RNA, 1997 Oct, 3(10), 1143 - 52 The yeast Prp3 protein is a U4/U6 snRNP protein necessary for integrity of the U4/U6 snRNP and the U4/U6.U5 tri-snRNP; Anthony JG et al.; Previously, yeast prp3 mutants were found to be blocked prior to the first catalytic step of pre-mRNA splicing . No splicing intermediates or products are formed from pre-mRNA in heat-inactivated prp3 mutants or prp3 mutant extracts . Here we show that Prp3p is a component of the U4/U6 snRNP and is also present in the U4/U6.U5 tri-snRNP . Heat inactivation of prp3 extracts results in depletion of free U6 snRNPs and U4/U6.U5 tri-snRNPs, but not U4/U6 snRNPs or U5 snRNPs . Free U4 snRNP, normally not present in wild-type extracts, accumulates under these conditions . Assays of in vivo levels of snRNAs in a prp3 mutant revealed that amounts of free U6 snRNA decreased, free U4 snRNA increased, and U4/U6 hybrids decreased slightly . These results suggest that Prp3p is required for formation of stable U4/U6 snRNPs and for assembly of the U4/U6.U5 tri-snRNP from its component snRNPs . Upon inactivation of Prp3p, spliceosomes cannot assemble from prespliceosomes due to the absence of intact U4/U6.U5 tri-snRNPs . Prp3p is homologous to a human protein that is a component of U4/U6 snRNPs, exemplifying the conservation of splicing factors between yeast and metazoans. J Biochem (Tokyo), 1997 Sep, 122(3), 525 - 30 Identification and subcellular localization of a novel mammalian dynamin-related protein homologous to yeast Vps1p and Dnm1p; Shin HW et al.; The dynamin family of GTP-binding proteins are implicated in vesicular transport . These include mammalian dynamins I, II, III, and yeast Vps1p and Dnm1p . Dynamin is involved in the formation of clathrin-coated vesicles at the plasma membrane . On the other hand, Vps1p and Dnm1p appear to be involved in transport from the late Golgi compartment to vacuoles and in an endocytic process, respectively . In this study, we identified a novel human protein, named Dnm1p/Vps1p-like protein (DVLP) . It resembled more closely Dnm1p and Vps1p than dynamins not only in the primary structure but also in the domain organization . DVLP mRNA was expressed ubiquitously, suggesting that this protein plays a fundamental role in cellular function . Immunofluorescence analysis of cells expressing epitope-tagged DVLP revealed that it showed a diffused perinuclear staining pattern that was not superimposed on that of the marker protein for the Golgi apparatus, trans-Golgi network, lysosomes, endosomes, or endoplasmic reticulum . These data suggest that DVLP is not involved in the formation of known coated vesicles. Genes Cells, 1997 Aug, 2(8), 487 - 98 Switching yeast from meiosis to mitosis: double-strand break repair, recombination and synaptonemal complex; Zenvirth D et al.; BACKGROUND: When Saccharomyces cerevisiae cells that have begun meiosis are transferred to mitotic growth conditions ('return-to-growth', RTG), they can complete recombination at high meiotic frequencies, but undergo mitotic cell division and remain diploid . It was not known how meiotic recombination intermediates are repaired following RTG . Using molecular and cytological methods, we investigated whether the usual meiotic apparatus could repair meiotically induced DSBs during RTG, or whether other mechanisms are invoked when the developmental context changes . RESULTS: Upon RTG, the rapid disappearance of meiotic features--double-strand breaks in DNA (DSBs), synaptonemal complex (SC), and SC related structures-was striking . In wild-type diploids, the repair of meiotic DSBs during RTG was quick and efficient, resulting in homologous recombination . Kinetic analysis of double-strand breakage and recombination indicated that meiotic DSB formation precedes the commitment to meiotic levels of recombination . DSBs were repaired in RTG in dmc1, but not rad51 mutants, hence repair did not occur by the usual meiotic mechanism which requires the Dmc1 gene product . In haploids, DSBs were also repaired quickly and efficiently upon RTG, showing that DSB repair did not require the presence of a homologous chromosome . In all strains examined, SC and related structures were not required for DSB repair or recombination following RTG . CONCLUSIONS: At least two pathways of DSB repair, which differ from the primary meiotic pathway(s), can occur during RTG: One involving interhomologue recombination, and another involving sister-chromatid exchange . DSB formation precedes commitment to recombination . SC elements appear to prevent sister chromatid exchange in meiosis. Eur J Biochem, 1997 Sep 1, 248(2), 441 - 4 Phosphorylation mutants of p53 show differential complex formation with putative dehydrogenase Tms1 of fission yeast; Wagner P; The yeast tms1 gene was originally identified as a multi-copy suppressor of a lethal growth arrest caused by expression of a tumour mutant cDNA of p53 in fission yeast . The tms1 gene product (Tms1) was found to form stable complexes with p53 in yeast and in vitro; using purified recombinant proteins, the interaction was mapped to the C-terminal region of p53 . This part is known to be modified by several protein kinases resulting in a transition of p53 from a latent to an activated state capable of transactivating various cellular genes involved in growth suppression or apoptosis . Since there is evidence for an evolutionary conservation of a Tms1-related protein in mammals, the effect of the phosphorylation status of the C-terminus of p53 on Tms1/p53 complex formation in vitro has been investigated . Whereas mutants changing the cdc2 phosphorylation site at position 315 of human p53 had only little effect on Tms1/p53 complex formation, we found that mutants involving the protein kinase CK2 site at position 392 showed a significantly decreased relative affinity for the Tms1 protein . The same result was obtained by using a C-terminal fragment of p53 which was phosphorylated by purified protein kinase CK2, suggesting that the complex formation of p53 with cellular C-terminal binding proteins like Tms1 impairs regulation by phosphorylation. Arch Biochem Biophys, 1997 Oct 1, 346(1), 37 - 44 Potassium collapses the deltaP in yeast mitochondria while the rate of ATP synthesis is inhibited only partially: modulation by phosphate; Castrejon V et al.; Addition of increasing concentrations of K+ to yeast mitochondria in the presence of 0 to 400 microM phosphate and 200 microM Mg2+ led to uncoupled respiration and decreased protonmotive force (deltaP):at 0 K+ deltaP = 213 mV, negative inside, where deltapsi = 180 mV and deltapH = 33 mV, while at 20 mM K+ deltaP = 28 mV, where deltapsi = 16 mV and deltapH = 12 mV . In contrast, the synthesis of ATP resulted in smaller values for the Km and the Vmax in 400 microM Pi and increasing ADP: in 0 K+, Km = 18.6 microM and Vmax = 75.4 nmol (min x mg protein)-1, while in 20 mM K+, Km = 5.2 microM and Vmax = 46.0 nmol (min x mg protein)-1, i.e., when K+ depleted most of the deltaP, and at ADP concentrations below the Km, the rate of ATP synthesis was essentially the same as in the absence of K+ . At saturating ADP, the rate of ATP synthesis in the presence of K+ was about 60% of the rate observed without K+ . The synthesis of ATP by yeast mitochondria was inhibited by oligomycin or uncouplers . K+ had no effects on rat liver mitochondria . Adenylate kinase activity was much smaller in yeast mitochondria than in rat liver mitochondria and thus did not account for the synthesis of ATP observed in the presence of K+ . The effects of K+ on the deltaP of yeast mitochondria were prevented by increasing concentrations of phosphate (1 to 4 mM) . At 4 mM phosphate, the deltaP was always above 200 mV and the kinetics of ATP synthesis were as follows: 0 K+ Km = 10.0 microM and Vmax = 88.3 nmol (min x mg protein)-1 . At 20 mM K+, Km = 7.4 microM and Vmax = 133 nmol (min x mg protein)-1. FEBS Lett, 1997 Sep 22, 415(1), 49 - 55 Formation of a yeast SNARE complex is accompanied by significant structural changes; Rice LM et al.; The evolutionarily conserved SNARE (SNAP receptor) proteins and their complexes are key players in the docking and fusion of secretory vesicles with their target membrane . Biophysical techniques were used to characterize structural and energetic properties of the cytoplasmic domains of the yeast SNAREs Snc1 and Sso1, of the SNAP-25-like domain of Sec9, and of the Sso1:Sec9 and Sso1:Sec9:Snc1 complexes . Individually, all three SNAREs are monomeric; Sso1 shows significant secondary structure while Snc1 and Sec9 are largely unstructured . Ternary SNARE complex formation (KD <50 nM) is accompanied by a more than two-fold increase in secondary structure . This binding induced structure, the large increase in thermal stability, and the self-association of the ternary complex represent conserved properties of SNAREs that are probably important in vesicle docking and fusion. FEBS Lett, 1997 Sep 22, 415(1), 29 - 32 Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-xL; Manon S et al.; The characteristics of mitochondria of yeast cells expressing the pro-apoptotic gene Bax or coexpressing Bax and the anti-apoptotic gene Bcl-xL have been investigated in whole cells, isolated mitochondria and permeabilized spheroplasts . It is found that Bax-induced growth arrest of yeast cells is related to two defects in the respiratory chain: (i) a decrease in the amount of cytochrome c oxidase, the terminal enzyme of the respiratory chain, and (ii) a dramatic increase in the release of cytochrome c to the cytosol . Other components of the inner mitochondrial membrane (bc1 complex and F0F1-ATPase) are unaffected . Coexpression of Bcl-xL almost fully prevented the effect of Bax . Surprisingly, these results obtained in yeast parallel similar observations reported in mammalian cells. FEBS Lett, 1997 Sep 22, 415(1), 6 - 10 Differential resistance to proteinase K digestion of the yeast prion-like (Ure2p) protein synthesized in vitro in wheat germ extract and rabbit reticulocyte lysate cell-free translation systems; Komar AA et al.; The Ure2p yeast prion-like protein was translated in vitro in the presence of labeled {35S}methionine in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE) cell-free systems . When subjected to proteinase K digestion, the Ure2p protein synthesized in WGE was proteolysed much more slowly compared to that synthesized in RRL; this displays fragments of about 31-34 kDa, persisting over 8 min . Thus, the digestion rate and pattern of the protein synthesized in WGE, unlike that synthesized in RRL, revealed characteristic features of the {URE3} prion-like isoform of the Ure2p protein {Masison, D.C . and Wickner, R.B . (1995) Science 270, 93-95} . Chloramphenicol acetyltransferase, synthesized under the same conditions, differed fundamentally in its proteolytic sensitivity toward proteinase K (PK); in the RRL system it was more slowly digested than in WGE, proving specific PK inhibitors to be absent in both systems . Posttranslational addition of the WGE to the RRL-synthesized Ure2p does not protect Ure2p from efficient PK