoacylates both yeast and mammalian tRNA(Lys) with similar catalytic efficiencies . Because this C-terminal polypeptide fragment is likely to make contacts with the acceptor stem of tRNA(Lys), we conclude that it should carry the protein determinants conferring specific recognition of the cognate tRNA acceptor stem and therefore contributes an essential role in the operational RNA code for amino acids.

EMBO J, 1996 Dec 2, 15(23), 6605 - 16
The fission yeast dma1 gene is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is compromised; Murone M et al.; Premature initiation of cytokinesis can lead to loss of chromosomes, and 'cutting' of the nucleus . Therefore, the proper spatial and temporal co-ordination of mitosis and cytokinesis is essential for maintaining the integrity of the genome . The fission yeast cdc16 gene is implicated both in the spindle assembly checkpoint and control of septum formation . To identify other proteins involved in these controls, we have isolated multicopy suppressors of the cdc16-116 mutation, and the characterization of one of these, dma1 (defective in mitotic arrest), is presented here . dma1 is not an essential gene, but in a dma1 null background (dma1-D1) the function of the spindle assembly checkpoint is compromised . If assembly of the spindle is prevented, dma1-D1 cells do not arrest, the activity of cdc2 kinase decays and cells form a division septum without completing a normal mitosis . dma1-D1 cells also show an increased rate of chromosome loss during exponential growth . Upon ectopic expression from an inducible promoter, dma1p delays progress through mitosis and inhibits septum formation, giving rise to elongated, multinucleate cells . We propose that dma1 is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is impaired.

EMBO J, 1996 Dec 2, 15(23), 6575 - 83
Yeast Gpi8p is essential for GPI anchor attachment onto proteins; Benghezal M et al.; Glycosylphosphatidylinositol (GPI) anchors are added onto newly synthesized proteins in the ER . Thereby a putative transamidase removes a C-terminal peptide and attaches the truncated protein to the free amino group of the preformed GPI . The yeast mutant gpi8-1 is deficient in this addition of GPIs to proteins . GPI8 encodes for an essential 47 kDa type I membrane glycoprotein residing on the luminal side of the ER membrane . GPI8 shows significant homology to a novel family of vacuolar plant endopeptidases one of which is supposed to catalyse a transamidation step in the maturation of concanavalin A and acts as a transamidase in vitro . Humans have a gene which is highly homologous to GPI8 and can functionally replace it.

EMBO J, 1996 Dec 2, 15(23), 6460 - 75
Two GTPase isoforms, Ypt31p and Ypt32p, are essential for Golgi function in yeast; Benli M et al.; In eukaryotic cells, monomeric GTPases of the Ypt/Rab family function as regulators at defined steps of vesicular transport in exo- and endocytosis . Here we report on the isolation and characterization of two genes (YPT31 and YPT32) of the yeast Saccharomyces cerevisiae which encode members of the Ypt family exhibiting >80% sequence identity . Whereas the disruption of one of the two genes was phenotypically neutral, the disruption of both YPT31 and YPT32 led to lethality . Depletion of wild-type Ypt31p or of a short-lived ubiquitin-Ypt31p in a ypt32 null background led to a massive accumulation of Golgi-like membranes, an inhibition of invertase secretion and defects in vacuolar protein maturation . Similar alterations were observed in a conditional-lethal ypt31-1 mutant at 30 min after shift to the non-permissive temperature . According to subcellular fractionation, a significant part of Ypt31p appeared to be located in Golgi-enriched membrane fractions . In accordance with this, indirect immunofluorescence using affinity-purified anti-Ypt31p antibodies gave a punctate staining similar to that observed with Golgi-located proteins . From the phenotypic alterations observed in ypt31 and ypt32 mutants, it seems likely that the two GTPases are involved in intra-Golgi transport or in the formation of transport vesicles at the most distal Golgi compartment.

EMBO J, 1996 Dec 2, 15(23), 6447 - 59
Kes1p shares homology with human oxysterol binding protein and participates in a novel regulatory pathway for yeast Golgi-derived transport vesicle biogenesis; Fang M et al.; The yeast phosphatidylinositol transfer protein (Sec14p) is required for biogenesis of Golgi-derived transport vesicles and cell viability, and this essential Sec14p requirement is abrogated by inactivation of the CDP-choline pathway for phosphatidylcholine biosynthesis . These findings indicate that Sec14p functions to alleviate a CDP-choline pathway-mediated toxicity to yeast Golgi secretory function . We now report that this toxicity is manifested through the action of yeast Kes1p, a polypeptide that shares homology with the ligand-binding domain of human oxysterol binding protein (OSBP) . Identification of Kes1p as a negative effector for Golgi function provides the first direct insight into the biological role of any member of the OSBP family, and describes a novel pathway for the regulation of Golgi-derived transport vesicle biogenesis.

FEBS Lett, 1996 Dec 2, 398(2-3), 135 - 40
Characterisation of a human homologue of a yeast cell division cycle gene, MCM6, located adjacent to the 5' end of the lactase gene on chromosome 2q21; Harvey CB et al.; Four exons of a human homologue of a yeast cell division cycle gene (MCM6/mis5, which is thought to encode a DNA replication licensing factor) have been identified 3.3 kb upstream from the start of transcription of the intestinal lactase gene on human chromosome 2q21, initially by similarity to a rat 'intestinal crypt-cell replication factor' . RT-PCR analysis shows, that unlike lactase, MCM6 is not restricted in its tissue distribution and does not show person-to-person variation in the level of expression in adult intestine.

Tohoku J Exp Med, 1996 Dec, 180(4), 297 - 308
Secretion of active Fc fragments of immunoglobulin E directed by the yeast invertase signal sequence in mammalian cells; Kamiya T et al.; The human Fc fragments of immunoglobulin E (IgE-Fc) expressed in the mammalian COS cells, those mainly consisting of C epsilon 3-C epsilon 4 chains with or without Cys-328 known to be responsible for interchain disulfide bonding, were secreted into the culture medium directed by the yeast Saccharomyces cerevisiae invertase (SUC2) signal sequence (SUC2/Ig2 or SUC2/Ig, respectively) as well as by the human interleukin 2 receptor alpha chain signal sequence (IL2R/Ig2 or IL2R/Ig, respectively) . For the binding activity to the human soluble alpha chain of high affinity receptor for IgE, IgE-Fc with Cys328, SUC2/Ig2 and IL2R/Ig2, were active but had smaller activity than native IgE . By comparison IgE-Fc without Cys328, SUC2/Ig and IL2R/Ig, showed lower activity than SUC2/Ig2 and IL2R/Ig2 . Immunoprecipitation analysis revealed both SUC2/Ig and SUC2/Ig2 had molecular weights (M(r)s) and degrees of glycosylation similar to IL2R/Ig and IL2R/Ig2, respectively . These results suggested that in mammalian cells the SUC2 signal sequence was functional in directing the heterologous multimeric proteins to the endoplasmic reticulum, resulting in secretion of the active proteins . This finding might show merits on heterologous protein secretion systems.

Yeast, 1996 Dec, 12(16), 1647 - 75
Yeast protein serine/threonine phosphatases: multiple roles and diverse regulation; Stark MJ; Since the isolation of the first yeast protein phosphatase genes in 1989, much progress has been made in understanding this important group of proteins . Yeast contain genes encoding all the major types of protein phosphatase found in higher eukaryotes and the ability to use genetic approaches will complement the wealth of biochemical information available from other systems . This review will summarize recent progress in understanding the structure, function and regulation of the PPP family of protein serine-threonine phosphatases, concentrating on the budding yeast Saccharomyces cerevisiae.

Glycobiology, 1996 Dec, 6(8), 851 - 9
Targeting of active rat alpha 2,3-sialyltransferase to the yeast cell wall by the aid of the hsp 150 delta-carrier: toward synthesis of sLe(x)-decorated L-selectin ligands; Mattila P et al.; Interactions between selectins and their oligosaccharide-decorated ligands play a crucial role in the initiation of leukocyte extravasation . We have shown that synthetic multivalent sialyl Lewis x glycans inhibit strongly the adhesion of lymphocytes to endothelium at sites of inflammation . However, enzyme-assisted synthesis of these oligosaccharides si hampered by the lack of sufficient amounts of specific glycosyltransferases . We report here the construction of Saccharomyces cerevisiae strains expressing the soluble catalytic ectodomain of rat Gal(beta)1-3/4GlcNac alpha 2,3-sialyltransferase (ST3Ne) fused to the C-terminus of the hsp150 delta-carrier polypeptide . The hsp150 delta-carrier, which is an N-terminal fragmented of a natural secretory protein of yeast, is able to confer secretion-competence to several heterologous proteins, which otherwise remain in the yeast endoplasmic reticulum . The ST3Ne portion of the hsp 150 delta-ST3Ne fusion protein adopted an enzymatically active conformation and was N-glycosylated and disulfide-bonded . Hsp150 delta-ST3Ne was secreted with a half-time of about 7.5 min and remained intercalated in the cell wall, which covers the yeast plasma membrane . About 110 mU of sialyltransferase per litre was produced in 16 h . Whole live yeast cells were able to transfer sialic acid from CMP-NeuNAc to N-acetyllactosamine yielding alpha 2,3-sialyl-N-acetyllactosamine, as evidenced by paper chromatography, cleavage by linkage-specific sialidase, and NMR analysis . Our data suggest that yeast cells externalizing mammalian glycosyltransferases with the aid of the hsp150 delta-carrier could provide a source of enzymes for synthesis of valuable oligosaccharides.

Pharmacogenetics, 1996 Dec, 6(6), 489 - 99
Contribution of human cytochrome P450 to benzo{a}pyrene and benzo{a}pyrene-7,8-dihydrodiol metabolism, as predicted from heterologous expression in yeast; Gautier JC et al.; The metabolism of benzo{a}pyrene (B{a}P) and its proximate mutagen B{a}P-7,8-dihydrodiol (7,8-diol) was investigated in the presence of human microsomal epoxide hydrolase and P450 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6 and 3A4 expressed in the yeast Saccharomyces cerevisiae . P450 1A1 had the highest turnover numbers for the formation of all B{a}P metabolites, including phenols and dihydrodiols . P450 1A2, 2C8, 2C9, 2C18, 2C19 and 3A4, which are well represented in the liver, gave rise to the formation of appreciable amounts of 3-hydroxy-B{a}P and of some dihydrodiols from B{a}P . When 7,8-diol was used as substrate, P450 1A1 also exhibited the highest turnover numbers for the formation of tetrols, the hydrolysis products of the diolepoxides, whereas P450 1A2, 2C8, 2C19 and 3A4 showed moderate activities . In order to test the validity of the yeast system, the contribution of each P450 isoform to B{a}P and 7,8-diol metabolism was evaluated as the product of the turnover numbers of recombinant P450s by specific contents of each P450 in human liver microsomes . Calculated formation rates for each B{a}P and 7,8-diol metabolite globally matched experimental values . There is evidence that P450 3A4 and 2C9 play a major role in the formation of 3-hydroxy-B{a}P from B{a}P . Accumulation of the proximate mutagen 7,8-diol was predicted to be mainly driven by P450 1A2, 2C9 and 2C19, while formation of the genotoxic diolepoxides from 7,8-diol appeared to be dependent on P450 1A2 and 3A4 in the liver.

Protein Eng, 1996 Dec, 9(12), 1203 - 10
Ligand-induced conformational changes in wild-type and mutant yeast pyruvate kinase; Collins RA et al.; A mutant form of pyruvate kinase in which serine 384 has been mutated to proline has been engineered in the yeast Saccharomyces cerevisiae . Residue 384 is located in a helix in a subunit interface of the tetrameric enzyme, and the mutation was anticipated to alter the conformation of the helix and hence destabilize the interface . Previous results indicate that the mutant favours the T quaternary conformation over the R conformation, and this is confirmed by the results presented here . Addition of phosphoenol-pyruvate (PEP), ADP and fructose-1, 6-bisphosphate (Fru-1.6-P2) singly to the wild-type and mutant enzymes results in a significant quenching of tryptophan fluorescence (12-44%), and for Fru-1,6-P2, a red shift of 15 nm in the emission maximum . Fluorescence titration experiments showed that PEP, ADP and Fru-1,6-P2 induce conformations which have similar ligand-binding properties in the wild-type and mutant enzymes . However, the Fru-1,6-P2 induced conformation is demonstrably different from those induced by either ADP or PEP . The enzymes differ in their susceptibility to trypsin digestion and N-ethylmaleimide inhibition . The thermal stability of the enzyme is unaltered by the mutation . Far-UV CD spectra show that both enzymes adopt a similar overall secondary structure in solution . Taken together, the results suggest that the Ser384-Pro mutation causes the enzyme to adopt a different tertiary and/or quaternary structure from the wild-type enzyme and affects the type and extent of the conformational changes induced in the enzyme upon ligand binding . A simplified minimal reaction mechanism is proposed in which the R and T states differ in both affinity and kcat . Thus, in terms of the models of cooperativity and allosteric interaction, pyruvate kinase is both a K and a V system.

Trends Biochem Sci, 1996 Dec, 21(12), 477 - 81
3'-end-forming signals of yeast mRNA; Guo Z et al.; The signals required for forming 3'-ends of mRNAs from the yeast Saccharomyces cerevisiae differ from the corresponding signals of higher eukaryotes . Yeast signals consist of three elements: (1) the efficiency element, which enhances the efficiency of downstream positioning elements; (2) the positioning element, which positions the poly(A) site; and (3) the actual poly(A) site . These three elements are not only necessary, but also sufficient for mRNA 3'-end formation in yeast.

Curr Opin Genet Dev, 1996 Dec, 6(6), 763 - 6
Exploiting the complete yeast genome sequence; Bassett DE Jr et al.; The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae marks the dawn of an exciting new era in eukaryotic biology that will bring with it a new understanding of yeast, other model organisms, and human beings . This body of sequence data benefits yeast researchers by obviating the need for piecemeal sequencing of genes, and allows researchers working with other organisms to tap into experimental advantages inherent in the yeast system and learn from functionally characterized yeast gene products which are their proteins of interest . In addition, the yeast post-genome sequence era is serving as a testing ground for powerful new technologies, and proven experimental approaches are being applied for the first time in a comprehensive fashion on a complete eukaryotic gene repertoire.

Curr Biol, 1996 Dec 1, 6(12), 1609 - 20
Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast; Minshull J et al.; BACKGROUND: Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28-cyclin B complexes . Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis . Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF . We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis . RESULTS: The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint . Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B . In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect . In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction . Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF . Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55 delta cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids . CONCLUSIONS: We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction . Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation . We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.

Can J Microbiol, 1996 Dec, 42(12), 1190 - 6
Segregation of yeast polymorphic STA genes in meiotic recombinants and analysis of glucoamylase production; Balogh I et al.; Hybrid yeast strains were constructed using haploid Saccharomyces cerevisiae and Saccharomyces cerevisiae var . diastaticus strains to get haploid meiotic recombinants having more than one copy of STA1, STA2, and STA3 genes . STA genes were localized on the chromosomes by pulsed field gel electrophoresis . Working gene dosage effects were found among STA genes in liquid starch medium, indicating low levels of glucose repression . Growth of strains, however, was not influenced by their STA copy number.

J Cell Biol, 1996 Dec, 135(6 Pt 1), 1485 - 500
A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15; Wendland B et al.; A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking . A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid . The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects . Detailed characterization of two of these mutants, dim1-1 and dim2-1, revealed defects in the endocytic pathway . Like other yeast endocytosis mutants, the temperature-sensitive dim mutant were unable to endocytose FM4-64 or radiolabeled alpha-factor as efficiently as wild-type cells . In addition, double mutants with either dim1-delta or dim2-1 and the endocytosis mutants end4-1 or act1-1 displayed synthetic growth defects, indicating that the DIM gene products function in a common or parallel endocytic pathway . Complementation cloning of the DIM genes revealed identity of DIM1 to SHE4 and DIM2 to PAN1 . Pan1p shares homology with the mammalian clathrin adaptor-associated protein, eps15 . Both proteins contain multiple EH (eps15 homology) domains, a motif proposed to mediate protein-protein interactions . Phalloidin labeling of filamentous actin revealed profound defects in the actin cytoskeleton in both dim mutants . EM analysis revealed that the dim mutants accumulate vesicles and tubulo-vesicular structures reminiscent of mammalian early endosomes . In addition, the accumulation of novel plasma membrane invaginations where endocytosis is likely to occur were visualized in the mutants by electron microscopy using cationized ferritin as a marker for the endocytic pathway . This new screening strategy demonstrates a role for She4p and Pan1p in endocytosis, and provides a new general method for the identification of additional endocytosis mutants.

