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