degradation either . The differences in Ure2p degradation may be ascribed to a specific structure or specific states of association of Ure2p synthesized in WGE; obviously, they yield a protein that mimics the behavior of the Ure2p in {URE3} yeast strains . The present data suggest that particular conditions of the Ure2p protein translation and/or certain cellular components (accessory proteins and extrinsic factors), as well as the nature of the translation process itself, could affect the intracellular folding pathway of Ure2p leading to the de novo formation of the prion {URE3} isoform. J Biol Chem, 1997 Oct 10, 272(41), 25928 - 34 VMA12 encodes a yeast endoplasmic reticulum protein required for vacuolar H+-ATPase assembly; Jackson DD et al.; The Saccharomyces cerevisiae vacuolar membrane proton-translocating ATPase (V-ATPase) can be divided into a peripheral membrane complex (V1) containing at least eight polypeptides of 69, 60, 54, 42, 32, 27, 14, and 13 kDa, and an integral membrane complex (V0) containing at least five polypeptides of 100, 36, 23, 17, and 16 kDa . Other yeast genes have been identified that are required for V-ATPase assembly but whose protein products do not co-purify with the enzyme complex . One such gene, VMA12, encodes a 25-kDa protein (Vma12p) that is predicted to contain two membrane-spanning domains . Biochemical analysis has revealed that Vma12p behaves as an integral membrane protein with both the N and C termini oriented toward the cytosol, and this protein immunolocalizes to the endoplasmic reticulum (ER) . In cells lacking Vma12p (vma12Delta), the 100-kDa subunit of the V0 complex (which contains six to eight putative membrane-spanning domains) was rapidly degraded (t1/2 approximately 30 min) . Protease protection assays revealed that the 100-kDa subunit was inserted/translocated correctly into the ER membrane of vma12Delta cells . These data indicate that Vma12p functions in the ER after the insertion of V0 subunits into the ER membrane . We propose that Vma12p functions directly in the assembly of the V0 subunits into a complex in the ER, and that assembly is required for the stability of the V0 subunits and their transport as a complex out of this compartment. J Biol Chem, 1997 Oct 10, 272(41), 25768 - 77 Mts4, a non-ATPase subunit of the 26 S protease in fission yeast is essential for mitosis and interacts directly with the ATPase subunit Mts2; Wilkinson CR et al.; We have isolated a fission yeast gene, mts4(+), by complementation of a temperature-sensitive mutation and show that it encodes subunit 2 (S2) of the 19 S regulatory complex of the 26 S protease . mts4(+) is an essential gene, and we show that loss of this subunit causes cells to arrest in metaphase, illustrating the importance of S2 for mitosis . The Mts4 protein is 48% identical to S2 of the human 26 S protease, and the lethal phenotype of the null mts4 allele can be rescued by the human cDNA encoding S2 . We provide genetic and physical evidence to suggest that the Mts4 protein interacts with the product of the mts2(+) gene, an ATPase which has previously been shown to be subunit 4 of the 26 S protease. Anal Biochem, 1997 Oct 1, 252(1), 33 - 9 Method to screen for relevant yeast two-hybrid-derived clones by coimmunoprecipitation and colocalization of epitope-tagged fragments--application to Bcl-xL; Wong C et al.; The yeast two-hybrid system is a powerful genetic method to screen cDNA libraries to identify protein-protein interactions . A screen with a typical bait may yield many clones, including ones that are not biologically relevant which need to be eliminated by evaluating each clone in an alternative assay to confirm the interaction with the bait protein . We have developed an efficient assay to quickly screen two-hybrid-derived clones for coimmunoprecipitation and colocalization with the bait protein in mammalian cells . Gene fragments derived from a two-hybrid screen are cloned into an epitope tagging vector that can generate high levels of epitope-tagged protein in mammalian cells . The vector expressing an epitope-tagged protein is then cotransfected into mammalian cells with an expression vector for the bait protein . Interaction between the bait protein and epitope-tagged protein is evaluated by coimmunoprecipitation and colocalization . We demonstrate the utility of this approach by applying it to clones isolated in a two-hybrid screen using Bcl-xL as bait, showing that two-hybrid-derived fragments of Bad and Bax, previously known to interact with Bcl-xL, both colocalize and coimmunoprecipitate with Bcl-xL. Mol Cell Biol, 1997 Nov, 17(11), 6555 - 62 Amino termini of histones H3 and H4 are required for a1-alpha2 repression in yeast; Huang L et al.; The Saccharomyces cerevisiae alpha2 repressor controls two classes of cell-type-specific genes in yeast through association with different partners . alpha2-Mcm1 complexes repress a cell-specific gene expression in haploid alpha cells and diploid a/alpha cells, while a1-alpha2 complexes repress haploid-specific genes in diploid cells . In both cases, repression is mediated through Ssn6-Tu1 corepressor complexes that are recruited via direct interactions with alpha2 . We have previously shown that nucleosomes are positioned adjacent to the alpha2-Mcm1 operator under conditions of repression and that Tupl interacts directly with histones H3 and H4 . Here, we examine the role of chromatin in a1-alpha2 repression to determine if chromatin is a general feature of repression by Ssn6-Tup1 . We find that mutations in the amino terminus of histone H4 cause a 4- to 11-fold derepression of a reporter gene under a1-alpha2 control, while truncation of the H3 amino terminus has a more modest (3-fold or less) effect . Strikingly, combination of the H3 truncation with an H4 mutation causes a 40-fold decrease in repression, clearly indicating a central role for these histones in a1-alpha2-mediated repression . However, in contrast to the ordered positioning of nucleosomes adjacent to the alpha2-Mcm1 operator, nucleosomes are not positioned adjacent to the a1-alpha2 operator in diploid cells . Our data indicate that chromatin is important to Ssn6-Tup1-mediated repression but that the degrees of chromatin organization directed by these proteins differ at different promoters. Mol Cell Biol, 1997 Nov, 17(11), 6339 - 47 A homolog of mammalian, voltage-gated calcium channels mediates yeast pheromone-stimulated Ca2+ uptake and exacerbates the cdc1(Ts) growth defect; Paidhungat M et al.; Previous studies attributed the yeast (Saccharomyces cerevisiae) cdc1(Ts) growth defect to loss of an Mn2+-dependent function . In this report we show that cdc1(Ts) temperature-sensitive growth is also associated with an increase in cytosolic Ca2+ . We identified two recessive suppressors of the cdc1(Ts) temperature-sensitive growth which block Ca2+ uptake and accumulation, suggesting that cytosolic Ca2+ exacerbates or is responsible for the cdc1(Ts) growth defect . One of the cdc1(Ts) suppressors is identical to a gene, MID1, recently implicated in mating pheromone-stimulated Ca2+ uptake . The gene (CCH1) corresponding to the second suppressor encodes a protein that bears significant sequence similarity to the pore-forming subunit (alpha1) of plasma membrane, voltage-gated Ca2+ channels from higher eukaryotes . Strains lacking Mid1 or Cch1 protein exhibit a defect in pheromone-induced Ca2+ uptake and consequently lose viability upon mating arrest . The mid1delta and cch1delta mutants also display reduced tolerance to monovalent cations such as Li+, suggesting a role for Ca2+ uptake in the calcineurin-dependent ion stress response . Finally, mid1delta cch1delta double mutants are, by both physiological and genetic criteria, identical to single mutants . These and other results suggest Mid1 and Cch1 are components of a yeast Ca2+ channel that may mediate Ca2+ uptake in response to mating pheromone, salt stress, and Mn2+ depletion. Annu Rev Microbiol, 1997, 51, 125 - 49 Getting started: regulating the initiation of DNA replication in yeast; Toone WM et al.; Initiation of DNA replication in yeast appears to operate through a two-step process . The first step occurs at the end of mitosis in the previous cell cycle, where, following the decrease in B cyclin-dependent kinase activity, an extended protein complex called the prereplicative complex (pre-RC) forms over the origin of replication . This complex is dependent on the association of the Cdc6 protein with the Origin Recognition Complex (ORC) and appears concomitantly with the nuclear entry of members of the Mcm family of proteins . The second step is dependent upon the cell passing through a G1 decision point called Start . If the environmental conditions are favorable, and the cells reach a critical size, then there is a rise in G1 cyclin-dependent kinase activity, which leads to the activation of downstream protein kinases; the protein kinases are, in turn, required for triggering initiation from the preformed initiation complexes . These protein kinases, Dbf4-Cdc7 and Clb5/6(B-cyclin)-Cdc28, are thought to phosphorylate targets within the pre-RC . The subsequent rise in B cyclin protein kinase activity following Start not only triggers origin firing, but also inhibits the formation of new pre-RCs, which ensures that there is only one S phase in each cell cycle . The destruction of B-cyclin protein kinase activity at the end of the cell cycle potentiates the formation of new pre-RCs-resetting origins for the next S phase. Annu Rev Microbiol, 1997, 51, 73 - 96 Molecular genetics of sulfur assimilation in filamentous fungi and yeast; Marzluf GA; The filamentous fungi Aspergillus nidulans and Neurospora crassa and the yeast Saccharomyces cerevisiae each possess a global regulatory circuit that controls the expression of permeases and enzymes that function both in the acquisition of sulfur from the environment and in its assimilation . Control of the structural genes that specify an array of enzymes that catalyze reactions of sulfur metabolism occurs at the transcriptional level and involves both positive-acting and negative-acting regulatory factors . Positive trans-acting regulatory proteins that contain a basic region, leucine zipper-DNA binding domain, are found in Neurospora and yeast . Each of these fungi contain a sulfur regulatory protein of the beta-transducin family that acts in a negative fashion to control gene expression . Sulfur regulation in yeast also involves the general DNA binding protein, centromere binding factor I . Sulfate uptake is a highly regulated step and appears to occur in fungi, plants, and mammals via a family of related transporter proteins . Recent developments have provided new insight into the nature and control of the enzymes ATP sulfurylase and APS kinase, which catalyze the early steps of sulfate assimilation, and of the Aspergillus enzyme, cysteine synthase, which produces cysteine from O-acetylserine. Zentralbl Veterinarmed A, 1997 Aug, 44(6), 373 - 80 Selenite and selenium yeast as feed supplements for dairy cows; Ortman K et al.; The availability of inorganic and organic forms of selenium to dairy cows was studied by giving 25 cows supplementary selenium for 9 months either as sodium selenite or as a selenium-containing yeast product . Group I (eight cows) received 3.0 mg selenium as sodium selenite daily, group II (nine cows) received 3.0 mg selenium as the selenium yeast product, and group III (eight cows) received 0.75 mg selenium as the selenium yeast product . The total selenium contents of the ration were 0.26-0.32 mg/kg feed dry matter for groups I and II, and 0.16-0.18 mg/kg for group III . The supplement of 0.75 mg selenium daily from the yeast product maintained the selenium concentrations of whole blood and milk at the same levels as 3.0 mg selenium as sodium selenite, and 3.0 mg selenium from the yeast product increased the selenium concentration of whole blood by approximately equal to 40% and that of milk by approximately equal to 100% . The activity of glutathione peroxidase in erythrocytes of the group given selenite was not significantly different from that in either of the groups given the yeast product . The concentrations of selenium in the tissues of two cows from each group were marginal to adequate, and there was a trend for the concentrations to be higher in the tissues of the cows supplemented with the yeast product. Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11827 - 32 Cloning of the cDNA for the TATA-binding protein-associated factorII170 subunit of transcription factor B-TFIID reveals homology to global transcription regulators in yeast and Drosophila; van der Knaap JA et al.; The human transcription factor B-TFIID is comprised of TATA-binding protein (TBP) in complex with one TBP-associated factor (TAF) of 170 kDa . We report the isolation of the cDNA for TAFII170 . By cofractionation and coprecipitation experiments, we show that the protein encoded by the cDNA encodes the TAF subunit of B-TFIID . Recombinant TAFII170 has (d)ATPase activity . Inspection of its primary structure reveals a striking homology with genes of other organisms, yeast MOT1, and Drosophila moira, which belongs to the Trithorax group . Both homologs were isolated in genetic screens as global regulators of pol II transcription . This supports our classification of B-TFIID as a pol II transcription factor and suggests that specific TBP-TAF complexes perform distinct functions during development. Eur J Immunol, 1997 Sep, 27(9), 2180 - 7 The X-linked immunodeficiency defect in the mouse is corrected by expression of human Bruton's tyrosine kinase from a yeast artificial chromosome transgene; Maas A et al.; Mutations in the gene for Bruton's tyrosine kinase result in the B cell differentiation defects X-linked agammaglobulinemia in man and X-linked immunodeficiency in mice . Here we describe the generation of two yeast artificial chromosome (YAC)-transgenic mouse strains in which high-level expression of human Btk is provided by endogenous regulatory cis-acting elements that are present on a 340-kb transgene, Yc340-hBtk . The expression pattern of the transgenic human Btk was found to parallel that of the endogenous murine gene . When the Yc340-hBtk-transgenic mice were mated onto a Btk-deficient background, the xid B cell defects were fully corrected: conventional and CD5+ B-1 B cells were present in normal numbers, serum IgM and IgG3 levels as well as responses to T cell-independent type II antigens were in the normal ranges . In vivo competition experiments in Btk+/- female mice demonstrated that in the conventional B cell population the Yc340-hBtk transgene could fully compensate the absence of expression of endogenous murine Btk . We conclude that in the YAC-transgenic mice Btk is appropriately expressed in the context of native regulatory sequences. Mol Gen Genet, 1997 Sep, 256(1), 7 - 17 Mutations at the human minisatellite MS32 integrated in yeast occur with high frequency in meiosis and involve complex recombination events; Appelgren H et al.; Minisatellites are composed of tandem repetitive DNA sequences and are present at many positions in the human genome . They frequently mutate to new length alleles in the germline, by complex and incompletely understood recombination mechanisms which may operate during meiosis . In several minisatellites the mutation events are restricted to one end of the repeat array, indicating a possible association with elements that act in cis . Mutant alleles do not show exchange of flanking regions . To construct a model system suitable for further investigations of the mutation process, we have integrated the human minisatellite MS32, flanked by synthetic markers, in the vicinity of a meiotic recombination hot spot upstream of the LEU2 locus in the yeast Saccharomyces cerevisiae . Here we provide direct evidence for a meiotic origin of MS32 mutations . Mutation events were polarised towards both ends of the minisatellite and varied from simple duplications and deletions to complex intra- and interallelic events . Interallelic events were frequently accompanied by exchange of regions flanking the minisatellite . The results also support the notion that cis-acting elements are involved in the mutational process . The fact that MS32 mutant structures are similar in yeast and human shows that meiotic recombination plays a crucial role in both organisms and emphasises the usefulness of yeast strains harbouring minisatellites as a model system for the study of minisatellite mutation. J Biol Chem, 1997 Oct 24, 272(43), 27107 - 15 Purification and characterization of the human SR 31747A-binding protein . A nuclear membrane protein related to yeast sterol isomerase; Jbilo O et al.; SR 31747A, defined as a sigma ligand, is a novel immunosuppressive agent that blocks proliferation of human and mouse lymphocytes . Using a radiolabeled chemical probe, we here purified a target of SR 31747A and called it SR 31747A-binding protein (SR-BP) . Purified SR-BP retained its binding properties and migrated on SDS-polyacrylamide gel as a Mr 28,000 protein . Cloning of the cDNA encoding human SR-BP shows an open reading frame for a 223-amino acid protein, which is homologous to the recently cloned sigma 1 receptor . Interestingly, the deduced amino acid sequence was found to be related to fungal C8-C7 sterol isomerase, encoded by the ERG2 gene . The ERG2 gene product has been identified recently as the molecular target of SR 31747A that mediates antiproliferative effects of the drug in yeast . Northern blot analysis of SR-BP gene expression revealed a single transcript of 2 kilobases which was widely expressed among organs, with the highest abundance in liver and the lowest abundance in brain . Subcellular localization analysis in various cells, using a specific monoclonal antibody raised against SR-BP, demonstrated that this protein was associated with the nuclear envelope . When studying the binding of SR 31747A on membranes from yeast expressing SR-BP, we found a pharmacological profile of sigma 1 receptors; binding was displaced by (+)-pentazocine, haloperidol, and (+)-SKF 10,047, with (+)-SKF 10, 047 being a more potent competitor than (-)-SKF 10,047 . Scatchard plot analysis revealed Kd values of 7.1 nM and 0.15 nM for (+)-pentazocine and SR 31747A, respectively, indicating an affinity of SR-BP 50-fold higher for SR 31747A than for pentazocine . Additionally, we showed that pentazocine, a competitive inhibitor of SR 31747A binding, also prevents the immunosuppressive effect of SR 31747A . Taken together, these findings strongly suggest that SR-BP represents the molecular target for SR 31747A in mammalian tissues, which could be critical for T cell proliferation. Mol Cells, 1997 Aug 31, 7(4), 526 - 31 Characterization of four yeast artificial chromosome clones mapped to human chromosome 21q22.1 with eight markers; Kim MY et al.; Yeast artificial chromosome (YAC) clones have been successfully utilized to generate a YAC contig map of the long arm of human chromosome 21 (Hu21q) . The chromosome subband of 21q22.1 where five genetic loci (IFNAR1, IFNAR2, CRFB4, AF-1, and GART) are mapped is a gene-rich region and needs to be characterized in further detail . YAC D142H8 and YAC F136C5, which were characterized previously by a functional YAC expression procedure, and two new YAC clones, YAC 872B5 and YAC 876D4 located at 21q22.1 whose YAC sizes are 800 kb and 1,500 kb, respectively, were used in this study . To obtain more markers useful for making a detailed physical map of the region, a purified yeast artificial chromosome (YAC D142H8) was used to screen the 3 x 1 S cDNA library . As a result three anonymous cDNA clones (Kmy1, Kmy2, and Qorf4) were obtained, and the nucleotide sequences of Kmy1 and Kmy2 were determined . In an attempt to make a detailed physical map of the region, the locations of five known genes as well as the three new markers were determined on the four YACs by Southern blot analysis . YAC 872B5 contained all markers except GART while YAC F136C5, YAC D142H8, and YAC 876D4 contained three markers (CRFB4, IFNAR1, and IFNAR2), four markers (Kmy1, Kmy2, Qorf4 and AF-1), and four markers (Kmy1, Kmy2, Qorf4 and GART), respectively . YAC 872B5 may represent 1,500 kb of the 21q22.1 subband and half of the 3 x 1 S region, so it should be very useful for studying the relevent region of the human chromosome functionally and physically. Mol Cells, 1997 Aug 31, 7(4), 489 - 94 Inducible expression of yeast mitochondrial citrate synthase in Aspergillus nidulans; Lee SK et al.; The coding region of the mitochondrial citrate synthase gene (CIT1) from Saccharomyces cerevisiae was amplified by PCR and cloned into an expression vector (pAL4) downstream of the alcohol dehydrogenase (alcA) promoter of Aspergillus nidulans to yield pALCS1 . Transformation of A . nidulans A773 with this construct gave stable transformants, AYC#1 and AYC#2, that were phenotypically stable for several mitotic divisions . Southern blot analysis showed that the CIT1 gene was successfully integrated into the chromosomes of the transformants . Western blot analysis and enzymatic assay for citrate synthase revealed that the integrated yeast gene was subject to inducible expression controlled by alcA promoter, which can be induced by threonine. Plasmid, 1997, 38(2), 91 - 6 Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast; Hua SB et al.; With efficient homologous recombination in Saccharomyces cerevisiae, a rapid in vivo cloning technique has been available . Here we demonstrated that 30 bp of a homologous sequence at each end of a DNA fragment is sufficient to integrate the fragment into a linearized plasmid in yeast . To obtain a high yield of recombination transformants, however, more than 60 bp are desirable . Interestingly, we observed that 20 bp of homology at one end of a DNA fragment is sufficient for efficient recombination provided that the other end contains 80 bp of homologous sequence . Some applications, including high-throughput transferring of EST inserts to the yeast expression systems for the Human Genome Project, are discussed. Curr