Genetics, 1996 Dec, 144(4), 1355 - 62
Yeast mutants deficient in ER-associated degradation of the Z variant of alpha-1-protease inhibitor; McCracken AA et al.; Saccharomyces cerevisiae mutants deficient in degradation of alpha-1-proteinase inhibitor Z (A1PiZ) have been isolated and genetically characterized . Wild-type yeast expressing A1PiZ synthesize an ER form of this protein that is rapidly degraded by an intracellular proteolytic process known as ER-associated protein degradation (ERAD) . The mutant strains were identified after treatment with EMS using a colony blot immunoassay to detect colonies that accumulated high levels of A1PiZ . A total of 120,000 colonies were screened and 30 putative mutants were identified . The level of A1PiZ accumulation in these mutants, measured by ELISA, ranged from two to 11 times that of A1PiZ in the parent strain . Further studies demonstrated that the increased levels of A1PiZ in most of the mutant strains was not the result of defective secretion or elevated A1PiZ mRNA . Pulse chase experiments indicated that A1PiZ was stabilized in several strains, evidence that these mutants are defective in ER-associated protein degradation . Genetic analyses revealed that most of the mutations were recessive, approximately 30% of the mutants characterized conformed to simple Mendelian inheritance, and at least seven complementation groups were identified.

Genome Res, 1996 Dec, 6(12), 1216 - 26
Isolation and genomic structure of a human homolog of the yeast periodic tryptophan protein 2 (PWP2) gene mapping to 21q22.3; Lafreniere RG et al.; As part of efforts to identify candidate genes for disease mapping to the 21q22.3 region, we have assembled a 770-kb cosmid and BAC contig containing eight tightly linked markers . These cosmids and BACs were restriction mapped using eight rare cutting enzymes, with the goal of identifying CpG-rich islands . One such island was identified by the clustering of NotI, EagI, SstII, and BssHII sites, and corresponded to the NotI linking clone LJ104 described previously . A 7.6-kb HindIII fragment containing this CpG-rich island was subcloned and partially sequenced . A homology search using the sequence obtained from either side of the NotI site identified an expressed sequence tag with homology to the yeast periodic tryptophan protein 2 (PWP2) . Several cDNAs corresponding to the human PWP2 gene were identified and partially sequenced . Northern blot analysis revealed a 3.3-kb transcript that was well expressed in all tissues tested . A cDNA consensus of 3157 bp was obtained, and an open reading frame potentially encoding 919 amino acid residues was identified . The predicted protein shows 42% identity and 57% similarity at the amino acid level to the yeast PWP2 protein, which is a member of the WD-repeat containing superfamily, and potentially encodes a G-protein beta subunit . The PWP2 gene is split into 21 exons, ranging in size from 53 to 516 bp, and spans an estimated 25 kb . The gene is transcribed in a 21cen-->21qter direction, with its 5' end mapping approximately 195 kb proximal to the 5' end of the phosphofructokinase-liver isoform gene . Four single base-pair polymorphisms were identified using single-stranded conformation polymorphism analysis . Possible functions of the protein based on homology to other members of the WD-repeat-containing family are discussed.

Nucleic Acids Res, 1996 Dec 1, 24(23), 4693 - 9
A shuttle system for transfer of YACs between yeast and mammalian cells; Simpson K et al.; The development of a system for shuttling DNA cloned as yeast artificial chromosomes (YACs) between yeast and mammalian cells requires that the DNA is maintained as extrachromosomal elements in both cell types . We have recently shown that circular YACs carrying the Epstein-Barr virus origin of plasmid replication (oriP) are maintained as stable, episomal elements in a human kidney cell line constitutively expressing the viral transactivator protein EBNA-1 . Here, we demonstrate that a 90-kb episomal YAC can be isolated intact from human cells by a simple alkaline lysis procedure and shuttled back into Saccharomyces cerevisiae by spheroplast transformation . In addition, we demonstrate that the 90-kb YAC can be isolated intact from yeast cells . The ability to shuttle large, intact fragments of DNA between yeast and human cells should provide a powerful tool in the manipulation and analysis of functional regions of mammalian DNA.

Nucleic Acids Res, 1996 Dec 1, 24(23), 4608 - 13
Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome; Sauer B; Variant lox sites having an altered spacer region (heterospecific lox sites) are not proficient for Cre-mediated recombination with the canonical 34 bp loxP site, but can recombine with each other . By placing different heterospecific lox sites at different genomic locations, Cre can catalyze independent DNA recombination events at multiple loci in the same cell without concern that unwanted inter-locus recombination events will be generated . Such heterospecific lox sites also allow Cre to specifically target efficient integration of exogenous DNA to endogenous lox-like sequences that naturally occur in the genome . Specific targeting occurs only with a DNA vector carrying a heterospecific lox site in which the spacer region has been redesigned to match the 'spacer' region of the targeted chromosomal element . Moreover, in cells expressing a catalytically active Cre recombinase, naturally occurring lox-like sequences can exhibit almost 20% mitotic recombination . Thus, in the same cell, heterospecific lox sites can be used independently at multiple loci for integration, for deletion and for enhanced mitotic recombination, thereby increasing the repertoire of genomic manipulations catalyzed by the Cre recombinase.

Yeast, 1996 Dec, 12(15), 1587 - 92
Analysis of a 23 kb region on the left arm of yeast chromosome IV; Delaveau T et al.; We have determined the complete nucleotide sequence of a 23 kb segment from the left arm of chromosome IV, which is carried by the cosmid 1L10 . This sequence contains the 3' coding region of the STE7 and RET1 (COP1) genes, and 13 complete open reading frames longer than 300 bp, of which ten correspond to putative new genes and three (CLB3, MSH5 and RPC53) have been sequenced previously . The sequence from cosmid IL10 was obtained entirely by a combined subcloning and walking primer strategy.

Yeast, 1996 Dec, 12(15), 1549 - 54
Analysis of a 26,756 bp segment from the left arm of yeast chromosome IV; Wolfl S et al.; The nucleotide sequence of a 26.7 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome IV is presented . An analysis of this segment revealed 11 open reading frames (ORFs) longer than 300 bp and one split gene . These ORFs include the genes encoding the large subunit of RNA polymerase II, the biotin apo-protein ligase, an ADP-ribosylation factor (ARF 2), the 'L35'-ribosomal protein, a rho GDP dissociation factor, and the sequence encoding the protein phosphatase 2A . Further sequence analysis revealed a short ORF encoding the ribosomal protein YL41B, an intron in a 5' untranslated region and an extended homology with another cosmid (X83276) located on the same chromosome . The potential biological relevance of these findings is discussed.

Yeast, 1996 Dec, 12(15), 1519 - 33
Identification of proteins of the yeast protein map using genetically manipulated strains and peptide-mass fingerprinting; Sagliocco F et al.; In this study we used genetically manipulated strains in order to identify polypeptide spots of the protein map of Saccharomyces cerevisiae . Thirty-two novel polypeptide spots were identified using this strategy . They corresponded to the product of 23 different genes . We also explored the possibilities of using peptide-mass fingerprinting for the identification of proteins separated on our gels . According to this strategy, proteins contained in spots are digested with trypsin and the masses of generated peptides are determined by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) . The peptide masses are then used to search a yeast protein database for proteins that match the experimental data . Application of this strategy to previously identified polypeptide spots gave evidence of the feasibility of this approach . We also report predictions on the identities of nine unknown spots using MALDI-MS.

FEMS Microbiol Lett, 1996 Dec 1, 145(2), 267 - 71
Influence of growth temperature on the acid phosphatase activity in the yeast Yarrowia lipolytica; Vasileva-Tonkova E et al.; The adaptation of the yeast Yarrowia lipolytica 76-18 to growth temperature was studied by measuring the levels of secreted and intracellular acid phosphatase activities during growth at five temperatures from 8 to 36 degrees C . The intracellular acid phosphatase activity is maximal at a growth temperature of 20 degrees C . The level of the secreted phosphatase activity decreases as growth temperature increases from 15 to 36 degrees C . It is the growth temperature itself and not the growth rate that regulates these activities . The observed dependence of the acid phosphatase activity on the growth temperature indicates a possible participation of acid phosphatases in the temperature adaptation of yeast cells.

J Bacteriol, 1996 Dec, 178(24), 7144 - 51
FLO11, a yeast gene related to the STA genes, encodes a novel cell surface flocculin; Lo WS et al.; We report the characterization of a gene encoding a novel flocculin related to the STA genes of yeast, which encode secreted glucoamylase . The STA genes comprise sequences that are homologous to the sporulation-specific glucoamylase SGA and to two other sequences, S2 and S1 . We find that S2 and S1 are part of a single gene which we have named FLO11 . The sequence of FLO11 reveals a 4,104-bp open reading frame on chromosome IX whose predicted product is similar in overall structure to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins . An amino-terminal domain containing a signal sequence and a carboxy-terminal domain with homology to GPI (glycosyl-phosphatidyl-inositol) anchor-containing proteins are separated by a central domain containing a highly repeated threonine- and serine-rich sequence . Yeast cells that express FLO11 aggregate in the calcium-dependent process of flocculation . Flocculation is abolished when FLO11 is disrupted . The product of STA1 also is shown to have flocculating activity . When a green fluorescent protein fusion of FLO11 was expressed from the FLO11 promoter on a single-copy plasmid, fluorescence was observed in vivo at the periphery of cells . We propose that FLO11 encodes a flocculin because of its demonstrated role in flocculation, its structural similarity to other members of the FLO gene family, and the cell surface location of its product . FLO11 gene sequences are present in all yeast strains tested, including all standard laboratory strains, unlike the STA genes which are present only in the variant strain Saccharomyces cerevisiae var . diastaticus . FLO11 differs from all other yeast flocculins in that it is located near a centromere rather than a telomere, and its expression is regulated by mating type . Repression of FLO11-dependent flocculation in diploids is conferred by the mating-type repressor al/alpha2.

J Cell Biol, 1996 Dec, 135(5), 1229 - 37
The refolding activity of the yeast heat shock proteins Ssa1 and Ssa2 defines their role in protein translocation; Bush GL et al.; Ssa1/2p, members of one of the yeast cytosolic hsp70 subfamilies, have been implicated in the translocation of secretory proteins into the lumen of the ER . The involvement of these hsp70s in translocation was tested directly by examining the effect of immunodepleting Ssa1/2p from yeast cytosol and subsequently testing the cytosol for its ability to support co- and post-translational translocation of prepro-alpha-factor . Depletion of Ssa1/2p had no effect on the efficiency of translocation in this in vitro assay . The system was used to examine the effect of the absence of Ssa1/2p on two other putative hsp70 functions: cotranslational folding of nascent luciferase and refolding of denatured luciferase . Depletion of Ssa1/2p had no effect on the ability of the yeast lysate to synthesize enzymatically active luciferase, but had a dramatic effect on the ability of the lysate to refold chemically denatured luciferase . These results demonstrate, for the first time, the refolding activity of Ssa1/2p in the context of the yeast cytosol, and define refolding activity as a chaperone function specific to Ssa1/2p, aprt from other cytosolic hsp70s . They also suggest that Ssa1/2p do not play a significant role in chaperoning the folding of nascent polypeptides . The implications of these findings for Ssa1/2p activity on their proposed role in the process of translocation are discussed.

Nat Genet, 1996 Dec, 14(4), 450 - 6
Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy; Shoemaker DD et al.; A quantitative and highly parallel method for analysing deletion mutants has been developed to aid in determining the biological function of thousands of newly identified open reading frames (ORFs) in Saccharomyces cerevisiae . This approach uses a PCR targeting strategy to generate large numbers of deletion strains . Each deletion strain is labelled with a unique 20-base tag sequence that can be detected by hybridization to a high-density oligonucleotide array . The tags serve as unique identifiers (molecular bar codes) that allow analysis of large numbers of deletion strains simultaneously through selective growth conditions . Hybridization experiments show that the arrays are specific, sensitive and quantitative . A pilot study with 11 known yeast genes suggests that the method can be extended to include all of the ORFs in the yeast genome, allowing whole genome analysis with a single selective growth condition and a single hybridization.

Mol Cell Biol, 1996 Dec, 16(12), 7161 - 72
A yeast acetyl coenzyme A carboxylase mutant links very-long-chain fatty acid synthesis to the structure and function of the nuclear membrane-pore complex; Schneiter R et al.; The conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1 (mtr7-1), displays a unique alteration of the nuclear envelope . Unlike nucleoporin mutants and other RNA transport mutants, the intermembrane space expands, protuberances extend from the inner membrane into the intermembrane space, and vesicles accumulate in the intermembrane space . MTR7 is the same gene as ACC1, encoding acetyl coenzyme A (CoA) carboxylase (Acc1p), the rate-limiting enzyme of de novo fatty acid synthesis . Genetic and biochemical analyses of fatty acid synthesis mutants and acc1-7-1 indicate that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is required for an essential pathway which is independent from de novo synthesis of fatty acids . We provide evidence that synthesis of very-long-chain fatty acids (C26 atoms) is inhibited in acc1-7-1, suggesting that very-long-chain fatty acid synthesis is required to maintain a functional nuclear envelope.

Mol Cell Biol, 1996 Dec, 16(12), 7004 - 17
Heat shock factor gains access to the yeast HSC82 promoter independently of other sequence-specific factors and antagonizes nucleosomal repression of basal and induced transcription; Erkine AM et al.; Transcription in eukaryotic cells occurs in the context of chromatin . Binding of sequence-specific regulatory factors must contend with the presence of nucleosomes for establishment of a committed preinitiation complex . Here we demonstrate that the high-affinity binding site for heat shock transcription factor (HSF) is occupied independently of other cis-regulatory elements and is critically required for preventing nucleosomal assembly over the yeast HSC82 core promoter under both noninducing (basal) and inducing conditions . Chromosomal mutation of this sequence, termed HSE1, erases the HSF footprint and abolishes both transcription and in vivo occupancy of the TATA box . Moreover, it dramatically reduces promoter chromatin accessibility to DNase I and TaqI, as the nuclease-hypersensitive region is replaced by a localized nucleosome . By comparison, in situ mutagenesis of two other promoter elements engaged in stable protein-DNA interactions in vivo, the GRF2/REB1 site and the TATA box, despite reducing transcription three- to fivefold, does not compromise the nucleosome-free state of the promoter . The GRF2-binding factor appears to facilitate the binding of proteins to both HSE1 and TATA, as these sequences, while still occupied, are less protected from in vivo dimethyl sulfate methylation in a deltaGRF2 strain . Finally, deletion of a consensus upstream repressor sequence (URS1), positioned immediately upstream of the GRF2-HSE1 region and only weakly occupied in chromatin, has no expression phenotype, even under meiotic conditions . However, deletion of URS1, like mutation of GRF2, shifts the translational setting of an upstream nucleosomal array flanking the promoter region . Taken together, our results argue that HSF, independent of and dominant among sequence-specific factors binding to the HSC82 upstream region, antagonizes nucleosomal repression and creates an accessible chromatin structure conducive to preinitiation complex assembly and transcriptional activation.

Mol Cell Biol, 1996 Dec, 16(12), 6783 - 93
Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription; Lauder S et al.; Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA . However, how MMS19 affects this process has remained unclear . Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes . Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription . mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription . Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair . However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins . Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract . Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme . These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element . It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected.

Mol Cell Biol, 1996 Dec, 16(12), 6698 - 706
Two human cDNAs, including a homolog of Arabidopsis FUS6 (COP11), suppress G-protein- and mitogen-activated protein kinase-mediated signal transduction in yeast and mammalian cells; Spain BH et al.; We have isolated two novel human cDNAs, gps1-1 and gps2, that suppress lethal G-protein subunit-activating mutations in the pheromone response pathway of the yeast Saccharomyces cerevisiae . Suppression of other pathway-activating events was examined . In wild-type cells, expression of either gps1-1 or gps2 led to enhanced recovery from cell cycle arrest induced by pheromone . Sequence analysis indicated that gps1-1 contains only the carboxy-terminal half of the gps1 coding sequence . The predicted gene product of gps1 has striking similarity to the protein encoded by the Arabidopsis FUS6 (COP11) gene, a negative regulator of light-mediated signal transduction that is known to be essential for normal development . A chimeric construct containing gps1 and FUS6 sequences also suppressed the yeast pheromone pathway, indicating functional conservation between these human and plant genes . In addition, when overexpressed in mammalian cells, gps1 or gps2 potently suppressed a RAS- and mitogen-activated protein kinase-mediated signal and interfered with JNK activity, suggesting that signal repression is part of their normal function . For gps1, these results are consistent with the proposed function of FUS6 (COP11) as a signal transduction repressor in plants.

Endocrinology, 1996 Dec, 137(12), 5441 - 6
Yeast aspartic protease 3 is sorted to secretory granules and activated to process proopiomelanocortin in PC12 cells; Cool DR et al.; The subcellular localization and functionality of transfected yeast aspartic protease 3 (YAP3p) in a mammalian cell line were investigated . The complementary DNAs encoding the prohormone-processing enzyme (YAP3p) and a prohormone, bovine POMC, were cotransfected into PC12 (rat pheochromocytoma) cells . Immunocytochemical analysis of the cells using a YAP3p antibody showed a perinuclear punctate distribution of YAP3p in the cell body as well as immunostaining in the tips of the neurites . This pattern of immunostaining indicates localization of YAP3p in secretory granules . Analysis of the processing of POMC showed that in cells transfected with the POMC complementary DNA alone, only POMC was found, indicating a lack of processing of the prohormone . However, in cells coexpressing YAP3p, the POMC was completely processed to yield ACTH-(1-39) and ACTH-(1-14), consistent with the specificity of YAP3p found in vitro . Pulse-chase studies showed that POMC was processed after 20 min of chase, suggesting that processing occurred in the late Golgi network and continued in the secretory granules . Western blot analysis determined that YAP3p was secreted from the cells in a regulated manner . This study provides the first demonstration that a yeast prohormone-processing enzyme (YAP3p) of the aspartic protease class can be sorted correctly to secretory granules and activated to process a prohormone (POMC) in a highly efficient manner in mammalian cells.

Chromosoma, 1996 Dec, 105(5), 276 - 84
Patterns of meiotic double-strand breakage on native and artificial yeast chromosomes; Klein S et al.; The preferred positions for meiotic double-strand breakage were mapped on Saccharomyces cerevisiae chromosomes I and VI, and on a number of yeast artificial chromosomes carrying human DNA inserts . Each chromosome had strong and weak double-strand break (DSB) sites . On average one DSB-prone region was detected by pulsed-field gel electrophoresis per 25 kb of DNA, but each chromosome had a unique distribution of DSB sites . There were no preferred meiotic DSB sites near the telomeres . DSB-prone regions were associated with all of the known "hot spots" for meiotic recombination on chromosomes I, III and VI.

Curr Genet, 1996 Dec, 30(6), 493 - 501
A probable cis-regulatory element on yeast mitochondrial DNA responsible for cAMP-mediated transcription; Iqbal J et al.; Studies from this laboratory have suggested that mitochondrial (mt) transcription in yeast (Saccharomyces cerevisiae) is governed by changing cellular cAMP levels, and that the mechanism of such transcriptional regulation requires cAMP-dependent protein kinase (PKA) activity; these observations, in turn, suggest a trans-activation process for nucleotide-dependent mt transcriptional control . Here we demonstrate a sequence-specific mtDNA-phosphorylated protein interaction, a requisite part of such a control mechanism, using filter-binding and gel mobility shift assays with mt protein extracts and mtDNA from rho- strains whose retained mt genes show cAMP-sensitive expression . We demonstrate that the protein-mt DNA interaction depends on PKA activity, that it specifically involves a tripartite GC-rich sequence element on yeast mtDNA, and that it does not involve mt coding or promoter sequences . Sequence analysis indicates that the GC-rich element undergoing protein interaction is present in ten copies on the yeast mt genome, and that each copy is located 5' to a strong mt promoter; the elements appear in both orientations relative to, and at varying distances upstream from, the putatively associated mt promoter elements . The mt element shows no sequence homology to relevant nuclear cis-elements examined and is unrelated to published vertebrate mt cis-elements . Several lines of evidence and argument strongly suggest that this GC-rich element functions as the cis-regulatory sequence involved in cAMP-mediated transcriptional control in yeast mitochondria.

Curr Genet, 1996 Dec, 30(6), 469 - 75
Novel aspects of pheromone-induced cell-cycle arrest in yeast; Dolan JW; Saccharomyces cerevisiae responds to pheromones by arresting cell-cycle progression at the G1/S boundary . This arrest has been shown to require the activation by phosphorylation of Far1, a cyclin-dependent kinase inhibitor . Arrest resembling pheromone-induced arrest can be triggered by high levels of Ste12, the pheromone-responsive transcriptional activator . In the present study, cell-cycle arrest induced by high levels of Ste12 is characterized . This arrest occurs predominately at the G1/S boundary and is independent of Far1 and upstream components of the pheromone response pathway . Since Ste12 is a transcriptional activator, it is proposed that cell-cycle arrest in response to high levels of Ste12 is due to transcription of a Ste12-dependent arrest factor regulated primarily, but not necessarily exclusively, at the level of transcription . The data presented here suggest that a novel component has a role in arrest in response to pheromone.

J Chromatogr A, 1996 Nov 29, 755(1), 75 - 80
Cell lipids of the Candida lipolytica yeast grown on methanol; Rupcic J et al.; Candida lipolytica yeast, grown on 1% methanol as the only carbon and energy source, synthesized 4.9% of dry cell mass as lipids, 52.3% of which were polar lipids . Polar lipids consisted mainly of phospholipids and sphingolipids as their minor components . The total long-chain bases content has been found to account only for 0.7% of the polar lipids . The long-chain bases composition determined by thin-layer and gas chromatography shows a preponderance of trihydroxy bases and a small amount of dihydroxy bases . The striking finding was the high content of 19-phytosphingosine (90.8% of total long-chain bases) . Fatty acid (FA) composition of polar lipids was characterized by the relatively high concentration of unsaturated fatty acids (66.4% of total FA) and by the predominance of fatty acids with 16 carbon atoms (85.0% of total FA).

J Biol Chem, 1996 Nov 29, 271(48), 30610 - 3
Cer1p, a novel Hsp70-related protein required for posttranslational endoplasmic reticulum translocation in yeast; Hamilton TG et al.; Proteins enter the secretory pathway by translocation across the endoplasmic reticulum (ER) membrane . In Saccharomyces cerevisiae, import of proteins into the ER occurs both cotranslationally and posttranslationally . Presumably, the cotranslational targeting to the ER membrane is directed by the signal recognition particle, as demonstrated in other eukaryotic systems . The deletion of a gene, called CER1, inhibits the translocation of proteins that enter the ER posttranslationally, but not those that enter cotranslationally . This translocation defect is more pronounced at lower temperatures . A strain possessing a null mutation of CER1 in combination with a kar2 temperature-sensitive mutation displays synthetic growth defects, whereas overexpression of the ER DnaJ homolog Scj1p suppresses the translocation defect in cer1Delta strains . CER1 is predicted to encode a 100-kDa polypeptide, residing in the ER lumen that is related to the hsp70 family of molecular chaperones.

Science, 1996 Nov 29, 274(5292), 1517 - 20
Dependence of yeast pre-mRNA 3'-end processing on CFT1: a sequence homolog of the mammalian AAUAAA binding factor; Stumpf G et al.; 3'-End formation of pre-mRNA in yeast and mammals follows a similar but distinct pathway . In Saccharomyces cerevisiae, the cleavage reaction can be reconstituted by two activities called cleavage factor I and II (CFI and CFII) . A CFII component, designated CFT1 (cleavage factor two) was identified by its sequence similarity to the AAUAAA-binding subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF), even though the AAUAAA signal sequence appears to play no role in yeast pre-mRNA 3' processing . Depletion of a yeast whole-cell extract with antibodies to CFT1 protein abolished cleavage and polyadenylation of pre-mRNAs . Addition of CFII restored cleavage activity, but not polyadenylation . Polyadenylation required the further addition of poly(A) polymerase and polyadenylation factor I, suggesting a close but not necessarily direct association of these two factors with the CFT1 protein.

Science, 1996 Nov 29, 274(5292), 1514 - 7
Sequence similarity between the 73-kilodalton protein of mammalian CPSF and a subunit of yeast polyadenylation factor I; Jenny A et al.; The 3' ends of most eukaryotic messenger RNAs are generated by endonucleolytic cleavage and polyadenylation . In mammals, the cleavage and polyadenylation specificity factor (CPSF) plays a central role in both steps of the processing reaction . Here, the cloning of the 73-kilodalton subunit of CPSF is reported . Sequence analyses revealed that a yeast protein (Ysh1) was highly similar to the 73-kD polypeptide . Ysh1 constitutes a new subunit of polyadenylation factor I (PFI), which has a role in yeast pre-mRNA 3'-end formation . This finding was unexpected because in contrast to CPSF, PFI is only required for the polyadenylation reaction . These results contribute to the understanding of how 3'-end processing factors may have evolved.

Science, 1996 Nov 29, 274(5292), 1511 - 4
Essential yeast protein with unexpected similarity to subunits of mammalian cleavage and polyadenylation specificity factor (CPSF); Chanfreau G et al.; The 3' ends of most eukaryotic messenger RNAs are generated by internal cleavage and polyadenylation . In mammals, there is a strict dependence of both reactions on the sequence AAUAAA, which occurs upstream of polyadenylation {poly(A)} sites and which is recognized by CPSF . In contrast, cis-acting signals for yeast 3'-end generation are highly divergent from those of mammals, suggesting that trans-acting factors other than poly(A) polymerase would not be conserved . The essential yeast protein Brr5/Ysh1 shows sequence similarity to subunits of mammalian CPSF and is required for 3'-end processing in vivo and in vitro . These results demonstrate a structural and functional conservation of the yeast and mammalian 3'-end processing machineries despite a lack of conservation of the cis sequences.

Gene, 1996 Nov 28, 181(1-2), 63 - 9
CADp44: a novel regulatory subunit of the 26S proteasome and the mammalian homolog of yeast Sug2p; Bauer VW et al.; We have identified a novel protein, CADp44, based on the analysis of cDNAs derived from the brainstem of the 13-lined ground squirrel, Spermophilus tridecemlineatus . CADp44 has an unmodified molecular mass of 44,178 Da and contains multiple functional domains, including a conserved ATPase domain (CAD) and a leucine zipper motif . We show that distinct regions of the CADp44 sequence are identical to a set of peptides prepared from a recently identified bovine protein, referred to as p42, which is found in the PA700 regulatory complex of the 26S proteasome (DeMartino et al., 1996) . We also show that CADp44 is the functional homolog of the newly characterized Sug2 protein from the budding yeast, Saccharomyces cerevisiae (Russell et al., 1996) . Consistent with its role as a component of the 26S proteasome, CADp44 mRNA is found in all ground squirrel tissues examined . Evolutionary relationships based on sequence analysis show that both CADp44 and yeast Sug2p are distinct from the other five CAD ATPases found in the PA700, and together comprise the sixth and newest CAD subunit of the regulatory complex of the 26S proteasome.

Mol Gen Genet, 1996 Nov 27, 253(1-2), 81 - 8
Complementation of a yeast top2ts mutation by a cDNA encoding rat DNA topoisomerase II alpha; Yoon JH et al.; A series of yeast expression plasmids which comprise segments of the cDNA sequences encoding rat topo II alpha have been constructed . The transcription of these constructs is under the control of the yeast GAL1 promoter . Galactose-dependent expression of the cloned rat topo II alpha cDNA complemented a yeast top2ts mutation, as well as a deletion mutation at the yeast TOP2 locus . Truncation of 12 N-terminal amino acids and/or 158 C-terminal amino acids of rat topo II alpha had no effect on its ability functionally to substitute for top2ts . Moreover, a cDNA construct with mutated putative leucine zipper domain (amino acids 993-1013) retained the complementation activity . These observations suggest that transformants capable of conditional topo II alpha expression can be exploited as a useful model system for studies on the structure-function relationships of wild-type and mutated topo II alpha, as well as the interplay of potential antitumor drugs with the enzyme.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13949 - 54
Lack of chromosome territoriality in yeast: promiscuous rejoining of broken chromosome ends; Haber JE et al.; Various studies suggest that eukarytoic chromosomes may occupy distinct territories within the nucleus and that chromosomes are tethered to a nuclear matrix . These constraints might limit interchromosomal interactions . We have used a molecular genetic test to investigate whether the chromosomes of Saccharomyces cerevisiae exhibit such territoriality . A chromosomal double-strand break (DSB) can be efficiently repaired by recombination between flanking homologous repeated sequences . We have constructed a strain in which DSBs are delivered simultaneously to both chromosome III and chromosome V by induction of the HO endonuclease . The arrangement of partially duplicated HIS4 and URA3 sequences around each HO recognition site allows the repair of the two DSBs in two alternative ways: (i) the creation of two intrachromosomal deletions or (ii) the formation of a pair of reciprocal translocations . We show that reciprocal translocations are formed approximately as often as the pair of intrachromosomal deletions . Similar results were obtained when one of the target regions was moved from chromosome V to any of three different locations on chromosome XI . These results argue that the broken ends of mitotic chromosomes are free to search the entire genome for appropriate partners; thus, mitotic chromosomes are not functionally confined to isolated domains of the nucleus, at least when chromosomes are broken.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13937 - 42
Two eukaryote-specific regions of Hsp82 are dispensable for its viability and signal transduction functions in yeast; Louvion JF et al.; The 90-kDa heat shock protein (Hsp90) is a molecular chaperone that is very abundant even at normal temperature . It is highly conserved and essential for viability in yeast . To delineate functional domains of Hsp90, we have performed a deletion analysis of one of the two Hsp90 isoforms from budding yeast, Hsp82 . The Hsp82 derivatives were tested for complementation of a Hsp90-deficient yeast strain and for their ability to function in two signal transduction pathways that depend on Hsp90 . Surprisingly, we found that two salient features of Hsp90 sequences from eukaryotes, the N-terminal charged domain and the extremely conserved C-terminal pentapeptide MEEVD, are dispensable for viability as well as for proper regulation of vertebrate steroid receptors and for pheromone signaling . Moreover, we describe, to our knowledge, the first dominant negative mutant of Hsp90; A Hsp82 derivative that lacks amino acids 538-552 fails to complement but has a dominant negative effect on viability of wild-type strains at moderately elevated temperatures . This mutant may become a valuable tool to study Hsp90 functions both in yeast and in mammalian cells.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13780 - 5
TOR2 is required for organization of the actin cytoskeleton in yeast; Schmidt A et al.; The Saccharomyces cerevisiae gene TOR2 encodes a putative phosphatidylinositol kinase that has two essential functions . One function is redundant with TOR1, a TOR2 homolog, and is required for signaling translation initiation and early G1 progression . The second essential function is unique to TOR2 . Here we report that loss of the TOR2-unique function disrupts polarized distribution of the actin cytoskeleton . A screen for dosage suppressors of a dominant negative TOR2 allele identified TCP20/CCT6, encoding a subunit of the cytosolic chaperonin TCP-1 that is involved in the biogenesis of actin structures . Overexpression of TCP20 restores growth and polarized distribution of the actin cytoskeleton in a tor2 mutant . TCP20 overexpression does not restore growth in a tor1 tor2 double mutant . We suggest that the unique function of the phosphatidylinositol kinase homolog TOR2 is required for signaling organization of the actin cytoskeleton during the cell cycle . TOR2, via its two functions, may thus integrate temporal and spatial control of cell growth.

Proc Natl Acad Sci U S A, 1996 Nov 26, 93(24), 13641 - 6
The essential yeast RNA binding protein Np13p is methylated; Siebel CW et al.; Arginine methylation is a prevalent modification found in many RNA binding proteins, yet little is known about its functional consequences . Using a monoclonal antibody, 1E4, we have shown that the yeast NPL3 gene product Np13p, an essential RNA binding protein with repeated RGG motifs, is arginine-methylated in vivo . The 1E4 epitope can be generated by incubating recombinant Np13p with partially purified bovine arginine methyltransferase block this reaction . Np13p methylation requires S-adenosyl-L-methionine and also occurs in yeast extracts . An Np13p deletion mutant lacking the RGG domain is not a substrate for methylation, suggesting that the methylation sites lie within the RGG motifs . The discovery of arginine methylation in a genetically tractable organism provides a powerful entree to understanding the function of this modification, particularly in view of the many roles postulated for Np13p in RNA processing and transport . The recent discovery of phosphorylated serine residues within the RGG domain suggests a hypothesis in which a molecular switch governed by methylation and phosphorylation regulates the biochemical properties of the Np13p RGG domain.

Biochemistry, 1996 Nov 26, 35(47), 14899 - 909
Comparative modeling of substrate binding in the S1' subsite of serine carboxypeptidases from yeast, wheat, and human; Elsliger MA et al.; Human cathepsin A ("lysosomal protective protein"; E.C.3.4.16.5) is a multifunctional lysosomal protein which forms a high-molecular-weight complex with beta-galactosidase and alpha-neuraminidase, protecting them against intralysosomal proteolysis . In addition to this protective function, cathepsin A is a serine carboxypeptidase and the understanding of its catalytic function requires a definition of its substrate specificity . For this purpose, we used a combined experimental {Pshezhetsky, A . V., Vinogradova, M . V., Elsliger, M.-A., El-Zein, F., Svedas, V.K., & Potier, M . (1995) Anal . Biochem . 230, 303-307} and theoretical approach comparing cathepsin A to two different homologous carboxypeptidases of the same family: yeast carboxypeptidase Y and wheat carboxypeptidase II . We computed the energies involved in substrate binding to the S1' subsite (C-terminal) of cathepsin A using a structural model based on the X-ray structure of the homologous wheat carboxypeptidase II . The binding energies of N-blocked Phe-Xaa dipeptide substrates to the active sites of cathepsin A, wheat carboxypeptidase II, and yeast carboxypeptidase Y were estimated using a molecular mechanics force field supplemented with a solvation energy term . This theoretical analysis showed a good correlation with the experimentally determined free energies of substrate binding . This result validates the use of this approach to analyze the energetics of substrate binding to the S1' subsite and provides a rational interpretation of serine carboxypeptidase-substrate interactions in molecular terms . We conclude that the three serine carboxypeptidases have similar affinities for substrates with hydrophobic P1' amino acid residues but that the wheat enzyme has an additional capacity for binding positively charged P1' residues . Finally, the substrate specificity of human cathepsin A is very similar to that of carboxypeptidase Y, with a high binding affinity for substrates with hydrophobic P1' residues, but the affinity of cathepsin A for P1; Phe residue is higher than for the Leu residue.