Genet, 1997 Sep, 32(3), 190 - 6 Homologous recombination partly restores the secretion defect of underglycosylated acid phosphatase in yeast; Praetorius-Ibba M et al.; The majority of secreted acid phosphatase in Saccharomyces cerevisiae is encoded by the PH05 gene . The secretion level of this acid phosphatase is directly determined by its level of glycosylation . Consequently, PHO5-11-encoded acid phosphatase which lacks 11 of 12 glycosylation sites is only poorly secreted . We have isolated and characterized both UV- and EMS-induced variants, which are partly able to restore the secretion of acid phosphatase . Our data indicate that the improved secretion is caused by mitotic intrachromosomal recombination between the PHO5-11 allele and the homologous tandemly repeated PHO3 sequences, resulting in the restoration of glycosylation sites in PHO5-11 . Two different recombination mechanisms, unequal sister-chromatid exchange and sister-chromatid gene conversion, are responsible for these alterations of the PHO5-11 locus . Thus, recombination between mutant and wild-type sequences are able to restore the ability of mutant yeast cells to secrete acid phosphatase. Curr Genet, 1997 Sep, 32(3), 182 - 9 The mcm17 mutation of yeast shows a size-dependent segregational defect of a mini-chromosome; Roy N et al.; Mini-chromosome-maintenance (mcm) mutants were described earlier as yeast mutants which could not stably maintain mini-chromosomes . Out of these, the ARS-specific class has been more extensively studied and is found to lose chromosomes and mini-chromosomes due to a defect in the initiation of DNA replication at yeast ARSs . In the present study we have identified a number of mcm mutants which show size-dependent loss of mini-chromosomes . When the size of the mini-chromosome was increased, from about 15 kb to about 60 kb, there was a dramatic increase in its mitotic stability in these mutants, but not in the ARS-specific class of mutants . One mutant, mcm17, belonging to the size-dependent class was further characterized . In this mutant, cells carried mini-chromosomes in significantly elevated copy numbers, suggesting a defect in segregation . This defect was largely suppressed in the 60-kb mini-chromosome . A non-centromeric plasmid, the TRP1ARS1 circle, was not affected in its maintenance . This mutant also displayed enhanced chromosome-III loss during mitosis over the wild-type strain, without elevating mitotic recombination . Cloning and sequencing of MCM17 has shown it to be the same as CHL4, a gene required for chromosome stability . This gene is non-essential for growth, as its disruption or deletion from the chromosome did not affect the growth-rate of cells at 23 degrees C or 37 degrees C . This work suggests that centromere-directed segregation of a chromosome in yeast is strongly influenced by its length. Am J Hum Genet, 1997 Sep, 61(3), 590 - 8 Characterization of two mutations associated with epimerase-deficiency galactosemia, by use of a yeast expression system for human UDP-galactose-4-epimerase; Quimby BB et al.; UDP-galactose-4-epimerase (GALE) is a highly conserved enzyme that catalyzes the interconversion of UDP-galactose and UDP-glucose . Impairment of this enzyme in humans results in one of two clinically distinct forms of epimerase-deficiency galactosemia-one benign, the other severe . The molecular and biochemical distinction between these disorders remains unknown . To enable structural and functional studies of both wild-type and patient-derived alleles of human GALE (hGALE), we have developed and applied a null-background yeast expression system for the human enzyme . We have demonstrated that wild-type hGALE sequences phenotypically complement a yeast gal10 deletion, and we have biochemically characterized the wild-type human enzyme isolated from these cells . Furthermore, we have expressed and characterized two mutant alleles, L183P-hGALE and N34S-hGALE, both derived from a patient with no detectable GALE activity in red blood cells but with approximately 14% activity in cultured lymphoblasts . Analyses of crude extracts of yeast expressing L183P-hGALE demonstrated 4% wild-type activity and 6% wild-type abundance . Extracts of yeast expressing N34S-hGALE demonstrated approximately 70% wild-type activity and normal abundance . However, yeast coexpressing both L183P-hGALE and N34S-hGALE exhibited only approximately 7% wild-type levels of activity, thereby confirming the functional impact of both substitutions and raising the intriguing possibility that some form of dominant-negative interaction may exist between the mutant alleles found in this patient . The results reported here establish the utility of the yeast-based hGALE-expression system and set the stage for more-detailed studies of this important enzyme and its role in epimerase-deficiency galactosemia. Int Arch Allergy Immunol, 1997 Oct, 114(2), 202 - 4 Lack of lysozyme activity of natural and yeast-derived recombinant Der p 2; Hakkaart GA et al.; For Dermatophagoides pteronyssinus a number of allergens have been reported . Although the function of several allergens is known, that of the house dust mite allergen Der p 2 is unknown . A role as lysozyme has been suggested . We tested a yeast-derived recombinant Der p 2 for its lysozymatic activity . This recombinant allergen and affinity-purified natural Der p 2 lacked this enzymatic activity . Whole-body mite extract retained its lysozymatic activity after removal of the group 2 allergen by monoclonal antibody depletion . Analysis of spent medium revealed the reciprocal: it contained group 2 allergen, but lacked lysozymatic activity . Together, these data demonstrate that mite lysozyme and Der p 2 are different components of mite extract. Cancer Surv, 1997, 29, 109 - 32 Yeast checkpoint controls and relevance to cancer; Weinert T; Checkpoint controls arrest cells with defects in DNA replication or DNA damage . For several reasons, checkpoint controls may be relevant to ontogeny and treatment of cancer . Firstly, mutations in two human genes, TP53 and ATM, give rise to cellular defects in cell cycle checkpoints and are associated with cancer . Secondly, although checkpoint defects potentially render the cell damage sensitive, they may do so only in combination with other defects in the cell's response to damage . Therefore, manipulation of checkpoint defects, requiring a description of normal and mutant pathways, will be required for this type of therapeutic approach . Those pathways are being described in yeast cells . In budding yeast, the study of checkpoint genes has led to the view that these genes have many roles in the cellular responses to DNA damage, including roles in arrest in multiple stages of cell cycle, in transcriptional induction of repair genes, in DNA repair itself and additionally some undefined role in DNA replication . The checkpoint pathways and proteins that carry out these responses may consist of sensor proteins that detect damage, signaller proteins that transduce an inhibitory signal and target proteins that are altered to arrest cell division (or cause other changes in cell behaviour) . Yeast genes that may act at each step have been identified, leading to a working model of checkpoint pathways . An initial step in the pathway may involve the processing of damage to an intermediate that signals arrest and acts in DNA repair . Human checkpoint pathways may have defects in processing damage as well. Biochem J, 1997 Aug 15, 326 ( Pt 1), 131 - 8 Purification and staining of intact yeast DNA chromosomes and real-time observation of their migration during gel electrophoresis; Gurrieri S et al.; In the past few years, fluorescence microscopy has been used successfully to characterize the motion of intermediate-size DNA molecules (50-500 kbp) during steady- and pulsed-field gel electrophoresis . However, experimental difficulties had prevented the application of this technique to the direct observation of longer DNA chromosomes (1-2 Mbp) . In the present study a particular procedure was followed for the purification and staining of chromosomal yeast DNA to protect it from shear forces . Also, a new highly fluorescent DNA-labelling dye, YOYO-1, was employed to improve brightness and contrast . Finally, the motion of such long DNA molecules (1-2 Mbp) was characterized under steady-field electrophoresis conditions . An accurate description of the molecular mechanisms of motion of such long molecules should provide the basis for a detailed analysis of the mechanisms responsible for DNA trapping. Cell, 1997 Oct 3, 91(1), 109 - 18 The AP-3 adaptor complex is essential for cargo-selective transport to the yeast vacuole; Cowles CR et al.; Three distinct adaptor protein (AP) complexes involved in protein trafficking have been identified . AP-1 and AP-2 mediate protein sorting at the trans-Golgi network and plasma membrane, respectively, whereas the function of AP-3 has not been defined . A screen for factors specifically involved in transport of alkaline phosphatase (ALP) from the Golgi to the vacuole/lysosome has identified Ap16p and Ap15p of the yeast AP-3 complex . Deletion of each of the four AP-3 subunits results in selective mislocalization of ALP and the vacuolar t-SNARE, Vam3p (but not CPS and CPY), while deletion of AP-1 and AP-2 subunits has no effect on vacuolar protein delivery . This study, therefore, provides evidence that the AP-3 complex functions in cargo-selective protein transport from the Golgi to the vacuole/lysosome. J Cell Biol, 1997 Oct 20, 139(2), 459 - 67 The yeast motor protein, Kar3p, is essential for meiosis I; Bascom-Slack CA et al.; The recognition and alignment of homologous chromosomes early in meiosis is essential for their subsequent segregation at anaphase I; however, the mechanism by which this occurs is unknown . We demonstrate here that, in the absence of the molecular motor, Kar3p, meiotic cells are blocked with prophase monopolar microtubule arrays and incomplete synaptonemal complex (SC) formation . kar3 mutants exhibit very low levels of heteroallelic recombination . kar3 mutants do produce double-strand breaks that act as initiation sites for meiotic recombination in yeast, but at levels severalfold reduced from wild-type . These data are consistent with a meiotic role for Kar3p in the events that culminate in synapsis of homologues. Genes Dev, 1997 Oct 1, 11(19), 2522 - 31 Coupling of cell division to cell growth by translational control of the G1 cyclin CLN3 in yeast; Polymenis M et al.; The eukaryotic cell cycle is driven by a cascade of cyclins and kinase partners including the G1 cyclin Cln3p in yeast . As the first step in this cascade, Cln3p is uniquely positioned to determine the critical growth-rate threshold for division . To analyze factors regulating CLN3 expression, we identified a short upstream open reading frame (uORF) in the 5' leader of CLN3 mRNA as a translational control element . This control element is critical for the growth-dependent regulation of Cln3p synthesis because it specifically represses CLN3 expression during