Biochemistry, 1996 Nov 26, 35(47), 14806 - 17
Partial constitutive activation of pheromone responses by a palmitoylation-site mutant of a G protein alpha subunit in yeast; Song J et al.; G protein alpha subunits are often myristoylated and/or palmitoylated near their amino terminus . The G protein alpha subunit in the yeast Saccharomyces cerevisiae (GPA1 gene product, Gpa1p) is known to be myristoylated, and this modification is essential for G protein activity in vivo . Here we examined whether Gpa1p is palmitoylated and determined the functional consequences of this modification . {3H}-Palmitic acid was incorporated into Gpa1p in cells expressing myc-tagged Gpa1p or Gpa1p-Gst . The label was released upon hydroxylamine treatment . Substitution of the conserved Cys 3 for Ser blocked incorporation of the label (Gpa1pC3S) . Palmitoylation was also blocked by a mutation that prevents myristoylation (Gly2Ala), whereas the palmitoylation-site mutation had no effect on myristoylation of Gpa1p . Gpa1pC3S complemented the gpa1 delta mutation in vivo and formed a complex with G beta gamma that was able to undergo nucleotide exchange in vitro . However, basal and pheromone-induced FUSl-lacZ transcription were 2-5-fold higher in the C3S mutant . Pheromone-induced growth arrest was also enhanced by the mutation, but recovery from arrest was not affected . Like wild-type Gpa1p, the C3S mutant was predominantly membrane-associated . Upon Triton X-114 partitioning or high pH treatment, no difference in the membrane-binding properties of the wild-type Gpa1p and the C3S mutant was detected . By sucrose density gradient centrifugation of membranes, however, most of the mutant protein was mislocalized to a non-plasma membrane compartment, whereas G beta gamma localization was unaltered . Taken together, our data suggest that Gpa1p is palmitoylated via a thioester bond at Cys 3 and that palmitoylation plays a role in modulating Gpa1p signaling and membrane localization.

FEBS Lett, 1996 Nov 25, 398(1), 113 - 9
Core promoter elements are essential as selective determinants for function of the yeast transcription factor GAL11; Sakurai H et al.; The GAL11 gene product, which copurifies with RNA polymerase II holoenzyme, is necessary for full expression of many, but not all, genes in yeast . Here we shows that the GAL11 dependence of a gene for expression is determined by the core promoter structure . In the GAL80 gene, a gal11 null mutation caused reduction of TATA-dependent transcription, but exerted no effect on initiator-mediated transcription . GAL11 stimulated TATA-dependent transcription, but did not affect the TATA-independent transcription in HIS4 . GAL11 was also required for transcription mediated by a canonical TATA sequence but not by a nonconsensus TATA sequence of HIS3 . These results suggest that GAL11 is specifically involved in the transcription machinery formed on the TATA element.

J Mol Biol, 1996 Nov 22, 264(1), 46 - 55
Very fast identification of RNA motifs in genomic DNA . Application to tRNA search in the yeast genome; el-Mabrouk N et al.; A common strategy characterises the various methods independently defined to identify almost unambiguously different types of RNA molecules in DNA fragments . So far, the good quality of detection of RNA motif has been the prior motivation and effectively delayed the optimisation of programs . As an illustration of possible improvements, a modified version of tRNAscan is described . The previous algorithm was altered to run 500 times faster and to lower both rates of false positives and false negatives . The newly sequenced genome of Saccharomyces cerevisiae is scanned both ways in less than three minutes and results match annotations found in databanks with three exceptions, two of which being arguably not real tRNAs.

J Biol Chem, 1996 Nov 22, 271(47), 29637 - 43
Amino acid and adenine cross-pathway regulation act through the same 5'-TGACTC-3' motif in the yeast HIS7 promoter; Springer C et al.; The HIS7 gene of Saccharomyces cerevisiae encodes a bifunctional glutamine amidotransferase:cyclase catalyzing two reactions that lead to the formation of biosynthetic intermediates of the amino acid histidine and the purine adenine . The HIS7 gene is activated by GCN4p under environmental conditions of amino acid starvation through two synergistic upstream sites GCRE1 and GCRE2 . The BAS1p-BAS2p complex activates the HIS7 gene in response to adenine limitation . For this activation the proximal GCN4p-binding site GCRE2 is required . GCN4p and BAS1p bind to GCRE2 in vitro . Under conditions of simultaneous amino acid starvation and adenine limitation the effects of GCN4p and BAS1/2p are additive and both factors are necessary for maximal HIS7 transcription . These results suggest that GCN4p and BAS1/2p are able to act simultaneously through the same DNA sequence in vivo and use this site independently from each other in a non-exclusive manner.

Nature, 1996 Nov 21, 384(6606), 279 - 82
G2 cyclins are required for the degradation of G1 cyclins in yeast; Blondel M et al.; Progression of the eukaryotic cell cycle is controlled by cyclin-dependent kinases (CDKs) . Cdc28, the budding yeast homologue of Cdc2 (Cdk1), is required for both the G1/S and G2/M transitions of the cell cycle . The functional specificity of the Cdc28 kinase is determined by its association with G1 or G2 cyclins . Alternation of cell cycle phases is thus mainly due to mechanisms that ensure that one cyclin family succeeds another . Here we show that the G2 cyclins Clb1, Clb2, Clb3 and Clb4 are required for the proteolysis of the G1 cyclins Cln1 and Cln2, providing a mechanism for coupling synthesis of G2 cyclins with the disappearance of G1 cyclins . Our data indicate that this pathway involves the Ubc9 ubiquitin-conjugating enzyme . The Cdc34 ubiquitin-conjugating activity may function redundantly with Ubc9, or it may only be involved in Cln1,2 turnover through its role in promoting the degradation of Sic1, a specific inhibitor of Cdc28-Clb complexes.

FEBS Lett, 1996 Nov 18, 397(2-3), 197 - 200
The intracellular cytoplasmic domain of the Alzheimer's disease amyloid precursor protein interacts with phosphotyrosine-binding domain proteins in the yeast two-hybrid system; McLoughlin DM et al.; We have used the yeast two-hybrid system to screen for proteins that interact with the carboxy-terminal domain of APP . Six different clones were isolated and sequence analyses revealed that they encoded domains of a previously described neuronal protein Fe65, a homologue of Fe65 and a homologue of protein X11 . All of these proteins contain one or more phosphotyrosine binding (PTB) domains . PTB domain proteins bind to the sequence Asn-Pro-X-Tyr when the Tyr is phosphorylated and are believed to function in signal transduction . APP contains such a motif . These results are consistent with a role for APP in signal transduction mechanisms.

Nucleic Acids Res, 1996 Nov 15, 24(22), 4479 - 86
The homeodomain protein Pho2 and the basic-helix-loop-helix protein Pho4 bind DNA cooperatively at the yeast PHO5 promoter; Barbaric S et al.; Two transcription factors, the bHLH protein Pho4 and the homeodomain protein Pho2, are required for transcriptional activation of the PHO5 promoter in Saccharomyces cerevisiae . There are two essential Pho4 binding sites, corresponding to the regulatory elements UASp1 and UASp2 at the PHO5 promoter, but only a single, dispensable Pho2 binding site had previously been identified . We have reinvestigated binding of Pho2 to the PHO5 promoter using purified recombinant protein and have found multiple Pho2 binding sites of different affinities along the promoter . One of the high affinity Pho2 sites largely overlaps the Pho4 binding site at UASp1 . Cooperative DNA binding of the two proteins to their overlapping sites, resulting in a high-affinity ternary complex, was demonstrated . Pho2 and Pho4 also bind DNA cooperatively at UASp2 where two Pho2 sites flank the Pho4 site . Finally, Pho2 facilitates binding of Pho4 to a third, cryptic Pho4 binding site which binds Pho4 with lower affinity than UASp1 or UASp2 . These results suggest that cooperative DNA binding with Pho4 is integral to the mechanism by which Pho2 regulates transcription of the PHO5 gene.

Anal Biochem, 1996 Nov 15, 242(2), 248 - 54
Separation by blue native and colorless native polyacrylamide gel electrophoresis of the oxidative phosphorylation complexes of yeast mitochondria solubilized by different detergents: specific staining of the different complexes; Grandier-Vazeille X et al.; Blue native polyacrylamide gel electrophoresis (BN-PAGE) or colorless native polyacrylamide gel electrophoresis (CN-PAGE) allowed separation of the oxidative phosphorylation complexes of yeast mitochondria . These complexes were characterized by specific staining related to their enzymatic activity . Solubilization of mitochondria by different nonionic detergents such as Triton X-100, dodecyl maltoside, Nonidet P-40, Lubrol, octyl glucoside, or Hecameg led to the separation of F1-FO ATPase complexes exhibiting distinct apparent molecular masses related to different contaminating proteins and lipids . All these different forms were active in ATP hydrolysis as revealed directly on the gel . Analysis of the subunit composition of these complexes was carried out by a two-dimensional Tricine-SDS-PAGE and showed that the purest F1-FO ATPase complex was obtained with Lubrol, whereas with Hecameg and octyl glucoside, only the F1 part of ATPase was solubilized.

J Biol Chem, 1996 Nov 15, 271(46), 29094 - 9
Structurally related Spc1p and Spc2p of yeast signal peptidase complex are functionally distinct; Mullins C et al.; Two subunits of the mammalian signal peptidase complex, SPC12 and SPC25, share similar membrane topologies with the majority of each protein oriented toward the cytoplasm . Such similarities may suggest that these proteins perform redundant functions in signal peptidase activity . In the present study, we addressed this issue through analysis of the yeast homologs to SPC12 and SPC25, Spc1p and Spc2p . We show that both Spc1p and Spc2p are nonessential for signal peptidase activity and growth of yeast cells and that null mutations in the genes encoding Spc1p and Spc2p are synthetically lethal with a conditional mutation affecting Sec11p, an essential subunit of yeast signal peptidase . However, a high copy plasmid encoding Spc1p suppresses the conditional sec11 mutation, whereas the corresponding plasmid encoding Spc2p does not suppress sec11 . Moreover, Spc2p, but not Spc1p, is important for signal peptidase activity and cell viability at high temperatures . These results indicate that although both Spc1p and Spc2p are noncatalytic, they are functionally distinct . Evidence is also presented that a double mutant lacking Spc1p and Spc2p grows well relative to wild type yeast cells, indicating that the signal peptidase complex missing at least two of its subunits is sufficient for signal peptidase activity in vivo.

J Biol Chem, 1996 Nov 15, 271(46), 29029 - 33
The yeast HAL2 nucleotidase is an in vivo target of salt toxicity; Murguia JR et al.; The yeast halotolerance gene HAL2 encodes a nucleotidase that dephosphorylates 3'-phosphoadenosine 5'-phosphate (PAP) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), intermediates of the sulfate assimilation pathway . This nucleotidase is inhibited by Na+ and Li+ but not by K+ . Incubation of wild-type yeast cells with NaCl and LiCl, but not with KCl, increased intracellular PAP to millimolar concentrations . No depletion of the pool of adenine nucleotides (AMP, ADP, ATP) was observed . Other stresses such as heat shock or oxidative stress did not result in PAP accumulation . PAPS concentrations also increased during salt stress but remained lower than 0.5 microM . S-Adenosylmethionine concentrations decreased by 50%, reflecting inhibition of sulfate assimilation during salt stress . Salt-induced PAP accumulation was attenuated in a yeast strain overexpressing HAL2 . This strain grew better than the wild type under salt stress . These results suggest that the cation sensitivity of the HAL2 nucleotidase is an important determinant of the inhibition of yeast growth by sodium and lithium salts . In addition to blocking sulfate assimilation by product inhibition of PAPS reductase, PAP accumulation may have other unidentified toxic effects.

J Biol Chem, 1996 Nov 15, 271(46), 28831 - 6
The yeast copper/zinc superoxide dismutase and the pentose phosphate pathway play overlapping roles in oxidative stress protection; Slekar KH et al.; In Saccharomyces cerevisiae, loss of cytosolic superoxide dismutase (Sod1) results in several air-dependent mutant phenotypes, including methionine auxotrophy and oxygen sensitivity . Here we report that these two sod1Delta phenotypes were specifically suppressed by elevated expression of the TKL1 gene, encoding transketolase of the pentose phosphate pathway . The apparent connection between Sod1 and the pentose phosphate pathway prompted an investigation of mutants defective in glucose-6-phosphate dehydrogenase (Zwf1), which catalyzes the rate-limiting NADPH-producing step of this pathway . We confirmed that zwf1Delta mutants are methionine auxotrophs and report that they also are oxygen-sensitive . We determined that a functional ZWF1 gene product was required for TKL1 to suppress sod1Delta, leading us to propose that increased flux through the oxidative reactions of the pentose phosphate pathway can rescue sod1 methionine auxotrophy . To better understand this methionine growth requirement, we examined the sulfur compound requirements of sod1Delta and zwf1Delta mutants, and noted that these mutants exhibit the same apparent defect in sulfur assimilation . Our studies suggest that this defect results from the impaired redox status of aerobically grown sod1 and zwf1 mutants, implicating Sod1 and the pentose phosphate pathway as being critical for maintenance of the cellular redox state.

Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 12986 - 91
Expression in yeast of binding regions of karyopherins alpha and beta inhibits nuclear import and cell growth; Enenkel C et al.; Using truncated forms of recombinant yeast karyopherins alpha and beta in in vitro binding assays, we mapped the regions of karyopherin alpha that bind to karyopherin beta and the regions of karyopherin beta that interact with karyopherin alpha and with Ran-GTP . Karyopherin alpha's binding region for karyopherin beta was localized to its N-terminal domain, which contains several clusters of basic residues, whereas karyopherin beta's binding region for karyopherin alpha was localized to an internal region containing two clusters of acidic residues . Karyopherin beta's binding region for Ran-GTP overlaps with that for karyopherin alpha and comprises at least one of the two acidic clusters required for karyopherin alpha binding in addition to further downstream determinants not required for karyopherin alpha binding . Overexpression in yeast of fragments containing either karyopherin beta's binding region for alpha and Ran-GTP or karyopherin alpha's binding region for beta resulted in sequestration of most of the cytosolic karyopherin alpha or karyopherin beta, respectively, in complexes containing the truncated proteins . As these binding region-containing fragments lack other domains required for function of the corresponding protein, the overexpression of either fragment also inhibited in vivo nuclear import of a model reporter protein as well as cell growth.

Proc Natl Acad Sci U S A, 1996 Nov 12, 93(23), 12845 - 50
Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3; Yang WM et al.; YY1 is a mammalian zinc-finger transcription factor with unusual structural and functional features . It has been implicated as a positive and a negative regulatory factor that binds to the CCATNTT consensus DNA element located in promoters of many cellular and viral genes . A mammalian cDNA that encodes a YY1-binding protein and possesses sequence homology with the yeast transcriptional factor RPD3 has been identified . A Gal4 DNA binding domain-mammalian RPD3 fusion protein strongly represses transcription from a promoter containing Gal4 binding sites . Association between YY1 and mammalian RPD3 requires a glycine-rich region on YY1 . Mutations in this region abolish the interaction with mammalian RPD3 and eliminate transcriptional repression by YY1 . These data suggest that YY1 negatively regulates transcription by tethering RPD3 to DNA as a cofactor and that this transcriptional mechanism is highly conserved from yeast to human.

J Biol Chem, 1996 Nov 8, 271(45), 28366 - 74
Binding of centrins and yeast calmodulin to synthetic peptides corresponding to binding sites in the spindle pole body components Kar1p and Spc110p; Geier BM et al.; Centrins contain four potential Ca2+ binding sites, known as EF-hands, and have essential functions in centrosome duplication and filament contraction . Here we report that centrins from yeast, green algae, and humans bound with high affinity to a peptide of the yeast centrosomal component Kar1p . Interestingly, centrin binding was regulated by physiological relevant changes in {Ca2+}, and this Ca2+ dependence was influenced by acidic amino acids within the Kar1p peptide, which also prevented efficient binding of the related yeast calmodulin . However, a hybrid protein with the third and fourth EF-hands from the yeast centrin Cdc31p and the amino-terminal half from yeast calmodulin behaved more like Cdc31p, indicating that the carboxyl-terminal half of Cdc31p influences binding specificity . Besides Kar1p, centrins bound to a yeast calmodulin binding site, explaining the dosage-dependent suppression of a calmodulin mutant by CDC31 . Consistent with an essential role of Ca2+ for centrin functions, mutations in the first or the fourth EF-hands of Cdc31p, impairing the Ca2+-induced conformational change of Cdc31p, resulted in nonfunctional proteins in vivo . Our results suggest that centrins are involved in Ca2+ signaling, likely by influencing the properties of target proteins in response to changes in {Ca2+}.

J Biol Chem, 1996 Nov 8, 271(45), 27987 - 90
Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair; Johnson RE et al.; DNA mismatch repair plays a key role in the maintenance of genetic fidelity . Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer . The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor . Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation . Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30-104 mutation impairs MSH2/MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair . MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches . Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.

J Biol Chem, 1996 Nov 8, 271(45), 27983 - 6
Yeast Rad51 recombinase mediates polar DNA strand exchange in the absence of ATP hydrolysis; Sung P et al.; Saccharomyces cerevisiae RAD51 gene is required for genetic recombination and recombinational repair of DNA strand breaks . Rad51 protein has a DNA-dependent ATPase activity, and it catalyzes ATP-dependent pairing and strand exchange between homologous DNA molecules . We show here that the rad51 Arg-191 protein, which is devoid of ATPase activity, mediates the pairing and strand exchange reaction upon binding ATP . In addition, the wild type Rad51 protein can catalyze pairing and strand exchange in the presence of the nonhydrolyzable ATP analogues adenylyl-imidodiphosphate and adenosine 5'-O-thiotriphosphate . Thus, homologous pairing and the unidirectional transfer of greater than 5 kilobases of DNA can occur efficiently without the need for nucleotide hydrolysis . Consistent with the results from the biochemical analyses, expression of the rad51 Arg-191 protein in a rad51 null mutant confers normal cellular resistance to the DNA damaging agent methylmethane sulfonate, suggesting that nucleotide binding by Rad51 is sufficient for biological function.

Oncogene, 1996 Nov 7, 13(9), 1859 - 66
ECA39 is regulated by c-Myc in human and by a Jun/Fos homolog, Gcn4, in yeast; Ben-Yosef T et al.; myc oncogenes are transcription factors regulating the level of expression of other genes . Using a subtraction/coexpression strategy, a murine genetic target for Myc regulation was isolated . To further characterize this target gene, named ECA39, we have recently isolated the human, nematode and budding yeast homologs of the mouse gene . The recognition site for Myc binding, located 3' to the start site of transcription in the mouse gene, is conserved in the human homolog . Transfection experiments demonstrated that the Myc binding site of the human gene, mediates activation of a reporter gene in response to over-expression of c-myc . The activation was better executed when the c-Myc binding element was positioned downstream to the promoter, which is the usual position of the c-Myc DNA binding element in its genetic targets . The tissue specific expression of human ECA39 during embryogenesis is similar to that of the mouse homolog . Moreover, ECA39 is expressed in c-myc induced human tumors . It is expressed in Burkitt's lymphoma (where c-myc is translocated and activated) but not in non Burkitt's B-cell lymphoma or in T-cell lymphoma . Thus, it seems that ECA39 is a target for c-myc oncogenesis in humans . In yeast, where c-myc is absent, the ECA39 sequences lack the c-Myc binding element . However, the promoter region of the yeast ECA39 harbors several Gcn4 binding elements . Moreover, ECA39 is markedly down regulated in cells deleted for gcn4, and deletion of Gcn4 binding elements down regulated the transcription from ECA39 promoter . We thus suggest that ECA39 is a target for c-Myc regulation in mammals, while in yeast the regulator is not c-Myc but the c-Jun/c-Fos homolog - Gcn4.

Mol Psychiatry, 1996 Nov, 1(5), 376 - 9
Mad cows meet mad yeast: the prion hypothesis; Lindquist S; Obscure work on the inheritance of two peculiar genetic traits in yeast has recently collided with work on infectious, neurodegenerative diseases in mammals . The impact illuminates both fields . The yeast work reveals a new mechanism of genetic variation that is based on differences in protein conformation rather than differences in nucleic acid . In the light of this work the prion hypothesis for transmissible neurodegenerative disease no longer seems so bizarre . Indeed, the mammalian prion hypothesis is now placed within a larger framework that is likely to be of universal importance in biology . Yeast prions, as the genetic elements are called, can be cured, offering new insights into the molecular processes involved in protein-based inheritance and new hope for the treatment of neurodegenerative diseases in man.

Genetika, 1996 Nov, 32(11), 1528 - 35
{Polymorphism of virus-like particles of retrotransposons in Drosophila and yeast cells}; Zolotova LI et al.; Data on the molecular arrangement of viruslike particles (VLPs) of yeast and Drosophila retrotransposons are presented . Two methods for identifying VLPs from specific retrotransposon families have been offered . The first method is based on VLPs fractionation by electrophoresis in agarose gel under strictly controlled conditions . VLPs of the Drosophila melanogaster retrotransposon families copia and gypsy and D . virilis retrotransposon Tv1 were identified by this method . The method based on heterologous induction of retrotransposons in cells of the mutant spt3 strain of Saccharomyces cerevisiae was used to identify VLPs of yeast retrotransposon Tyl and D . melanogaster gypsy retrotransposon.

Biofizika, 1996 Nov-Dec, 41(6), 1193 - 200
{Study of yeast phosphoglycerate kinase by electron paramagnetic resonance . 1 . Modification of protein SH-group by spin labels . Conformational changes of enzyme}; Vlasova II et al.; The conditions for modifications of a single SH-group of phosphoglycerate kinase from yeast by maleimid spin label and the mercury-containing spin labels were defined . The time course of immobilization of mercury containing paramagnetic radicals was obtained . Based on changes in EPR spectra we calculated dissociation constants for complexes of phosphoglycerate kinase with 3-phosphoglycerate and sulfate ions . Distinct substances were shown to induce different changes in enzyme conformation . It was concluded that approach of enzyme domains is caused by the binding of 3-phosphoglycerate-a specific substrate of this enzyme.

Bioorg Med Chem, 1996 Nov, 4(11), 1949 - 61
Macromolecular recognition: effect of multivalency in the inhibition of binding of yeast mannan to concanavalin A and pea lectins by mannosylated dendrimers; Page D et al.; The synthesis and binding properties of a new family of high affinity alpha-D-mannopyranoside ligands are described . The synthesis of the new multivalent ligands is based on the scaffolding of multiantennary branches of L-lysine residues having electrophilic N-chloroacetylated end groups as core structures . An alpha-D-mannopyranoside with p-substituted aryl aglycon ending with a thiol group was prepared and covalently attached to each of the branches of the dendritic structures . The resulting glycodendrimers with 2 (12), 4 (14), 8 (16), and 16 (18) mannoside residues were tested for their relative inhibitory potency by solid-phase enzyme-linked lectin assays (ELLA) using methyl and p-nitrophenyl alpha-D-mannopyranosides as standards . Concentrations necessary for 50% inhibition (IC50s) of binding of yeast mannan to Jack bean phytohemagglutinin (Canavalia ensiformis, concanavalin A) and to pea lectin (Pisum sativum) were determined . Analogous mannosylated copolyacrylamides were also prepared for comparison . The IC50 values were also plotted as a function of dendrimer valencies . The inhibitions showed 16-mer 18 to be approximately 600- and 2000-fold more potent than methyl alpha-D-mannopyranoside, and 66- and 1383-fold more potent than p-nitrophenyl alpha-D-mannopyranosides with Con A and pea lectins, respectively . Even when these numbers are expressed relative to single mannopyranoside residues per dendrimers, the relative potencies against the aromatic mannoside are still 4- and 86-fold better against Con A and pea lectins . These results unequivocally indicate that the optimum inhibitory binding properties of the new mannosylated dendrimers vary with both dendrimers and lectin valencies.

Appl Microbiol Biotechnol, 1996 Nov, 46(4), 365 - 70
Overexpression of binding protein and disruption of the PMR1 gene synergistically stimulate secretion of bovine prochymosin but not plant thaumatin in yeast; Harmsen MM et al.; When the heterologous proteins thaumatin and bovine prochymosin are produced in yeast cells as a fusion with the yeast invertase secretory signal peptide, less than 2% of the product is secreted in a biologically active form into the medium . The remainder accumulates intracellularly in a misfolded conformation . We investigated whether this poor secretion can be improved by overexpression of binding protein (BiP) one of the major chaperones in eukaryotic cells . Indeed, a tenfold increase in the level of binding protein, as a result of the introduction of extra copies of the kar2 gene into yeast cells containing a single, integrated copy of the invertase/prochymosin fusion gene, caused more than a 20-fold increase in the amount of extracellular prochymosin . By additional disruption of the PMR1 gene of these cells we were able to obtain secretion of virtually all of the prochymosin produced . Export of thaumatin, on the other hand, was not significantly stimulated by binding protein overexpression.

Trends Biochem Sci, 1996 Nov, 21(11), 433 - 8
Making sense of nonsense in yeast; Ruiz-Echevarria MJ et al.; Messenger RNA (mRNA) degradation is a process that plays an important role in the regulation of gene expression and can be linked to translation . Study of the nonsense-mediated mRNA decay pathway has greatly aided our understanding of the link between these processes . Evidence indicates that this pathway regulates the abundance of both aberrant and wild-type transcripts . Factors involved in this pathway have been identified and recent results indicate that they might also be involved in modulating translation . Here, we discuss the mechanism of nonsense-mediated mRNA decay in the yeast Saccharomyces cerevisiae and the potential role that this pathway can have on the regulation of gene expression.

Trends Genet, 1996 Nov, 12(11), 467 - 71
Maintenance and inheritance of yeast prions; Tuite MF et al.; The unusual genetic behaviour of two yeast extrachromosomal elements {PSI} and {URE3} is entirely consistent with a prion-like mechanism of inheritance involving an autocatalytic alteration in the conformation of a normal cellular protein . In the case of both yeast determinants the identity of the underlying cellular prion protein is known . The discovery that the molecular chaperone Hsp104 is essential for the establishment and maintenance of the {PSI} determinant provides an explanation for several aspects of the puzzling genetic behaviour of these determinants . What remains to be explained is whether these determinants represent 'disease states' of yeast or represent the first examples of a unique mechanism for producing a heritable change in phenotype without an underlying change in genotype.

J Microsc, 1996 Nov, 184 ( Pt 2), 81 - 7
Improved ultrastructural preservation of yeast cells for scanning electron microscopy; Hanschke R et al.; The processing of yeast cells for scanning electron microscopy by conventional sequential fixation with glutaraldehyde and osmium tetroxide and subsequent dehydration and critical point-drying caused pronounced deformation and visible shrinkage in all basidiomycetous and ascomycetous yeast strains studied . The mean cell diameter decreased to nearly 60 and 70%, respectively . After an additional sequential fixation with 1% tannic acid and 0.5% uranyl acetate the cell shrinkage was significantly reduced, but the most important result was a considerable reduction of wrinkling and deformation of the yeast cells.

Protein Eng, 1996 Nov, 9(11), 1055 - 61
A novel yeast expression/secretion system for the recombinant plant thiol endoprotease propapain; Ramjee MK et al.; A new high-yield yeast expression/secretion system has been adapted for the plant thiol endoprotease papain . The propapain gene, obtained from Carica papaya fruit, is expressed in the yeast Saccharomyces cerevisiae . The gene was cloned into a FLAG epitope-tagging expression vector downstream of the yeast alpha mating factor (alpha-factor) secretion signal sequence . Expression of the heterologous propapain in yeast is controlled by the glucose-repressible alcohol dehydrogenase isoenzyme II promoter (ADH2) . Glycosylated FLAG-tagged propapain is secreted by a so-called 'super secretor' strain, pmr1 (ssc1), into the culture supernatant where it accumulates to approximately 1.7 mg/l . The proregion contains three consensus N-linked glycosylation sites, whereas there are only two such sites in previously reported cDNA sequences . Removal of this third N-linked glycosylation site results in a drastic reduction in the level of protease activity present in the culture supernatant . Two different types of affinity chromatography were used to purify either propapain or papain . The propapain precursor is autoproteolytically activated to mature papain (M(r) = 24 kDa) using conditions reported previously . The kinetic parameters obtained agree well with the literature values . The yields of active papain are 10-fold higher than those previously reported for propapain in other yeast or bacterial expression systems . This, together with the ease with which mutant proteins can be made, makes this yeast advantageous for a structure-function analysis of recombinant wild-type and mutant papain, and possibly for other related cysteine proteases as well.

Plant Cell, 1996 Nov, 8(11), 2079 - 91
An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase; Ueda T et al.; The Arabidopsis Ara proteins belong to the Rab/Ypt family of small GTPases, which are implicated in intracellular vesicular traffic . To understand their specific roles in the cell, it is imperative to identify molecules that regulate the GTPase cycle . Such molecules have been found and characterized in animals and yeasts but not in plants . Using a yeast system, we developed a novel method of functional screening to detect interactions between foreign genes and identified this Rab regulator in plants . We found that the expression of the ARA4 gene in yeast ypt mutants causes exaggeration of the mutant phenotype . By introducing an Arabidopsis cDNA library into the ypt1 mutant, we isolated a clone whose coexpression overcame the deleterious effect of ARA4 . This gene encodes an Arabidopsis homolog of the Rab GDP dissociation inhibitor (GDI) and was named AtGDI1 . The expression of AtGDI1 complemented the yeast sec19-1 (gdi1) mutation . AtGDI1 is expressed almost ubiquitously in Arabidopsis tissues . The method described here indicates the physiological interaction of two plant molecules, Ara4 and GDI, in yeast and should be applicable to other foreign genes.

Yeast, 1996 Nov, 12(14), 1483 - 92
The sequence of 55 kb on the left arm of yeast chromosome XVI identifies a small nuclear RNA, a new putative protein kinase and two new putative regulators; Purnelle B et al.; We have sequenced and analysed a 55786 bp fragment located on the left arm of chromosome XVI of Saccharomyces cerevisiae . The sequence contains 29 non-overlapping open reading frames (ORFs) longer than 300 bp, among which 12 genes have previously been sequenced: OYE3, REV3, SVS1, BEM4, CDC60, KIP2, PEP4, SPK1, PAL1, KES1, SNR17B and RPL37A . Three new ORFs, P2591, P2594 and P2597 are highly homologous to the human phosphotyrosyl phosphatase activator PTPA, to the pleiotropic regulator PRL1 of PP1 and PP2a protein phosphatases in plants and to the protein kinase PAR-1 in Caenorhabditis elegans, respectively . Three other ORFs, P2545, P2567 and P2578 have significant homology with ORFs of unknown function located on yeast chromosomes VIII, XVI and IV respectively.

Yeast, 1996 Nov, 12(14), 1475 - 81
Nucleotide sequence analysis of a 40 kb segment on the right arm of yeast chromosome XV reveals 18 open reading frames including a new pyruvate kinase and three homologues to chromosome I genes; Purnelle B et al.; We have determined the nucleotide sequence of a 40 kb fragment from the right arm of chromosome XV of Saccharomyces cerevisiae . Subsequent analysis revealed 18 non-overlapping open reading frames (ORFs) numbered from 06257 to 06357, an ARS, two tRNA genes and a Ty2 with its flanking elements . Ten ORFs have been sequenced previously: TEA1, RPA43, RPA190, SGC1 (also called TYE7) REV1, PUT4, CIN1, MNE and MRE4 (also called MEK1) . Among the others, two seem to code for a new pyruvate kinase and for a new ubiquitin-conjugating enzyme; three have interesting homology with genes located on the left arm of chromosome I . This similarity with chromosome I extends to the left of the sequence presented here (Parle et al., submitted to Yeast) . The homologous genes on the two chromosomes are placed in the same relative order.

Yeast, 1996 Nov, 12(14), 1471 - 4
Sequencing analysis of a 40.2 kb fragment of yeast chromosome X reveals 19 open reading frames including URA2 (5' end), TRK1, PBS2, SPT10, GCD14, RPE1, PHO86, NCA3, ASF1, CCT7, GZF3, two tRNA genes, three remnant delta elements and a Ty4 transposon; Cziepluch C et al.; The complete sequence of a 40247 bp DNA segment located on the left arm of chromosome X of Saccharomyces cerevisiae has been determined and analysed . The sequence encodes the 5' coding region of the URA2 gene and 18 open reading frames of at least 100 amino acids . Ten of these correspond to known genes, whereas eight correspond to new genes . In addition, the sequence contains a tRNA-Ala gene, a tRNA-Asp gene, a Ty4 transposable element and three delta elements.

Yeast, 1996 Nov, 12(14), 1439 - 57
New vectors for combinatorial deletions in yeast chromosomes and for gap-repair cloning using 'split-marker' recombination; Fairhead C et al.; New tools are needed for speedy and systematic study of the numerous genes revealed by the sequence of the yeast genome . We have developed a novel transformation strategy, based on 'split-marker' recombination, which allows generation of chromosomal deletions and direct gene cloning . For this purpose, pairs of yeast vectors have been constructed which offer a number of advantages for large-scale applications such as one-step cloning of target sequence homologs and combinatorial use . Gene deletions or gap-repair clonings are obtained by cotransformation of yeast by a pair of recombinant plasmids . Gap-repair vectors are based on the URA3 marker . Deletion vectors include the URA3, LYS2 and kanMX selection markers flanked by I-Scel sites, which allow their subsequent elimination from the transformant without the need for counter-selection . The application of the "split-marker' vectors to the analysis of a few open reading frames of chromosome XI is described.

Yeast, 1996 Nov, 12(14), 1427 - 38
The yeast Rad6 protein: a mediator of homologous recombination across the scaffold attached region at the replication origin ARS1; Markvart MB et al.; Here we show that the ubiquitin-conjugating enzyme Rad6p plays a crucial role in locus-specific replacement recombination in the TRP1-ARS1 region . In rad6-1 strains, where this ubiquitination activity is modified, homologous recombination across a 150 bp continuous region is completely abolished . Our results unambiguously identified the ARS1 scaffold attached region (SAR) as being the region where this impediment for replacement recombination is located, since a merging of the location of the recombination impediment and binding properties in a scaffold exchange assay with deletion mutations was observed . Our observations strongly support the notion of torsionally separated chromosomal domains being organized by SARs and scaffold proteins, and being dynamically realigned as a consequence of ubiquitination and proteolysis.

Yeast, 1996 Nov, 12(14), 1421 - 5
Cloning and identification of HEM14, the yeast gene for mitochondrial protoporphyrinogen oxidase; Glerum DM et al.; A respiratory-defective mutant (C54) of Saccharomyces cerevisiae was found to have a phenotype consistent with a mutation in either mitochondrial protoporphyrinogen oxidase or ferrochelatase . The mutant is grossly deficient in hemes, accumulates protoporphyrin and is rescued by exogenous heme . The increased levels of protoporphyrin at the expense of heme is indicative of a block in one of the two last steps of the heme biosynthetic pathway . Complementation of C54 by a known ferrochelatase mutant suggested that the defect was most likely in HEM14 encoding protoporphyrinogen oxidase . A plasmid capable of complementing C54 was obtained by transformation with a yeast genomic plasmid library . A partial sequence of the insert identified the gene as reading frame YER014 of yeast chromosome V (GenBank Accession Number U18778) . This reading frame codes for a protein homologous to human protoporphyrinogen oxidase . Disruption of this gene elicits a respiratory defect and accumulation of protoporphyrin . The phenotype of the null mutant together with the homology of YER014p to human protoporphyrinogen oxidase provide compelling evidence that YER014 is HEM14.

Genes Dev, 1996 Nov 1, 10(21), 2769 - 81
Yeast HMG proteins NHP6A/B potentiate promoter-specific transcriptional activation in vivo and assembly of preinitiation complexes in vitro; Paull TT et al.; Nonhistone proteins 6A and 6B (NHP6A/B) are nonsequence-specific DNA-binding proteins from Saccharomyces cerevisiae that are related structurally and functionally to the mammalian high mobility group proteins 1 and 2 . These DNA architectural proteins distort DNA structure severely and have been shown to promote assembly of specialized recombination complexes . Here we show that the yeast NHP6A/B proteins are required for the induction of a subset of genes transcribed by RNA polymerase II (pol II) . Activation of the CUP1, CYC1, GAL1, and DDR2 genes was decreased or abolished completely in the delta nhp6A/B strain . No significant change in basal expression was observed for any of the 10 genes examined . Analysis of chimeric gene constructs localized the regions dependent on NHP6A/B to be primarily at the core promoters, although the GAL1 UAS also requires NHP6A/B for activity . In vitro, NHP6A stimulated transcription by pol II at the GAL1 promoter three- to fivefold above the level of activation by GAL4-VP16 alone . Gel mobility shift assays showed that NHP6A promotes the formation of a complex with TBP and TFIIA at the TATA box that has enhanced affinity for TFIIB.

Genes Chromosomes Cancer, 1996 Nov, 17(3), 166 - 71
Identification of a yeast artificial chromosome spanning the 8q12 translocation breakpoint in pleomorphic adenomas with t(3;8)(p21;q12); Roijer E et al.; A subgroup of pleomorphic adenomas of the salivary glands is characterized by translocations involving chromosome 8, with consistent breakpoints at 8q12 . As part of a positional cloning effort to isolate the gene(s) affected by these translocations we now report the mapping of the 8q12 breakpoint in two primary pleomorphic adenomas with the recurrent t(3;8)(p21;q12) . Yeast artificial chromosome (YAC) clones corresponding to eight different loci in 8q11-12 were isolated and mapped by fluorescence in situ hybridization (FISH) . The t(3;8) breakpoint was mapped within a 1 Mb region flanked by MOS proximally and by the genetic marker D8S166 distally . One YAC within this region was shown to span the t(3;8) breakpoint in two tumors . This YAC will provide an excellent tool for isolating the gene(s) at the breakpoint(s) in adenomas with t(3;8).

Eur J Biochem, 1996 Nov 1, 241(3), 797 - 804
Oxidation of tienilic acid by human yeast-expressed cytochromes P-450 2C8, 2C9, 2C18 and 2C19 . Evidence that this drug is a mechanism-based inhibitor specific for cytochrome P-450 2C9; Jean P et al.; Oxidation of tienilic acid by human cytochromes P-450 (CYP) 2C9, 2C18, 2C8 and 2C19 was studied using recombinant enzymes expressed in yeast . CYP 2C9 was the best catalyst for 5-hydroxylation of tienilic acid (K(m) = 5 +/- 1 microM, kcat = 1.7 +/- 0.2 min-1), 30-fold more potent in terms of kcat/K(m) than CYP 2C18 (K(m) = 150 +/- 15 microM, kcat = 1.8 +/- 0.2 min-1) and 300-fold more potent than CYP 2C8 (K(m) = 145 +/- 15 microM, kcat = 0.2 +/- 0.1 min-1) . CYP 2C19 was unable to catalyze this hydroxylation under our experimental conditions . During this study, a marked effect of the ionic strength on the activities (hydroxylations of tienilic acid and tolbutamide) of these cytochromes P-450 expressed in the yeast strain 334 was observed . The effect was particularly great in the case of CYP 2C18, with a tenfold decrease of activity upon increasing ionic strength from 0.02 to 0.1 . Specific-covalent binding of tienilic acid metabolites to cytochrome P-450 (incubations in the presence of 5 mM glutathione) was markedly higher upon tienilic acid oxidation by CYP 2C9 than by CYP 2C18 and CYP 2C8 . Mechanism-based inactivation of cytochrome P-450 during tienilic acid oxidation was observed in the case of CYP 2C9 but was not detectable with CYP 2C18 and CYP 2C8 . Tienilic acid thus appears to be a mechanism-based inhibitor specific for CYP 2C9 in human liver . Experiments performed with human liver microsomes confirmed that tienilic acid 5-hydroxylase underwent a time-dependent inactivation (apparent t1/2 = 10 +/- 5 min) during 5-hydroxylation of tienilic acid.

Curr Biol, 1996 Nov 1, 6(11), 1426 - 34
Glucose repression/derepression in budding yeast: SNF1 protein kinase is activated by phosphorylation under derepressing conditions, and this correlates with a high AMP:ATP ratio; Wilson WA et al.; BACKGROUND: Genetic studies of Saccharomyces cerevisiae have shown that Snf1p and Snf4p, which together form the SNF1 complex, are essential for gene derepression on removal of glucose from the medium . However the metabolic signal(s) involved, and the exact role of SNF1, have remained enigmatic . Recently, the AMP-activated protein kinase (AMPK) was shown to be the mammalian homologue of SNF1 . AMPK is activated by the elevation of the cellular AMP:ATP ratio, which occurs during cellular stress in mammalian cells . The mechanism of activation involves phosphorylation of AMPK by an upstream protein kinase (AMPKK) . We have investigated whether a similar mechanism might explain the role of SNF1 in yeast in the response to the stress of glucose starvation . RESULTS: The protein kinase activity of SNF1 was dramatically and rapidly activated by phosphorylation on removal of glucose from the medium . SNF1 was not activated directly by AMP, but could be inactivated by protein phosphatases and reactivated by mammalian AMPKK . We also demonstrated that an endogenous SNF1-reactivating factor, most likely an upstream protein kinase, is present in yeast extracts . Under a variety of different growth conditions, there was a correlation between cellular adenine nucleotide levels and the activation state of SNF1 . CONCLUSIONS: Apart from the lack of direct allosteric activation of SNF1 by AMP, the regulation of the mammalian AMPK and yeast SNF1 protein kinase cascades is highly conserved . Adenine nucleotides are now good candidates for metabolic signals which indicate the lack of glucose in the medium, triggering activation of SNF1 and derepression of glucose-repressed genes.

Plant Physiol, 1996 Nov, 112(3), 1101 - 9
A1 toxicity in yeast . A role for Mg?
MacDiarmid CW, Gardner RC.
We have established conditions in which soluble Al is toxic to the yeast Saccharomyces cerevisiae . The major modifications to a standard synthetic medium were lowering the pH and the concentration of Mg ions . Alterations to the PO4, Ca, or K concentration had little effect on toxicity . Organic acids known to chelate Al reduced its toxicity, suggesting that Al3+ is the toxic Al species . The unique ability of Mg ions to ameliorate Al toxicity led us to investigate the hypothesis that Al inhibits Mg uptake by yeast . Yeast cells accumulate Mg, Co, Zn, Ni, and Mn ions via the same transport system (G.F . Fuhrmann, A . Rothstein {1968} Biochim Biophys Acta 163: 325-330) . Al3+ inhibited the accumulation of 57Co2+ by yeast cells more effectively than Ga, La, or Mg . In addition, a mutant yeast strain with a defect in divalent cation uptake proved to be more sensitive to Al than a wild-type strain . Taken together, these results suggest that Al may cause Mg deficiency in yeast by blocking Mg transport . We discuss the relevance of yeast as a model for the study of Al toxicity in plant systems.

Nucleic Acids Res, 1996 Nov 1, 24(21), 4304 - 12
mRNA turnover in yeast promoted by the MATalpha1 instability element; Caponigro G et al.; The decay rates of eukaryotic transcripts can be determined by sequence elements within an mRNA . One example of this phenomenon is the rapid degradation of the yeast MATalpha1 mRNA, which is promoted by a 65 nt segment of its coding region termed the MATalpha1 instability element (MIE) . The MIE is also capable of destabilizing the stable PGK1 transcript . To determine how the MIE accelerates mRNA turnover we examined the mechanism of degradation of the MATalpha1 transcript . These experiments indicated that the MATalpha1 mRNA was degraded by a deadenylation-dependent decapping reaction which exposed the transcript to 5'-->3' exonucleolytic digestion . Deletion of the MIE from the MATalpha1 mRNA decreased the rate at which this mRNA was decapped . In contrast, insertion of the MIE into the PGK1 transcript caused an increase in the rate of deadenylation of the resulting chimeric mRNA . These observations suggest that the MIE promotes rapid mRNA decay by increasing the rates of deadenylation and decapping, with its primary effect on mRNA turnover depending on additional features of a given transcript . These results also strengthen the hypothesis that deadenylation-dependent decapping is a common pathway of mRNA decay in yeast and indicate that an instability element within the coding region of an mRNA can effect nucleolytic events that occur at both the 5'- and 3'-ends of an mRNA.

Nucleic Acids Res, 1996 Nov 1, 24(21), 4192 - 6
A method for linking yeast artificial chromosomes; Larin Z et al.; A method for linking any standard yeast artificial chromosomes (YAC) is described . YACs are introduced into the same cell and joined by mitotic recombination between the vector arms and the homologous sequence in a linking vector; several YACs can be recombined sequentially . The linking vectors also contain the beta-galactosidase gene as an expression reporter in mammalian cells.

Mol Biol Cell, 1996 Nov, 7(11), 1723 - 35
Interaction between yeast Cdc6 protein and B-type cyclin/Cdc28 kinases; Elsasser S et al.; During purification of recombinant Cdc6 expressed in yeast, we found that Cdc6 interacts with the critical cell cycle, cyclin-dependent protein kinase Cdc28 . Cdc6 and Cdc28 can be coimmunoprecipitated from extracts, Cdc6 is retained on the Cdc28-binding matrix p13-agarose, and Cdc28 is retained on an affinity column charged with bacterially produced Cdc6 . Cdc6, which is a phosphoprotein in vivo, contains five Cdc28 consensus sites and is a substrate of the Cdc28 kinase in vitro . Cdc6 also inhibits Cdc28 histone H1 kinase activity . Strikingly, Cdc6 interacts preferentially with B-type cyclin/Cdc28 complexes and not Cln/Cdc28 in log-phase cells . However, Cdc6 does not associate with Cdc28 when cells are blocked at the restrictive temperature in a cdc34 mutant, a point in the cell cycle when the B-type cyclin/Cdc28 inhibitor p40Sic1 accumulates and purified p40Sic1 inhibits the Cdc6/Cdc28 interaction . Deletion of the Cdc28 interaction domain from Cdc6 yields a protein that cannot support growth . However, when overproduced, the mutant protein can support growth . Furthermore, whereas overproduction of wild-type Cdc6 leads to growth inhibition and bud hyperpolarization, overproduction of the mutant protein supports growth at normal rates with normal morphology . Thus, the interaction may have a role in the essential function of Cdc6 in initiation and in restraining mitosis until replication is complete.

Mol Biol Cell, 1996 Nov, 7(11), 1657 - 66
Cdc28 tyrosine phosphorylation and the morphogenesis checkpoint in budding yeast; Sia RA et al.; A morphogenesis checkpoint in budding yeast delays nuclear division (and subsequent cell cycle progression) in cells that have failed to make a bud . We show that the ability of this checkpoint to delay nuclear division requires the SWE1 gene, encoding a protein kinase that inhibits the master cell cycle regulatory kinase Cdc28 . The timing of nuclear division in cells that cannot make a bud is exquisitely sensitive to the dosage of SWE1 and MIH1 genes, which control phosphorylation of Cdc28 at tyrosine 19 . In contrast, the timing of nuclear division in budded cells does not rely on Cdc28 phosphorylation, suggesting that the morphogenesis checkpoint somehow turns on this regulatory pathway . We show that SWE1 mRNA levels fluctuate during the cell cycle and are elevated in cells that cannot make a bud . However, regulation of SWE1 mRNA levels by the checkpoint is indirect, acting through a feedback loop requiring Swe1 activity . Further, the checkpoint is capable of delaying nuclear division even when SWE1 transcription is deregulated . We propose that the checkpoint delays nuclear division through post-translational regulation of Swe1 and that transcriptional feedback loops enhance the efficacy of the checkpoint.

Curr Genet, 1996 Nov, 30(5), 410 - 6
Genomic complexity and chromosomal rearrangements in wine-laboratory yeast hybrids; Ibeas JI et al.; In this report we describe the genomic complexity of a number of Saccharomyces yeast strains isolated from sherry wine (flor yeasts), and the genomic stability of a yeast hybrid derived from one of these and a laboratory strain . Flor yeast strains largely differed in their DNA content, but showed very few variations their molecular karyotype . These strains contained a large number of Ty2 sequences, but lacking the Ty1 elements commonly found in laboratory strains . The genetic analysis of a flor-laboratory hybrid indicated that flor yeasts were aneuploid . Hybridization patterns obtained with Ty1 and Ty2 probes in the meiotic progeny of this hybrid suggested that recombination may occur not only among homologous chromosomes of similar length, but also among polymorphic partners with different sizes . New chromosomal variants were frequently observed in the meiotic products, suggesting that polymorphism in chromosome length may itself be a major source of karyotypic variation . The genetic analysis of such variants indicated that recombinational events leading to new chromosomal forms may occur both mitotically and meiotically.

Curr Genet, 1996 Nov, 30(5), 381 - 8
Stimulation of mitotic recombination upon transcription from the yeast GAL1 promoter but not from other RNA polymerase I, II and III promoters; Bratty J et al.; Homologous recombination in Saccharomyces cerevisiae and other organisms can be stimulated by transcription . Consistent with this, we find that recombination of a chromosomal ade1 allele with a plasmid-borne ADE1 ORF under the control of the GAL1 promoter increased from 6.1x10(-6) to 1.7x10(-4) when transcription of the plasmid locus was induced by growing the cells in the presence of galactose . Recombination could also be stimulated by over-expressing the Gal4 transcription factor in the presence of the GAL1-ADE1 plasmid, while culturing the cells in dextrose medium . However, when transcription of the same ORF was driven from the highly active promoters of the rDNA (RNA polymerase I), and ADH1 (RNA polymerase II) genes, only background levels of recombination (5-10x10(-6)) were observed, irrespective of the carbon source . Recombination was found to involve integration of the whole plasmid and to depend on RAD51, RAD52 and RAD54 . The results indicate that increased accessibility of transcriptionally active chromatin is not sufficient to cause increased rates of this kind of reciprocal exchange.

Anal Biochem, 1996 Nov 1, 242(1), 68 - 72
A yeast three-hybrid method to clone ternary protein complex components; Zhang J et al.; The yeast two-hybrid system is a powerful technique to detect binary protein interactions . In order to facilitate ternary complex analysis, we have developed the yeast three-hybrid system . Using epidermal growth factor (EGF) receptor, Grb2 and Sos proteins as an example, we demonstrated the use of the three-hybrid system to detect formation of a three-component complex, i.e., the Grb2-mediated interactions between EGF receptor and Sos . We then used the three-hybrid system to screen a library with Gal4-DNA binding domain -EGF receptor and Gal4 activation domain-Sos fusion proteins and isolated a plasmid encoding Grb2 . Thus, the three-hybrid system is useful for characterizing and cloning components of ternary protein complexes.

Mol Endocrinol, 1996 Nov, 10(11), 1358 - 70
Identification of single amino acid substitutions of Cys-736 that affect the steroid-binding affinity and specificity of the glucocorticoid receptor using phenotypic screening in yeast; Lind U et al.; In this study a yeast-screening system has been developed for the isolation of rarely occurring change-of-function missense mutations in defined protein segments that have potential to give more information about the function of mutated residues . Mutagenesis of cysteine-736 was chosen for this initial study because it has been shown previously, by photoaffinity labeling, to lie in close proximity to the bound hormone molecule . After randomization of residue 736 by oligonucleotide-directed mutagenesis, two functional substitutions with serine (C736S) and threonine (C736T) were found . These were further analyzed using transactivation assays in both yeast and mammalian cells and by steroid-binding assays using wild type and mutant proteins expressed in mammalian cells . The C736S protein showed reduced sensitivity to all hormones tested in transactivation assays and a reduced affinity of hormone binding . A correspondence between sensitivity to hormones in transactivation assays and hormone-binding affinity was also observed for the C736T protein . However, in this case the sensitivity to the synthetic hormone triamcinolone acetonide was higher than that for wild type whereas the sensitivity to endogenous hormones was somewhat lower . To test the efficacy of the yeast-screening system in relation to the two informative mutations identified, all 20 alternative substitutions at position 736 were constructed and analyzed . In addition to Ser and Thr, which resulted in change of function, alanine was the only other substitution that resulted in significant activity . The activity of this mutant was indistinguishable from wild type in yeast . Thus we conclude that very conservative substitutions of cysteine-736 (C736A, C736S, and C736T) cause variable effects on hormone binding that distinguish between different glucocorticoid steroid hormones.

Hum Mol Genet, 1996 Nov, 5(11), 1743 - 8
A novel human homologue of yeast nucleosome assembly protein, 65 kb centromeric to the p57KIP2 gene, is biallelically expressed in fetal and adult tissues; Hu RJ et al.; Three genes on 11p15.5 are known to undergo genomic imprinting . The gene for insulin-like growth factor II (IGF2) is normally expressed from the paternal allele, while H19 and p57KIP2, a cyclin-dependent kinase inhibitor, are expressed from the maternal allele . Five germline balanced chromosomal rearrangement breakpoints from patients with Beckwith-Wiedemann syndrome (BWS) have been mapped to 11p15.5 between p57KIP2 and IGF2, and all are derived from the maternal chromosome . By positional cloning from BWS breakpoints, we have isolated a gene 100 kb and 65 kb centromeric to the proximal end of this BWS breakpoint cluster and p57KIP2, respectively . This gene is homologous to yeast nucleosome assembly protein (NAP1) and to a human homologue of NAP1, and we designate it hNAP2 (human nucleosome assembly protein 2) . hNAP2 diverges in its expression pattern from IGF2, H19, and p57KIP2, and it shows biallelic expression in all tissues tested . Thus, hNAP2 is functionally insulated from the imprinting domain of 11p15.

Biochem J, 1996 Nov 1, 319 ( Pt 3), 705 - 12
Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme; Christgau S et al.; Seventeen full-length cDNAs encoding pectin methyl esterase I (PME I) have been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast . Yeast colonies expressing functional PME I were identified on agar plates containing highly esterified pectin, and a cDNA encoding PME I was isolated . The deduced amino acid sequence of PME I is highly similar (74% identity) to the PME from Aspergillus niger . A full-length cDNA encoding PME I was cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for heterologous expression, purification and characterization of the recombinant enzyme . The recombinant PME I had a molecular mass of 36.2 kDa, an isoelectric point of pH 3.8, a pH optimum of 4.6 and a temperature optimum of 45 degrees C . The authentic PME I was purified from A . aculeatus culture supernatant and subjected to amino acid sequencing . The peptide sequences covered 138 amino acid residues and were in complete agreement with the deduced PME I sequence . Both recombinant and authentic PME I were glycosylated, but the composition of the glycan moieties was different . PME I was able to remove 75-85% of the methyl groups in highly methylated pectin, and it did not remove acetyl groups from acetylated polysaccharides . When the enzyme was added together with polygalacturonases to pectin, a rapid depolymerization was observed . By comparison, polygalacturonases alone showed a very limited degradation of the methylated substrate . This demonstrates that PME I acts in synergy with polygalacturonases in the degradation of plant cell wall pectin.

Biochem J, 1996 Nov 1, 319 ( Pt 3), 675 - 81
Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes; Renaud JP et al.; An approach to the quantitative spectral analysis of substrate binding and inactivation of cytochrome P-450 in microsomes is described . The method is based on the application of the principal component analysis technique on the Soret-region spectra measured at different temperatures at various concentrations of substrate . This approach allowed us to study the thermodynamic parameters of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast (Saccharomyces cerevisiae) microsomes . These parameters are discussed in comparison with the values reported earlier by Ristau et al . {(1979) Acta Biol . Med . Ger . 38, 177-185} for rabbit liver cytochrome P-450 2B4 in solution with benzphetamine as a substrate . Our analysis shows the substrate-free states of 2B4 and 3A4 to be very similar . However, substrate binding seems to perturb haem-protein interactions in 3A4 in contrast with 2B4, where the effect of substrate binding on the thermodynamic parameters of spin transitions was insignificant . The implication of the results for the mechanism of substrate-induced spin shift is discussed.

Steroids, 1996 Nov, 61(11), 642 - 6
Differential interaction of natural and synthetic estrogens with extracellular binding proteins in a yeast estrogen screen; Arnold SF et al.; We have used the yeast estrogen (YES) consisting of the human estrogen receptor and a reporter containing two estrogen response elements linked to the lacZ gene to evaluate the interaction between ovarian, phyto-, and synthetic estrogens with extracellular binding proteins . YES was incubated with charcoal-stripped human serum, human sex hormone-binding globulin, or human alpha-fetoprotein in the presence of concentrations of various estrogens that induced a 100% estrogenic response, as measured by beta-galactosidase activity . The activity of estradiol and coumestrol, a phytoestrogen, was reduced 75% with physiological levels of serum, sex hormone-binding globulin, or alpha-fetoprotein . The beta-galactosidase activity of genistein, another phytoestrogen, also decreased with extracellular proteins but to a lower extent than estradiol . In contrast, the activity of the synthetic estrogens diethylstilbestrol, kepone, and p,'p-DDD was only minimally reduced with extracellular proteins . These results indicate a potential fundamental difference in the interaction of estrogens from diverse sources with extracellular binding proteins . This suggests that the capacity for various estrogens to induce estrogen-associated responses is in part regulated by their affinity for extracellular bindings proteins.

Arch Biochem Biophys, 1996 Nov 1, 335(1), 173 - 82
Urea-induced equilibrium unfolding of single tryptophan mutants of yeast phosphoglycerate kinase: evidence for a stable intermediate; Szpikowska BK et al.; Several single-tryptophan mutants of yeast phosphoglycerate kinase (PGK) have been used in the present study to characterize the urea-induced unfolding of PGK . A possibility that residual structures might be present in the urea-unfolded state was also investigated . The urea-induced unfolding transitions were monitored using circular dichroism (CD) and fluorescence techniques . The presence of stable intermediate(s) during urea-induced unfolding is suggested by biphasic transitions detected for the mutants containing tryptophans in the N-terminal domain and by the noncoincidence of transitions detected by various methods for other mutants . The N-terminal tryptophan probes exhibit hyperfluorescent properties in the intermediate state and a wavelength of maximum emission that lies between that of the native and unfolded state . This unfolding intermediate exhibits a major decrease in the ellipticity at 220 nm, but only a minor decrease at 278 nm, relative to the native state . These results suggest a significant loss of secondary structure content and a relatively small change in the asymmetric environment of tyrosine residues . Increased 1-anilinonaphthalene-8-sulfonic acid binding in the denaturant concentration range corresponding to the N --> I transition indicates the presence of a partially folded structure with exposed hydrophobic surfaces . These results demonstrate that the partially folded intermediates detected during urea-induced denaturation are structurally similar to those detected previously during guanidine-induced denaturation . No significant differences were detected between the urea- and guanidine-unfolded proteins on the basis of their fluorescence and CD properties.

Genetics, 1996 Nov, 144(3), 979 - 89
Yeast mutants that produce a novel type of ascus containing asci instead of spores; Xue Z et al.; Tetraploid yeast cells lacking BFR1 or overexpressing an essential gene BBP1 produce a novel type of ascus that contains asci instead of spores . We show here that the asci within an ascus likely arise because a/alpha spores undergo a second round of meiosis . Cells depleted of Bbp1p or lacking Bfr1p are defective in a number of processes such as nuclear segregation, bud formation, cytokinesis and nuclear spindle formation . Furthermore, deletion of BFR1 or overexpression of BBP1 leads to an increase in cell ploidy, indicating that Bfr1p and Bbp1p play roles in both the mitotic cell cycle and meiosis . Bfr1p and Bbp1p interact with each other in a two hybrid assay, further suggesting that they might form a complex important for cell cycle coordination.

Genetics, 1996 Nov, 144(3), 947 - 55
Induction of Ty recombination in yeast by cDNA and transcription: role of the RAD1 and RAD52 genes; Nevo-Caspi Y et al.; In the yeast Saccharomyces cerevisiae ectopic recombination has been shown to occur at high frequencies for artificially created repeats, but at relatively low frequencies for a natural family of repeated sequences, the Ty family . Little is known about the mechanism(s) that prevent recombination between repeated sequences . We have previously shown that nonreciprocal recombination (gene conversion) of a genetically marked Ty can be induced either by the presence of high levels of Ty cDNA or by transcription of the marked Ty from a GAL1 promoter . These two kinds of induction act in a synergistic manner . To further characterize these two kinds of Ty recombination, we have investigated the role played by the RAD52 and RAD1 genes . We have found that the RAD52 and RAD1 gene products are essential to carry out transcription-induced Ty conversion whereas cDNA-mediated conversion can take place in their absence.

J Biol Chem, 1996 Nov 1, 271(44), 27509 - 16
Endoplasmic reticulum glucosidase II is composed of a catalytic subunit, conserved from yeast to mammals, and a tightly bound noncatalytic HDEL-containing subunit; Trombetta ES et al.; Trimming of glucoses from N-linked core glycans on newly synthesized glycoproteins occurs sequentially through the action of glucosidases I and II in the endoplasmic reticulum (ER) . We isolated enzymatically active glucosidase II from rat liver and found that, in contrast with previous reports, it contains two subunits (alpha and beta) . Sequence analysis of peptides derived from them allowed us to identify their corresponding human cDNA sequences . The sequence of the alpha subunit predicted a soluble protein (104 kDa) devoid of known signals for residence in the ER . It showed homology with several other glucosidases but not with glucosidase I . Among the homologues, we identified a Saccharomyces cerevisiae gene, which we showed by gene disruption experiments to be the functional catalytic subunit of glucosidase II . The disrupted yeast strains had no detectable growth defect . The sequence of the beta subunit (58 kDa) showed no sequence homology with other known proteins . It encoded a soluble protein rich in glutamic and aspartic acid with a putative ER retention signal (HDEL) at the C terminus . This suggested that the beta subunit is responsible for the ER localization of the enzyme.

J Cell Biol, 1996 Nov, 135(3), 741 - 51
Role of the ABC transporter Ste6 in cell fusion during yeast conjugation; Elia L et al.; Though early stages of yeast conjugation are well-mimicked by treatment with pheromones, the final degradation of the cell wall and membrane fusion of mating that leads to cytoplasmic mixing may require separate signals . Mutations that blocked cell fusion during mating in Saccharomyces cerevisiae were identified in a multipartite screen . The three tightest mutations proved to be partial-function alleles of the ABC-transporter gene STE6 required for transport of a-factor . The ste6(cefl-1) allele was recovered and sequenced . The ste6(cefl-1) allele contained a stop codon predicted to truncate Ste6 at amino acid residue 862 (of 1290) . The ste6(cef) mutations reduced, but did not eliminate, expression of a-factor . Light and electron microscopy revealed that unlike ste6 null mutations which block mating before the formation of mating pairs, the ste6(cef) (cell fusion) alleles permitted early steps in mating to proceed normally but blocked at a late stage in conjugation where mating partners were encased by a single cell wall and separated by only a thin layer of cell wall material we term the fusion wall . Morphologically the prezygotes appeared symmetrical with successful cell wall fusion at the periphery of the region of cell contact . Responses to a-factor were efficiently induced in partner cells under mating conditions as expected given the symmetric appearance of the prezygotes . A strain expressing a ste6(K1093A) mutation that conferred export of a twofold to fourfold higher level of a-factor than ste6(cef) did not accumulate prezygotes during mating which could indicate a tight threshold of a-factor signaling required for mating . However, mating to an sst2 partner which has a greatly increased sensitivity to a-factor did not suppress the fusion defect of a ste6(cef) strain . Overexpression of the structural gene for a-factor also did not suppress the fusion defect . It is possible that a-factor or STE6 play more complex roles in cell fusion.

J Cell Biol, 1996 Nov, 135(3), 725 - 39
Pea2 protein of yeast is localized to sites of polarized growth and is required for efficient mating and bipolar budding; Valtz N et al.; Saccharomyces cerevisiae exhibits polarized growth during two phases of its life cycle, budding and mating . The site for polarization during vegetative growth is determined genetically: a and alpha haploid cells exhibit an axial budding pattern, and a/alpha diploid cells exhibit a bipolar pattern . During mating, each cell polarizes towards its partner to ensure efficient mating . SPA2 is required for the bipolar budding pattern (Snyder . M 1989 . J . Cell Biol . 108:1419-1429; Zahner, J.A., H.A . Harkins, and J.R . Pringle . 1996 . Mol . Cell . Biol . 16:1857-1870) and polarization during mating (Snyder, M., S . Gehrung, and B.D . Page . 1991 . J . Cell Biol . 114: 515-532) . We previously identified mutants defective in PEA2 and SPA2 which alter cell polarization in the presence of mating pheromone in a similar manner (Chenevert, J., N . Valtz, and I . Herskowitz . 1994 . Genetics, 136:1287-1297) . Here we report the further characterization of these mutants . We have found that PEA2 is also required for the bipolar budding pattern and that it encodes a novel protein with a predicted coiled-coil domain . Pea2p is expressed in all cell types and is localized to sites of polarized growth in budding and mating cells in a pattern similar to Spa2p, Pea2p and Spa2p exhibit interdependent localization: Spa2p is produced in pea2 mutants but fails to localize properly; Pea2p is not stably produced in spa2 mutants . These results suggest that Pea2p and Spa2p function together as a complex to generate the bipolar budding pattern and to guarantee proper polarization during mating.

J Cell Biol, 1996 Nov, 135(3), 623 - 33
A pathway for targeting soluble misfolded proteins to the yeast vacuole; Hong E et al.; We have evaluated the fate of misfolded protein domains in the Saccharomyces cerevisiae secretory pathway by fusing mutant forms of the NH2-terminal domain of lambda repressor protein to the secreted protein invertase . The hybrid protein carrying the wild-type repressor domain is mostly secreted to the cell surface, whereas hybrid proteins with amino acid substitutions that cause the repressor domain to be thermodynamically unstable are retained intracellularly . Surprisingly, the retained hybrids are found in the vacuole, where the repressor moiety is degraded by vacuolar proteases . The following observations indicate that receptor-mediated recognition of the mutant repressor domain in the Golgi lumen targets these hybrid fusions to the vacuole . (a) The invertase-repressor fusions, like wild-type invertase, behave as soluble proteins in the ER lumen . (b) Targeting to the vacuole is saturable since overexpression of the hybrids carrying mutant repressor increases the fraction of fusion protein that appears at the cell surface . (c) Finally, deletion of the VPS10 gene, which encodes the transmembrane Golgi receptor responsible for targeting carboxypeptidase Y to the vacuole, causes the mutant hybrids to be diverted to the cell surface . Together these findings suggest that yeast have a salvage pathway for degradation of nonnative luminal proteins by receptor-mediated transport to the vacuole.

RNA, 1996 Nov, 2(11), 1110 - 23
Interactions of the yeast U6 RNA with the pre-mRNA branch site; McPheeters DS; The small nuclear RNA (snRNA) components of the spliceosome have been proposed to catalyze the excision of introns from nuclear pre-mRNAs . If this hypothesis is correct, then the snRNA components of the spliceosome may interact directly with the reactive groups of pre-mRNA substrates . To explore this possibility, a genetic screen has been used to identify potential interactions between the U6 RNA and the pre-mRNA branch site . Notably, the selection yielded mutants in two regions of the yeast U6 RNA implicated previously in the catalytic events of splicing . These mutants significantly increase the splicing of pre-mRNA substrates containing non-adenosine branch sites . U6 mutants in U2/U6 helix Ia show strong allele-specific interactions with the branch site nucleotide and interact with PRP16, a factor implicated previously in branch site utilization . The other mutants cluster in the intramolecular helix of U6 and suppress the effects of branch site mutations in a nonallele-specific fashion . The locations of these mutants may define positions important for binding of the U6 intramolecular helix to the catalytic core of the spliceosome.

Endocrinology, 1996 Nov, 137(11), 5135 - 43
Immunological identification and localization of yeast aspartic protease 3-like prohormone-processing enzymes in mammalian brain and pituitary; Cawley NX et al.; The novel aspartic proteases, yeast aspartic protease 3 and the mammalian POMC-converting enzyme (PCE), can process prohormones at specific basic residue cleavage sites . We show that an antibody against yeast aspartic protease 3 (YAP3p) cross-reacted with purified bovine PCE on Western blot, indicating structural homology between these two enzymes, but not with other aspartic proteases, such as renin or cathepsin D . A PCE-sized anti-YAP3p-immunoreactive band was detected on Western blots of bovine intermediate lobe where PCE activity has been found . YAP3p antiserum also cross-reacted with a protein of approximately 90 kDa from mouse hypothalamus and anterior pituitary, and bovine anterior pituitary secretory granules . Distribution studies showed the presence of anti-YAP3p-immunopositive cells in bovine pituitary and peptide-rich brain regions, including the mouse arcuate nucleus and hippocampus and the rat supraoptic nucleus, paraventricular nucleus, cortex, striatum, and reticular nucleus . In the bovine intermediate pituitary, a subpopulation of cells was intensely stained with the YAP3p antiserum, and in combination with in situ hybridization, these cells were shown to contain POMC messenger RNA (mRNA) . Only a subpopulation of cells was immunopositive for anti-YAP3p in bovine anterior pituitary, and most of these cells were identified by double immunostaining with ACTH antiserum as corticotrophs . In situ hybridization in combination with immunocytochemistry provided evidence for the localization of arginine vasopressin mRNA in YAP3p-immunopositive neurons in the rat supraoptic nucleus, whereas cholecystokinin mRNA was detected in YAP3p-immunopositive cells in the rat cortex and hippocampus . These results support the hypothesis that YAP3p-like aspartic proteases, including PCE, play a role in prohormone processing in endocrine/neuroendocrine cells in vivo.

Endocrinology, 1996 Nov, 137(11), 4944 - 52
Interaction of SH2-containing protein tyrosine phosphatase 2 with the insulin receptor and the insulin-like growth factor-I receptor: studies of the domains involved using the yeast two-hybrid system; Rocchi S et al.; Activated insulin and insulin-like growth factor-I receptors transmit downstream signals via the insulin receptor substrate (IRS-1 and IRS-2) and a series of proteins containing Src homology-2 (SH2) domains, including SH2-containing protein tyrosine phosphatase 2 (SHP-2) . In the present study, we analyzed in the yeast two-hybrid system the interaction between both receptors and SHP-2 . We found that a catalytically inactive SHP-2 is able to bind to tyrosine-phosphorylated IR beta-subunit and IGF-I R beta-subunit . However, with wild-type SHP-2, we were unable to detect an interaction with these receptors, which is likely to be due to dephosphorylation of the receptors by the phosphatase . Further, our results demonstrate that tyrosine 1322 of the IR, and the corresponding tyrosine 1316 of the IGF-I R are implicated in the interaction with the SHP-2 SH2 domain . At the level of SHP-2, it would appear that both SH2 domains of SHP-2 are necessary for optimal association with either receptor . Finally, using several insulin and IGF-I receptor mutants, we found that the kinase regulatory autophosphorylation sites play an important role in the interaction of these receptors with the SHP-2 SH2 domain . These sites are also necessary for the interaction with full-length IRS-1 . We conclude that 1) the IR and IGF-I R directly interact with SHP-2; 2) the C-terminus autophosphorylation of these receptors sites are involved in this process; and 3) the receptors' kinase autophosphorylation sites are necessary for the interaction with SHP-2 and also with IRS-1.

Mol Cell Biol, 1996 Nov, 16(11), 6524 - 31
Yeast mitochondria lacking the phosphate carrier/p32 are blocked in phosphate transport but can import preproteins after regeneration of a membrane potential; Zara V et al.; Two different functions have been proposed for the phosphate carrier protein/p32 of Saccharomyces cerevisiae mitochondria: transport of phosphate and requirement for import of precursor proteins into mitochondria . We characterized a yeast mutant lacking the gene for the phosphate carrier/p32 and found both a block in the import of phosphate and a strong reduction in the import of preproteins transported to the mitochondrial inner membrane and matrix . Binding of preproteins to the surface of mutant mitochondria and import of outer membrane proteins were not inhibited, indicating that the inhibition of protein import occurred after the recognition step at the outer membrane . The membrane potential across the inner membrane of the mutant mitochondria was strongly reduced . Restoration of the membrane potential restored preprotein import but did not affect the block of phosphate transport of the mutant mitochondria . We conclude that the inhibition of protein import into mitochondria lacking the phosphate carrier/p32 is indirectly caused by a reduction of the mitochondrial membrane potential (delta(gamma)), and we propose a model that the reduction of delta(psi) is due to the defective phosphate import, suggesting that phosphate transport is the primary function of the phosphate carrier/p32.

Mol Cell Biol, 1996 Nov, 16(11), 6494 - 508
Structure-function comparisons of the proapoptotic protein Bax in yeast and mammalian cells; Zha H et al.; Expression of the proapoptotic protein Bax under the control of a GAL10 promoter in Saccharomyces cerevisiae resulted in galactose-inducible cell death . Immunofluorescence studies suggested that Bax is principally associated with mitochondria in yeast cells . Removal of the carboxyl-terminal transmembrane (TM) domain from Bax {creating Bax (deltaTM)} prevented targeting to mitochondrial and completely abolished cytotoxic function in yeast cells, suggesting that membrane targeting is crucial for Bax-mediated lethality . Fusing a TM domain from Mas70p, a yeast mitochondrial outer membrane protein, to Bax (deltaTM) restored targeting to mitochondria and cytotoxic function in yeast cells . Deletion of four well-conserved amino acids (IGDE) from the BH3 domain of Bax ablated its ability to homodimerize and completely abrogated lethality in yeast cells . In contrast, several Bax mutants which retained ability to homodimerize (deltaBH1, deltaBH2, and delta1-58) also retained at least partial lethal function in yeast cells . In coimmunoprecipitation experiments, expression of the wild-type Bax protein in Rat-1 fibroblasts and 293 epithelial cells induced apoptosis, whereas the Bax (deltaIGDE) mutant failed to induce apoptosis and did not associate with endogenous wild-type Bax protein . In contrast to yeast cells, Bax (deltaTM) protein retained cytotoxic function in Rat-1 and 293 cells, was targeted largely to mitochondria, and dimerized with endogenous Bax in mammalian cells . Thus, the dimerization-mediating BH3 domain and targeting to mitochondrial membranes appear to be essential for the cytotoxic function of Bax in both yeast and mammalian cells.

Mol Cell Biol, 1996 Nov, 16(11), 6169 - 77
Fission yeast mal2+ is required for chromosome segregation; Fleig U et al.; By a screen designed to isolate new fission yeast genes required for chromosome segregation, we have identified mal2+ . The conditionally lethal mal2-1 allele gives rise to increased loss of a nonessential minichromosome at the permissive temperature and leads to severe missegregation of the chromosomes at the nonpermissive temperature . Cloning by complementation and subsequent sequence analysis revealed that mal2 is a novel protein with a mass of 34 kDa . Cells containing a mal2 null allele were inviable, indicating that mal2+ is an essential gene . Fusion of mal2 protein to the green fluorescent protein (GFP) showed that mal2 was predominantly localized in the nucleus . Sensitivity to microtubule-destabilizing drugs and strong genetic interactions with alpha1-tubulin suggest an interaction of the mal2 protein with the microtubule system . Spindle formation and elongation were not detectably affected in the mal2-1 mutant as determined by indirect immunofluorescence . However, anomalous chromosome movement on the spindle leading to aberrant distribution of the chromosomal material was observed.

Mol Cell Biol, 1996 Nov, 16(11), 6096 - 102
A novel DNA binding motif for yeast zinc cluster proteins: the Leu3p and Pdr3p transcriptional activators recognize everted repeats; Hellauer K et al.; The Gal4, Put3, and Ppr1 yeast zinc cluster proteins bind as homodimers to DNA sequences composed of palindromic CGG triplets . Spacing between the triplets specifies the target site for a given zinc cluster protein . In addition, Hap1p, another zinc cluster protein, also recognizes CGG triplets but only when oriented as a direct repeat . Unexpectedly, our results show that Leu3p, another member of this family, also recognizes CGG triplets but oriented in opposite directions and spaced by 4 nucleotides (an everted repeat or inverted palindrome: CCG-N4-CGG) . This constitutes a novel DNA motif for zinc cluster proteins . Moreover, the presence of this motif was shown to be essential for in vivo activation by Leu3p of a minimal reporter containing one copy of a target site for this activator . We also provide evidence that another member of this family, Pdr3p, binds to an everted repeat spaced by 0 nucleotides (CCGCGG) . Thus, our results show that three CGG motifs are used by members of the zinc cluster family: palindromes, direct repeats, and everted repeats.

Mol Cell Biol, 1996 Nov, 16(11), 5985 - 96
Rpo26p, a subunit common to yeast RNA polymerases, is essential for the assembly of RNA polymerases I and II and for the stability of the largest subunits of these enzymes; Nouraini S et al.; Eukaryotic nuclear RNA polymerases (RNAPs) are composed of two large subunits and a number of small polypeptides, some of which are common among these enzymes . To understand the function of Rpo26p, one of the five subunits common to yeast RNAPs, 34 different mutations have been isolated in RP026 that cause cell death in a strain carrying a temperature-sensitive (ts) mutation in the gene (RP021) encoding the largest subunit of RNAPII . These mutant alleles were grouped into three phenotypic classes (null, ts, and neutral) on the basis of the phenotype they imposed in combination with wild-type RP021 . The function of Rpo26p was addressed by biochemical analysis of the ts rpo26-31 allele . The steady-state level of rpo26-31p was reduced at high temperature; this was accompanied by a decrease in the level of at least two other subunits, the largest subunits of RNAPI (A190p) and RNAPII (Rpo21p) . Pulse-chase metabolic labeling and immunoprecipitation of RNAPII showed that at high temperature, rpo26-31 did not lead to dissociation of Rpo26p from the polymerase but prevented the assembly of RNAPII . Overexpression of rpo26-31 partially suppressed the ts phenotype and led to accumulation of the mutant subunit . However, overexpression only marginally suppressed the assembly defect of RNAPII . Furthermore, A190p and Rpo21p continued to accumulate at low levels under these conditions . We suggest that Rpo26p is essential for the assembly of RNAPI and RNAPII and for the stability of the largest subunits of these enzymes.

DNA Res, 1996 Oct 31, 3(5), 291 - 6
Physical mapping of rice chromosome 1 with yeast artificial chromosomes (YACs); Wang ZX et al.; We have constructed a physical map of rice chromosome 1 using yeast artificial chromosomes (YACs) . A YAC library of 350 kb average insert size, covering about 6 rice haploid genome equivalents, was screened using 182 DNA markers which we had previously located on chromosome 1, by colony hybridization and polymerase chain reaction (PCR) amplification . One hundred and sixty-two DNA markers identified at least one YAC each carrying one, two or more marker sequences, for a total of 476 clones . Of these identified YACs, 284 were located in their original positions on chromosome 1 . These 284 YACs defined 69 YAC contigs or islands which are estimated to cover more than 60% of the total chromosome length . The use of mapped DNA markers in constructing a physical map facilitates the integration of genetic and physical maps, as well as fine ordering of the DNA markers, especially at sites where the markers are clustered tightly on the genetic map . Our high density molecular map has been proven, by chromosome landing with YACs using mapped DNA markers, to cover more than half of the entire length of chromosome 1 . The remaining 192 YACs were selected by other copies of DNA markers that mapped on chromosome 1 . This description of the YAC contigs formed on chromosome 1 constitutes the second report of rice physical mapping, following that for chromosome 6.

Gene, 1996 Oct 31, 178(1-2), 187 - 9
Cloning of a human cDNA encoding a protein with high homology to yeast methionyl-tRNA synthetase; Lage H et al.; A composite 2779-bp cDNA that encodes human cytoplasmic methionyl-tRNA synthetase (MetRS) has been constructed from partial cDNA clones derived from the gastric carcinoma cell line EPG85-257RNOV and the nucleotide sequence has been determined . The open reading frame (ORF) encodes a 900 amino acid (aa) protein with a predicted molecular mass of 101 kDa . Northern blotting analysis of total RNA extracted from human gastric carcinoma cells demonstrated a single band with a mobility corresponding to a size of 3.0 kb.

Gene, 1996 Oct 31, 178(1-2), 139 - 43
Structural and functional analyses of APG5, a gene involved in autophagy in yeast; Kametaka S et al.; The APG5 gene of Saccharomyces cerevisiae was cloned from a yeast genomic library by complementation of autophagy defective phenotype of apg5-1 mutant . Structural analysis of the obtained genomic fragment showed that the APG5 gene encodes a novel hydrophilic protein of 294 amino-acid residues without apparent structural similarities to other proteins in the database . To examine its function, a null allele for APG5 (delta apg5) was constructed and introduced into yeast . delta apg5 cells germinated and grew normally in nutrient-rich condition, however, their viability reduced significantly upon the nutrient starvation . They were also shown to be defective in autophagy: they could not sequester autophagic bodies in the vacuole under nitrogen-starvation conditions . These phenotypes are identical to those found in the apg5-1 mutant . The lack of apparent phenotype in rich medium suggests that APG5 function is required only under nutrient starvation condition, however, Northern blot analysis showed that its expression levels remained unchanged after nutrient depletion.

Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12304 - 8
Cytoplasm-to-vacuole targeting and autophagy employ the same machinery to deliver proteins to the yeast vacuole; Scott SV et al.; The vacuolar protein aminopeptidase I (API) uses a novel cytoplasm-to-vacuole targeting (Cvt) pathway . Complementation analysis of yeast mutants defective for cytoplasm-to-vacuole protein targeting (cvt) and autophagy (apg) revealed seven overlapping complementation groups between these two sets of mutants . In addition, all 14 apg complementation groups are defective in the delivery of API to the vacuole . Similarly, the majority of nonoverlapping cvt complementation groups appear to be at least partially defective in autophagy . Kinetic analyses of protein delivery rates indicate that autophagic protein uptake is induced by nitrogen starvation, whereas Cvt is a constitutive biosynthetic pathway . However, the machinery governing Cvt is affected by nitrogen starvation as targeting defects resulting from API overexpression can be rescued by induction of autophagy.

J Biol Chem, 1996 Oct 25, 271(43), 26554 - 60
Initiation of glycogen synthesis in yeast . Requirement of multiple tyrosine residues for function of the self-glucosylating Glg proteins in vivo; Mu J et al.; The self-glucosylating proteins, Glg1p and Glg2p, are required for glycogen synthesis in Saccharomyces cerevisiae (Cheng, C., Mu., J., Farkas, I., Huang, D., Goebl M . G., and Roach, P . J . (1995) Mol . Cell . Biol . 15, 6632-6640) . Glg2p was shown to be associated with carbohydrate in vivo and was released from the high molecular weight glycogen fraction by treatment with alpha-amylase . In addition, some Glg2p exists as a protein of Mr approximately 43,000, whose proportion is increased in cells lacking glycogen synthase . Unlike the mammalian counterpart, glycogenin, the yeast Glg proteins appear to require multiple Tyr residues for functionality . In Glg2p, mutation of both Tyr230 and Tyr232 is necessary to suppress self-glucosylation of purified protein in vitro . The mutant protein is still capable of transferring glucose to an exogeneous acceptor, n-dodecyl beta-D-maltoside . A small COOH-terminal region, conserved between Glg1p and Glg2p, is also important for function; mutation of Tyr367 or truncation at residue 362 impairs the ability of primed Glg2p to be elongated by glycogen synthase . Complete suppression of glycogen accumulation in vivo requires mutation of all three Tyr residues . In Glg1p, two Tyr residues are implicated, Tyr232 and Tyr600, mutation of both being required to eliminate glycogen accumulation in vivo.

Gene, 1996 Oct 24, 177(1-2), 253 - 5
The SPO1 gene product required for meiosis in yeast has a high similarity to phospholipase B enzymes; Tevzadze GG et al.; The SPO1 gene of Saccharomyces cerevisiae has been cloned and sequenced . The Spo1 protein reveals significant similarity with fungal phospholipase B (PLB) enzymes . Features of the SPO1 gene sequence are presented.

Gene, 1996 Oct 24, 177(1-2), 195 - 201
Assembly and extension of yeast artificial chromosomes to build up a large locus; Popov AV et al.; For the assembly of a large human locus, overlapping regions on yeast artificial chromosomes (YACs) and cosmids were linked up using their regions of homology . By site-specific recombination a YAC of 410 kb was created accommodating the major part of the human lambda light chain locus in authentic configuration with 28 variable (V) genes, all joining (J) segments, all constant (C) genes and the downstream enhancer . A contiguous region was first constructed from three overlapping cosmids . Each of these was linearized at unique sites in the vectors and YAC arms were ligated to the 5' and 3' ends . After cells of Saccharomyces cerevisiae were transformed with the three cosmids, YACs of 120 kb were obtained which contained the reassembled 3' J-C region in authentic configuration . The assembled YAC was further extended by mitotic recombination with a YAC containing a 280-kb region of the C-proximal part of the V gene cluster with a 15-kb 3' overlap . For this, a simple three-way selection procedure was developed involving the integration of different selectable marker genes at specific sites in the left and right YAC arms . Rare recombination events between two overlapping YACs could be identified in yeast clones able to grow in lysine- and adenine-deficient medium in the presence of 5-fluoro-orotic acid which is toxic for yeast cells containing a YAC with a functional URA3 gene . This approach made it possible to assemble and extend large YACs from an unlimited number of smaller overlapping YACs by positive-negative selection.

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