conditions of diminished protein synthesis or slow growth . Inactivation of the uORF accelerates the completion of Start and entry into the cell cycle suggesting that translational regulation of CLN3 provides a mechanism coupling cell growth and division. J Biol Chem, 1997 Oct 17, 272(42), 26787 - 93 V1-situated stalk subunits of the yeast vacuolar proton-translocating ATPase; Tomashek JJ et al.; The proton-translocating ATPase of the yeast vacuole is an enzyme complex consisting of a large peripheral membrane sector (V1) and an integral membrane sector (V0), each composed of multiple subunits . The V1 sector contains subunits that hydrolyze ATP, whereas the V0 sector contains subunits that translocate protons across the membrane . Additional subunits in both sectors couple these activities . Here we have continued our examination of intermediate subunits primarily associated with the V1 but also implicated in interactions with the V0 . Interactions between Vma7p (F) and Vma8p (D) and between Vma4p (E) and Vma10p (G) are described . Although Vma7p and Vma10p have been observed to interact with the V0 sector, our results indicate that these subunits behave primarily as canonical V1 sector subunits . We categorize these four subunits as "stalk" subunits to distinguish them from the known catalytic (A and B) and proton-translocating (c, c', and Vma16p) subunits and to highlight their intermediate nature . Furthermore, we show that the in vivo stability of Vma4p is dependent upon interaction with Vma10p . This may be important in the regulation of assembly, since these two subunits add to the V1 during later stages of V1 assembly . This is the first demonstration of interdependence between ATPase subunits for structural stability. J Biol Chem, 1997 Oct 17, 272(42), 26550 - 4 Identification of the region in yeast S-II that defines species specificity in its interaction with RNA polymerase II; Shimoaraiso M et al.; Yeast S-II was found to stimulate yeast RNA polymerase II only and not mouse RNA polymerase II . To identify the molecular region of S-II that defines species specificity, we constructed six hybrid S-II molecules consisting of three regions from yeast and/or Ehrlich cell S-II and examined their activity in terms of RNA polymerase II specificity and suppression of 6-azauracil sensitivity in the yeast S-II null mutant . We found that the region 132-270 (amino acid positions) of yeast S-II is indispensable for specific interaction with yeast RNA polymerase II in vitro and for suppression of 6-azauracil sensitivity in vivo . The corresponding region of Ehrlich cell S-II, the region 132-262, was also shown to be essential for its interaction with mouse RNA polymerase II . This region is known to be less conserved than the N- and C-terminal regions in the S-II family suggesting that it is important in the interaction with transcription machinery proteins in a tissue and/or species-specific manner. J Biol Chem, 1997 Oct 17, 272(42), 26318 - 24 The adjacent yeast genes ARO4 and HIS7 carry no intergenic region; Springer C et al.; The region between the open reading frames of the adjacent yeast genes ARO4 and HIS7 consists of 417 base pairs (bp) . Termination of ARO4 transcription and initiation of HIS7 transcription has to take place within this interval, because both genes are transcribed into the same direction . We show that the ARO4 terminator and the HIS7 promoter are spatially separated, nonoverlapping units . The ARO4 terminator includes 84 bp of the ARO4 3'-untranslated region with several redundant ARO4 3' end processing signals . Deletion of the ARO4 terminator does reduce but not completely shut down its expression . The adjacent region of 40 bp is neither required for correct ARO4 3' end formation nor for HIS7 initiation but contains the nucleotides corresponding to the wild type mRNA 3' ends . The following 280 bp are required for the HIS7 promoter . Replacement of the housekeeping ARO4 promoter by the stronger ACT1 promoter leads to reduced HIS7 expression due to transcriptional interference . This underlines the compactness of the yeast genome carrying virtually no intergenic regions between adjacent genes. J Biol Chem, 1997 Oct 17, 272(42), 26145 - 52 Intracellular sequestration of sodium by a novel Na+/H+ exchanger in yeast is enhanced by mutations in the plasma membrane H+-ATPase . Insights into mechanisms of sodium tolerance; Nass R et al.; Sodium tolerance in yeast is disrupted by mutations in calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, which is required for modulation of Na+ uptake and efflux mechanisms . Five Na+-tolerant mutants were isolated by selecting for suppressors of calcineurin mutations, and mapped to the PMA1 gene, encoding the plasma membrane H+-ATPase . One mutant, pma1-alpha4, which has the single amino acid change Glu367 --> Lys at a highly conserved site within the catalytic domain of the ATPase, was analyzed in detail to determine the mechanism of Na+ tolerance . After exposure to Na+ in the culture medium, 22Na influx in the pma1 mutant was reduced 2-fold relative to control, consistent with a similar decrease in ATPase activity . Efflux of 22Na from intact cells was relatively unchanged in the pma1 mutant . However, selective permeabilization of the plasma membrane revealed that mutant cells retained up to 80% of intracellular Na+ within a slowly exchanging pool . We show that NHX1, a novel gene homologous to the mammalian NHE family of Na+/H+ exchangers, is required for Na+ sequestration in yeast and contributes to the Na+-tolerant phenotype of pma1-alpha4. Biochemistry (Mosc), 1997 Jul, 62(7), 779 - 86 Synthesis and some aspects of topogenesis of bovine cytochrome P450scc in yeast; Savelev AS et al.; A plasmid for effective expression of recombinant DNA encoding a hybrid protein composed of the N-terminal targeting presequence of subunit IV of yeast cytochrome c oxidase preceding the mature polypeptide chain of bovine cytochrome P450scc (pCoxIV-CYP11A1) in yeast has been constructed . It has been shown that this protein, when synthesized in yeast cells, in imported into mitochondria and undergoes proteolytic processing, thus yielding a product of molecular mass corresponding to that of mature cytochrome P450scc . However, only insignificant portion of the imported protein proves to be inserted into the inner membrane of heterologous mitochondria . The membrane-bound cytochrome P450scc exhibits cholesterol hydroxylase activity towards 22R-hydroxycholesterol in the presence of exogenous adrenodoxin and adrenodoxin reductase . This fact indicates that the foreign protein is correctly folded and oriented in the membrane . Thus, insertion into the inner membrane is a limiting step of the pCoxIV-CYP11A1 topogenesis in yeast cells, whereas its import into mitochondria and proteolytic processing proceed without significant impediments. Structure, 1997 Sep 15, 5(9), 1173 - 85 The structure of an essential splicing element: stem loop IIa from yeast U2 snRNA; Stallings SC et al.; BACKGROUND: Eukaryotic genes are usually transcribed as precursor mRNAs which are then spliced, removing introns to produce functional mRNAs . Splicing is performed by the spliceosome and provides an important level of post-translational control of gene expression . Stem loop IIa from U2 small nuclear (sn)RNA is required for the efficient association of the U2 small nuclear ribonuclear protein (snRNP) with the nascent spliceosome in yeast . Genetic analysis suggests that stem loop IIa is involved in RNA-protein interactions early in splicing, and it may also interact with other RNA sequences in U2 . The sequence of loop IIa is well conserved, consistent with the idea that this loop is important for function . RESULTS: We have solved the structure of U2A, a 20-base analogue of stem loop IIa from Saccharomyces cerevisiae, using NMR and restrained molecular dynamics . In the process, we have demonstrated the efficacy of a new structure calculation protocol, torsion angle molecular dynamics . The structure that has emerged, which is consistent with the in vivo chemical protection data available for stem loop IIa in the context of intact U2 snRNA, contains a sheared GA pair followed by a U-turn in the loop . The U-turn conformation, which resembles the U-turns in tRNA anticodon loops, makes this stretch of U2 snRNA an obvious target for interactions with proteins and/or other RNA sequences . CONCLUSIONS: The phenotypes of many stem loop IIa mutants can be rationalized assuming that the U-turn conformation in the loop must be preserved for efficient splicing . This observation, combined with the phylogenetic conservation of its sequence, suggests that the conformation of the loop of stem loop IIa is essential for its function in pre-mRNA splicing. Genome Res, 1997 Oct, 7(10), 1006 - 19 The largest subunit of human RNA polymerase III is closely related to the largest subunit of yeast and trypanosome RNA polymerase III; Sepehri S et al.; In both yeast and mammalian systems, considerable progress has been made toward the characterization of the transcription factors required for transcription by RNA polymerase III . However, whereas in yeast all of the RNA polymerase III subunits have been cloned, relatively little is known about the enzyme itself in higher eukaryotes . For example, no higher eukaryotic sequence corresponding to the largest RNA polymerase III subunit is available . Here we describe the isolation of cDNAs that encode the largest subunit of human RNA polymerase III, as suggested by the observations that (1) antibodies directed against the cloned protein immunoprecipitate an active enzyme whose sensitivity to different concentrations of alpha-amanitin is that expected for human RNA polymerase III; and (2) depletion of transcription extracts with the same antibodies results in inhibition of transcription from an RNA polymerase III, but not from an RNA polymerase II, promoter . Sequence comparisons reveal that regions conserved in the RNA polymerase I, II, and III largest subunits characterized so far are also conserved in the human RNA polymerase III sequence, and thus probably perform similar functions for the human RNA polymerase III enzyme. Mutat Res, 1997 Sep, 384(3), 205 - 11 Cell cycle-dependent protein expression of mammalian homologs of yeast DNA double-strand break repair genes Rad51 and Rad52; Chen F et al.; Recently, human and rodent homologs of yeast repair genes Rad51 and Rad52 have been identified and proposed to play roles in DNA double-strand break (DSB) repair . In this study, cell cycle-dependent expression of human and rodent RAD51 and RAD52 proteins was monitored using two approaches . First, flow cytometric measurements of DNA content and immunofluorescence were used to determine the phase-specific levels of RAD51 and RAD52 protein expression in irradiated and control populations . The expression of both proteins was lowest in G0/G1, increased in S and reached a maximum in G2/M . No difference was found in the whole-cell level of RAD51 or RAD52 protein expression between gamma-irradiated and control cell populations . Second, cell cycle-dependent protein expression was confirmed by Western analysis of populations synchronized in G0, G1 and G2 phases . Analysis of V3, a hamster equivalent of SCID, indicates that the protein level increases of RAD51 and RAD52 from G0 to G1/S/G2 do not require DNA-PK. Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11190 - 5 Three Ever Shorter Telomere (EST) genes are dispensable for in vitro yeast telomerase activity; Lingner J et al.; Telomerase is a specialized reverse transcriptase consisting of both RNA and protein components . Previous characterization of yeast telomerase function in vivo identified four EST (for ever shorter telomeres) genes that, when mutated, result in the phenotypes expected for a defect in telomerase . Consistent with this genetic prediction, the EST2 gene has recently been shown to encode the catalytic component of telomerase . Using an in vitro assay, we show here that telomerase activity is present in extracts prepared from yeast strains carrying est1-Delta, est3-Delta, and cdc13-2(est) mutations . Therefore, while these three genes are necessary for telomerase function in vivo, they do not encode components essential for core catalytic activity . When Est2p, the one EST gene product found to be essential for catalytic activity, was immunoprecipitated from extracts, the telomerase RNA subunit was also specifically precipitated, supporting the conclusion that these two components are in a stable complex. Proc Natl Acad Sci U S A, 1997 Oct 14, 94(21), 11173 - 8 A yeast sterol auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme; Gachotte D et al.; Genetic disruption of the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxotrophy . We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain . One suppressor mutation is erg11, which is blocked in 14alpha-demethylation of lanosterol and is itself an auxotroph . The second suppressor mutation required is either slu1 or slu2 (suppressor of lanosterol utilization) . These mutations are leaky versions of HEM2 and HEM4, respectively; addition of exogenous hemin reverses the suppressing effects of slu1 and slu2 . Suppression of erg25 by erg11 slu1 (or erg11 slu2) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates . This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth . In the triple mutants, all but 1 (ERG6) of the 13 subsequent reactions of the ergosterol pathway are inactive . Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the ERG11 gene product, the 14alpha-demethylase . In this investigation, we demonstrate that treatment of the sterol auxotrophs erg25 slu1 or erg25 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system we have described. Biochem J, 1997 Sep 15, 326 ( Pt 3), 683 - 92 NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis . Isolation, characterization and biochemical properties of the enzyme; Neuhauser W et al.; During growth on d-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme . This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76% . Aldose reductase consists ofa single 43 kDa polypeptide with an isoelectric point of 4.70 . Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last . The catalytic efficiency (kcat/Km) in d-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants . The enzyme prefers NADP(H) approx . 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme . NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 microM), whereas NAD+ is not . Unlike mammalian aldose reductase, the enzyme from C . tenuis is not subject to oxidation-induced activation . Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5'-phosphate . The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase. Nucleic Acids Res, 1997 Oct 1, 25(19), 3795 - 800 RNA polymerase II transcription inhibits DNA repair by photolyase in the transcribed strand of active yeast genes; Livingstone-Zatchej M et al.; Yeast uses nucleotide excision repair (NER) and photolyase (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs) generated by ultraviolet light . In active genes, NER preferentially repairs the transcribed strand (TS) . In contrast, we recently showed that photolyase preferentially repairs the non-transcribed strands (NTS) of the URA3 and HIS3 genes in minichromosomes . To test whether photoreactivation depends on transcription, repair of CPDs was investigated in the transcriptionally regulated GAL10 gene in a yeast strain deficient in NER {AMY3 (rad1Delta)} . In the active gene (cells grown in galactose), photoreactivation was fast in the NTS and slow in the TS demonstrating preferential repair of the NTS . In the inactive gene (cells grown in glucose), both strands were repaired at similar rates . This suggests that RNA polymerases II blocked at CPDs inhibit accessibility of CPDs to photolyase . In a strain in which both pathways are operational {W303-1a (RAD1)}, no strand bias was observed either in the active or inactive gene, demonstrating that photoreactivation of the NTS compensates preferential repair of the TS by NER . Moreover, repair of the NTS was more quickly in the active gene than in the repressed gene indicating that transcription dependent disruption of chromatin facilitates repair of an active gene. Cell, 1997 Sep 19, 90(6), 1097 - 106 CDC5 and CKII control adaptation to the yeast DNA damage checkpoint; Toczyski DP et al.; A single double-stranded DNA (dsDNA) break will cause yeast cells to arrest in G2/M at the DNA damage checkpoint . If the dsDNA break cannot be repaired, cells will eventually override (that is, adapt to) this checkpoint, even though the damage that elicited the arrest is still present . Here, we report the identification of two adaptation-defective mutants that remain permanently arrested as large-budded cells when faced with an irreparable dsDNA break in a nonessential chromosome . This adaptation-defective phenotype was entirely relieved by deletion of RAD9, a gene required for the G2/M DNA damage checkpoint arrest . We show that one mutation resides in CDC5, which encodes a polo-like kinase, whereas a second, less penetrant, adaptation-defective mutant is affected at the CKB2 locus, which encodes a nonessential specificity subunit of casein kinase II. Proc Int Conf Intell Syst Mol Biol, 1997, 5, 65 - 74 Data mining for regulatory elements in yeast genome; Brazma A et al.; We have examined methods and developed a general software tool for finding and analyzing combinations of transcription factor binding sites that occur relatively often in gene upstream regions (putative promoter regions) in the yeast genome . Such frequently occurring combinations may be essential parts of possible promoter classes . The regions upstream to all genes were first isolated from the yeast genome database MIPS using the information in the annotation files of the database . The ones that do not overlap with coding regions were chosen for further studies . Next, all occurrences of the yeast transcription factor binding sites, as given in the IMD database, were located in the genome and in the selected regions in particular . Finally, by using a general purpose data mining software in combination with our own software, which parametrizes the search, we can find the combinations of binding sites that occur in the upstream regions more frequently than would be expected on the basis of the frequency of individual sites . The procedure also finds so-called association rules present in such combinations . The developed tool is available for use through the WWW. EMBO J, 1997 Oct 15, 16(20), 6263 - 71 Interplay of yeast global transcriptional regulators Ssn6p-Tup1p and Swi-Snf and their effect on chromatin structure; Gavin IM et al.; Transcriptional regulation in yeast involves a number of general trans-acting factors affecting chromatin structure . The Swi-Snf complex is required for expression of a large number of genes and has the ability to remodel chromatin in vitro . The Ssn6p-Tup1p repressor complex may be involved in chromatin organization through the interaction with pathway-specific DNA-binding proteins . To study the interplay of these factors and their effect on chromatin we have analyzed SUC2 chromatin structure in wild-type cells and in strains bearing combinations of ssn6/tup1 and swi1 mutations . We have mapped nucleosome positioning of the repressed gene in wild-type cells using primer extension methodology, allowing base pair resolution, and have analyzed details of chromatin remodeling in the derepressed state . In ssn6 or tup1 mutants under repressing conditions the observed changes in SUC2 chromatin structure may be suppressed by the swi1 mutation, suggesting that Ssn6p-Tup1p is not required for the establishment of nucleosome positioning at the SUC2 promoter . Our data indicate the involvement of chromatin remodeling factors distinct from the Swi-Snf complex in SUC2 transcriptional regulation and suggest that Swi-Snf may antagonize Ssn6p-Tup1p by controlling remodeling activity . We also show that a relatively high level of SUC2 transcription can coexist with positioned nucleosomes. EMBO J, 1997 Oct 15, 16(20), 6237 - 49 Yrb4p, a yeast ran-GTP-binding protein involved in import of ribosomal protein L25 into the nucleus; Schlenstedt G et al.; Gsp1p, the essential yeast Ran homologue, is a key regulator of transport across the nuclear pore complex (NPC) . We report the identification of Yrb4p, a novel Gsp1p binding protein . The 123 kDa protein was isolated from Saccharomyces cerevisiae cells and found to be related to importin-beta, the mediator of nuclear localization signal (NLS)-dependent import into the nucleus, and to Pse1p . Like importin-beta, Yrb4p and Pse1p specifically bind to Gsp1p-GTP, protecting it from GTP hydrolysis and nucleotide exchange . The GTPase block of Gsp1p complexed to Yrb4p or Pse1p is released by Yrb1p, which contains a Gsp1p binding domain distinct from that of Yrb4p . This might reflect an in vivo function for Yrb1p . Cells disrupted for YRB4 are defective in nuclear import of ribosomal protein L25, but show no defect in the import of proteins containing classical NLSs . Expression of a Yrb4p mutant deficient in Gsp1p-binding is dominant-lethal and blocks bidirectional traffic across the NPC in wild-type cells . L25 binds to Yrb4p and Pse1p and is released by Gsp1p-GTP . Consistent with its putative role as an import receptor for L25-like proteins, Yrb4p localizes to the cytoplasm, the nucleoplasm and the NPC. EMBO J, 1997 Oct 15, 16(20), 6171 - 81 Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle; Herman PK et al.; Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division . Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division . Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division . In addition, germination did not require many key regulators of cell cycle progression including the cyclin-dependent kinase, Cdc28p . However, two processes essential for cell growth, protein synthesis and signaling through the Ras protein pathway, were required for spore germination . Moreover, increasing Ras protein activity in spores resulted in an accelerated rate of germination and suggested that activation of the Ras pathway was rate-limiting for entry into the germination program . An early step in germination, commitment, was identified as the point at which spores became irreversibly destined to complete the uncoating process even if the original stimulus for germination was removed . Spore commitment to germination required protein synthesis and Ras protein activity; in contrast, post-commitment events did not require ongoing protein synthesis . Altogether, these data suggested a model for Ras function during transitions between periods of quiescence and cell cycle progression. Mol Cell Biol, 1997 Oct, 17(10), 6175 - 83 The yeast nucleolar protein Cbf5p is involved in rRNA biosynthesis and interacts genetically with the RNA polymerase I transcription factor RRN3; Cadwell C et al.; Yeast Cbf5p was originally isolated as a low-affinity centromeric DNA binding protein (W . Jiang, K . Middleton, H.-J . Yoon, C . Fouquet, and J . Carbon, Mol . Cell . Biol . 13:4884-4893, 1993) . Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1) . However, Cbf5p was found to be nucleolar and is highly homologous to the rat nucleolar protein NAP57, which coimmunoprecipitates with Nopp140 and which is postulated to be involved in nucleolar-cytoplasmic shuttling (U . T . Meier, and G . Blobel, J . Cell Biol . 127:1505-1514, 1994) . The temperature-sensitive cbf5-1 mutant demonstrates a pronounced defect in rRNA biosynthesis at restrictive temperatures, while tRNA transcription and pre-rRNA and pre-tRNA cleavage processing appear normal . The cbf5-1 mutant cells are deficient in cytoplasmic ribosomal subunits at both permissive and restrictive temperatures . A high-copy-number yeast genomic library was screened for genes that suppress the cbf5-1 temperature-sensitive growth phenotype . SYC1 (suppressor of yeast cbf5-1) was identified as a multicopy suppressor of cbf5-1 and subsequently was found to be identical to RRN3, an RNA polymerase I transcription factor . A cbf5delta null mutant is not rescued by plasmid pNOY103 containing a yeast 35S rRNA gene under the control of a Pol II promoter, indicating that Cbf5p has one or more essential functions in addition to its role in rRNA transcription. Mol Cell Biol, 1997 Oct, 17(10), 5905 - 14 Rfc5, a replication factor C component, is required for regulation of Rad53 protein kinase in the yeast checkpoint pathway; Sugimoto K et al.; The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae . We have previously shown that a temperature-sensitive (ts) rfc5-1 mutation is impaired in the S-phase checkpoint . In this report, we show that the rfc5-1 mutation is sensitive to DNA-damaging agents . RFC5 is necessary for slowing the S-phase progression in response to DNA damage . The phosphorylation of the essential central transducer, Rad53 protein kinase, is reduced in response to DNA damage in rfc5-1 mutants during the S phase . Furthermore, the inducibility of RNR3 transcription in response to DNA damage is dependent on RFC5 . It has been shown that phosphorylation of Rad53 is controlled by Mec1 and Tel1, members of the subfamily of ataxia-telangiectasia mutated (ATM) kinases . We also demonstrate that overexpression of TEL1 suppresses the ts growth defect and DNA damage sensitivity of rfc5-1 mutants and restores phosphorylation of Rad53 and RNR3 induction in response to DNA damage in rfc5-1 . Our results, together with the observation that overexpression of RAD53 suppresses the defects of the rfc5-1 mutation, suggest that Rfc5 is part of a mechanism transducing the DNA damage signal to the activation of the central transducer Rad53. Mol Cell Biol, 1997 Oct, 17(10), 5727 - 38 Multicopy suppressors of phenotypes resulting from the absence of yeast VDAC encode a VDAC-like protein; Blachly-Dyson E et al.; The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel) . Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1 . However, cells missing the POR1 gene (delta por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37 degrees C) . If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function . To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of delta por1 yeast grown on glycerol at 37 degrees C . This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1) . YVDAC2 can functionally complement defects present in delta por1 strains only when it is overexpressed . Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30 degrees C, albeit more slowly than delta por1 single mutants . Like delta por1 single mutants, they could not grow on glycerol at 37 degrees C . Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane . However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems . Therefore, mitochondrial membranes made from wild-type cells, delta por1 cells, delta por1 delta por2 cells, and delta por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined . The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel . In contrast, when the VDAC gene from Drosophila melanogaster was expressed in delta por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37 degrees C . Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels. Biochem Mol Biol Int, 1997 Sep, 43(1), 53 - 61 Modulation of the electrophoretic ATP-induced K(+)-transport in yeast mitochondria by delta pH; Roucou X et al.; The modulation of the ATP-induced K(+)-transport pathway of the yeast inner mitochondrial membrane by delta pH was investigated in two ways . First, the inhibitory effect of phosphate was compared to the effect of other permeant acids, demonstrating that a part of the effect of phosphate was linked to its electroneutral transport down delta pH . However, an additional effect specific for phosphate also occurred inside the matrix . Second, the stimulation of the respiration by ATP in the presence of K+ was compared to the effects of the protonophore ClCCP2 and of the K(+)-ionophore valinomycin . Quite unexpectedly, the effect of ATP looked more like the effect of ClCCP than the effect of valinomycin . This and previous results (Manon et al., Biochimica et Biophysica Acta 1231, 282-288 (1995)) show that the rate of electrophoretic K(+)-entry is limited by the rate of electroneutral K(+)-exit via the K+/H+ exchange and is therefore delta pH-dependent. Oncogene, 1997 Sep, 15(11), 1255 - 62 Identification and characterization of two novel SH2 domain-containing proteins from a yeast two hybrid screen with the ABL tyrosine kinase; Oda T et al.; To further our understanding of the molecular mechanism of Bcr-Abl mediated transformation, a yeast two hybrid screen was used to identify proteins binding to the Abl tyrosine kinase . Two partial cDNAs encoding novel SH2 domain-containing proteins were cloned and designated Shd and She . Both have homology to Shb, a previously reported SH2 domain-containing protein . Northern blot analysis showed that She is expressed in heart, lung, brain, and skeletal muscle, while expression of Shd is restricted to the brain . The deduced amino acid sequence of the full length mouse Shd cDNA contains an amino-terminal proline-rich region, and a carboxyterminal SH2 domain . A bacterially expressed Shd domain bound multiple tyrosine-phosphorylated proteins with relative molecular weights of 200, 170, 130, 100, 90, 78, 72 and 32 kDa from K562 cell lysates . Shd contains five YXXP motifs, a substrate sequence preferred by Abl tyrosine kinases . Shd was tyrosine phosphorylated in COS-7 cells co-transfected with Shd and c-Abl or Bcr-Abl . These results suggest that Shd may be a physiological substrate of c-Abl and may function as an adapter protein in the central nervous system. Genome Res, 1997 Sep, 7(9), 879 - 86 Construction of P1-derived artificial chromosome and yeast artificial chromosome contigs encompassing the DFNB7 and DFNB11 region of chromosome 9q13-21; Greinwald JH Jr et al.; DFNB7 and DFNB11, two loci for autosomal recessive nonsyndromic hearing loss (ARNSHL), have been mapped to chromosome 9q13-21 in separate consanguineous families . Using a radiation hybrid map, we have determined the correct marker order in the DFNB7/11 region and have demonstrated that the DFNB11 locus resides within a redefined DFNB7 interval . The gene(s) responsible for ARNSHL at these loci resides within an approximately 1 cM interval bounded by markers D9S1806 (centromeric) and D9S769 (telomeric) . A recently discovered Indian family confirms the new telomeric boundary . To assist in the identification and cloning of candidate genes, YAC and PAC contigs were constructed . A total of 19 YAC and 23 PAC clones were utilized to span the affected region and ensure double coverage throughout . Twenty-two previously published STSs and 21 new STSs were used to determine marker order and confirm the integrity of the contig . Using a positional cloning strategy we have identified three cochlear expressed genes that map to the DFNB7/11 interval. J Biol Chem, 1997 Oct 3, 272(40), 25345 - 52 The yeast nucleolar protein Nop4p contains four RNA recognition motifs necessary for ribosome biogenesis; Sun C et al.; The Saccharomyces cerevisiae nucleolar protein Nop4p is necessary for processing of rRNA and assembly of 60 S ribosomal subunits . Nop4p is unusual in that it contains four RNA recognition motifs (RRMs) including one noncanonical RRM, as well as several auxiliary motifs, two acidic regions between the RRMs, and a carboxyl-terminal domain rich in lysines and arginines . To examine the functional importance of these motifs, we isolated random and site-directed mutations in NOP4 and assayed Nop4p function in vivo . Our results indicate that each RRM is essential for Nop4p function; mutations in conserved aromatic residues of Nop4p cause a temperature-sensitive lethal phenotype and diminished 60 S ribosomal subunit production . The carboxyl-terminal 68 amino acids are important but apparently not essential; carboxyl-terminal truncation of Nop4p causes slow growth, decreased ribosome production, and mislocalization of Nop4p . Deletion of both acidic motifs is lethal but replacement of most of the acidic residues with alanine has no apparent phenotype . These acidic residues may serve as spacers or tethers to separate the RRMs. Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10780 - 5 The unique hetero-oligomeric nature of the subunits in the catalytic cooperativity of the yeast Cct chaperonin complex; Lin P et al.; The structural and functional organization of the Cct complex was addressed by genetic analyses of subunit interactions and catalytic cooperativity among five of the eight different essential subunits, Cct1p-Cct8p, in the yeast Saccharomyces cerevisiae . The cct1-1, cct2-3, and cct3-1 alleles, containing mutations at the conserved putative ATP-binding motif, GDGTT, are cold-sensitive, whereas single and multiple replacements of the corresponding motif in Cct6p are well tolerated by the cell . We demonstrated herein that cct6-3 (L19S), but not the parolog cct1-5 (R26I), specifically suppresses the cct1-1, cct2-3, and cct3-1 alleles, and that this suppression can be modulated by mutations in a putative phosphorylation motif, RXS, and the putative ATP-binding pocket of Cct6p . Our results suggest that the Cct ring is comprised of a single hetero-oligomer containing eight subunits of differential functional hierarchy, in which catalytic cooperativity of ATP-binding/hydrolysis takes place in a sequential manner different from the concerted cooperativity proposed for GroEL. Glycobiology, 1997 Sep, 7(6), 829 - 34 Cloning and characterization of human phosphomannomutase, a mammalian homologue of yeast SEC53; Hansen SH et al.; Phosphomannomutase (PMM) catalyzes the conversion of mannose-6-phosphate to mannose-1-phosphate, which is a substrate for the synthesis of GDP-mannose . This nucleotide sugar is then used in the synthesis of dolichol-phosphate-mannose, which is essential for N-linked glycosylation and thus the secretion of several glycoproteins as well as for the synthesis of glycosyl-phosphatidyl-inositol (GPI) anchored proteins . In the yeast Saccharomyces cerevisiae, SEC53, a gene required for viability, encodes PMM . Given the importance of PMM in glycoprotein synthesis, it is surprising that very little is known about the enzyme in higher eukaryotes . Recently, an autosomal recessive human disease, Carbohydrate-deficient glycoprotein syndrome type I (CDGS-I) has been correlated with severely reduced PMM activity . Here we report the isolation of a cDNA encoding human PMM, a protein of 29 kDa that is 55% identical and 66% similar to yeast Sec53p . Northern blot analysis shows a single 1.4 kb transcript that is ubiquitously expressed, although levels vary markedly among tissues . Expression of the human cDNA in a temperature-sensitive mutant sec53 yeast strain confers growth at the restrictive temperature, strongly suggesting that this gene encodes a functional PMM . Finally, when expressed in BHK cells, PMM is localized exclusively to the cytosol corresponding to its localization in yeast. EMBO J, 1997 Oct 1, 16(19), 5988 - 97 Is the yeast anaphase promoting complex needed to prevent re-replication during G2 and M phases? Pichler S, Piatti S, Nasmyth K. The Anaphase Promoting Complex (APC) is required for anaphase progression and B-type cyclin proteolysis . The recent finding that inactivation of the APC allows 'over-replication' of DNA has led to the proposal that the APC might also be required for preventing reduplication of chromosomes during G2 and M phases . In this report we re-investigate the phenotype of apc mutant cells and find that they do not re-replicate their DNA during the period taken for wild-type cells to traverse G2 and M phases . apc mutants do, however, gradually increase their DNA content after long periods of cell cycle arrest . Such DNA synthesis occurs almost exclusively in the cytoplasm and neither occurs in cells lacking mitochondrial DNA nor depends on Cdc6, a protein which is essential for the initiation of chromosomal but not mitochondrial DNA replication . ARS1, a chromosomal replication origin, is not re-fired in cells deprived of APC function, confirming that the 'over-replicated' DNA in apc mutant cells is of mitochondrial origin . Furthermore, we find that APC function is required to promote but not to prevent re-replication in ndc10 mutant cells . We therefore propose that the APC is not involved in preventing re-duplication of chromosomes during G2 and M phases. EMBO J, 1997 Oct 1, 16(19), 5966 - 76 The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast; Drury LS et al.; The budding yeast Cdc6 protein (Cdc6p) is essential for formation of pre-replicative complexes (pre-RCs) at origins of DNA replication . Regulation of pre-RC assembly plays a key role in making initiation of DNA synthesis dependent upon passage through mitosis and in limiting DNA replication to once per cell cycle . Cdc6p is normally only present at high levels during the G1 phase of the cell cycle . This is partly because the CDC6 gene is only transcribed during G1 . In this article we show that rapid degradation of Cdc6p also contributes to this periodicity . Cdc6p degradation rates are regulated during the cell cycle, reaching a peak during late G1/early S phase . Removal of a 47-amino-acid domain near the N-terminus of Cdc6p prevents degradation of Cdc6p . Likewise, mutations in the Cdc4/34/53 pathway involved in ubiquitin-mediated degradation block proteolysis and genetic evidence is presented indicating that the N-terminus of Cdc6p interacts with the Cdc4/34/53 pathway, probably through Cdc4p . A stable Cdc6p mutant which is no longer degraded by the Cdc4/34/53 pathway is, none the less, fully functional . Constitutive overexpression of either wild-type or stable Cdc6p does not induce re-replication and does not induce assembly of pre-replicative complexes after DNA replication is complete. EMBO J, 1997 Oct 1, 16(19), 5847 - 54 Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein; Galan JM et al.; We have recently reported that the yeast plasma membrane uracil permease undergoes cell-surface ubiquitination, which is dependent on the Npi1/Rsp5 ubiquitin-protein ligase . Ubiquitination of this permease, like that of some other transporters and receptors, signals endocytosis of the protein, leading to its subsequent vacuolar degradation . This process does not involve the proteasome, which binds and degrades ubiquitin-protein conjugates carrying Lys48-linked ubiquitin chains . The data presented here show that ubiquitination and endocytosis of uracil permease are impaired in yeast cells lacking the Doa4p ubiquitin-isopeptidase . Both processes were rescued by overexpression of wild-type ubiquitin . Mutant ubiquitins carrying Lys-->Arg mutations at Lys29 and Lys48 restored normal permease ubiquitination . In contrast, a ubiquitin mutated at Lys63 did not restore permease polyubiquitination . Ubiquitin-permease conjugates are therefore extended through the Lys63 of ubiquitin . When polyubiquitination through Lys63 is blocked, the permease still undergoes endocytosis, but at a reduced rate . We have thus identified a natural target of Lys63-linked ubiquitin chains . We have also shown that monoubiquitination is sufficient to induce permease endocytosis, but that Lys63-linked ubiquitin chains appear to stimulate this process. EMBO J, 1997 Sep 15, 16(18), 5520 - 30 Essential functions and actin-binding surfaces of yeast cofilin revealed by systematic mutagenesis; Lappalainen P et al.; Cofilin stimulates actin filament turnover in vivo . The phenotypes of twenty yeast cofilin mutants generated by systematic mutagenesis were determined . Ten grew as well as the wild type and showed no cytoskeleton defects, seven were recessive-lethal and three were conditional-lethal and caused severe actin organization defects . Biochemical characterization of interactions between nine mutant yeast cofilins and yeast actin provided evidence that F-actin binding and depolymerization are essential cofilin functions . Locating the mutated residues on the yeast cofilin molecular structure allowed several important conclusions to be drawn . First, residues required for actin monomer binding are proximal to each other . Secondly, additional residues are required for interactions with actin filaments; these residues might bind an adjacent subunit in the actin filament . Thirdly, despite striking structural similarity, cofilin interacts with actin in a different manner from gelsolin segment-1 . Fourthly, a previously unrecognized cofilin function or interaction is suggested by identification of spatially proximal residues important for cofilin function in vivo, but not for actin interactions in vitro . Finally, mutation of the cofilin N-terminus suggests that its sequence is conserved because of its critical role in actin interactions, not because it is sometimes a target for protein kinases. EMBO J, 1997 Sep 1, 16(17), 5216 - 26 The novel DNA damage checkpoint protein ddc1p is phosphorylated periodically during the cell cycle and in response to DNA damage in budding yeast; Longhese MP et al.; The DDC1 gene was identified, together with MEC3 and other checkpoint genes, during a screening for mutations causing synthetic lethality when combined with a conditional allele altering DNA primase . Deletion of DDC1 causes sensitivity to UV radiation, methyl methanesulfonate (MMS) and hydroxyurea (HU) . ddc1Delta mutants are defective in delaying G1-S and G2-M transition and in slowing down the rate of DNA synthesis when DNA is damaged during G1, G2 or S phase, respectively . Therefore, DDC1 is involved in all the known DNA damage checkpoints . Conversely, Ddc1p is not required for delaying entry into mitosis when DNA synthesis is inhibited . ddc1 and mec3 mutants belong to the same epistasis group, and DDC1 overexpression can partially suppress MMS and HU sensitivity of mec3Delta strains, as well as their checkpoint defects . Moreover, Ddc1p is phosphorylated periodically during a normal cell cycle and becomes hyperphosphorylated in response to DNA damage . Both phosphorylation events are at least partially dependent on a functional MEC3 gene. Science, 1997 Oct 3, 278(5335), 103 - 6 Ste5 RING-H2 domain: role in Ste4-promoted oligomerization for yeast pheromone signaling; Inouye C et al.; Ste5 is a scaffold for the mitogen-activated protein kinase (MAPK) cascade components in a yeast pheromone response pathway . Ste5 also associates with Ste4, the beta subunit of a heterotrimeric guanine nucleotide-binding protein, potentially linking receptor activation to stimulation of the MAPK cascade . A RING-H2 motif at the Ste5 amino terminus is apparently essential for function because Ste5(C177S) and Ste5(C177A C180A) mutants did not rescue the mating defect of a ste5Delta cell . In vitro Ste5(C177A C180A) bound each component of the MAPK cascade, but not Ste4 . Unlike wild-type Ste5, the mutant did not appear to oligomerize; however, when fused to a heterologous dimerization domain (glutathione S-transferase), the chimeric protein restored mating in an ste5Delta cell and an ste4Delta ste5Delta double mutant . Thus, the RING-H2 domain mediates Ste4-Ste5 interaction, which is a prerequisite for Ste5-Ste5 self-association and signaling. J Virol, 1997 Oct, 71(10), 8041 - 7 Conserved interaction of the papillomavirus E2 transcriptional activator proteins with human and yeast TFIIB proteins; Benson JD et al.; Papillomavirus early gene expression is regulated by the virus gene-encoded E2 proteins . The best-characterized E2 protein, encoded by bovine papillomavirus type 1 (BPV-1), has been shown to interact with basal transcription factor IIB (TFIIB) and the TATA binding protein basal transcription factor (N . M . Rank and P . F . Lambert, J . Virol . 69:6323-6334, 1995) . We demonstrate that the potent E2 transcriptional activator protein encoded by a gene of human PV type 16 also interacts with TFIIB in vitro . Moreover, a direct comparison of domains within human TFIIB (hTFIIB) required for VP16 and BPV-1 E2 indicates that these acidic activators interact with hTFIIB in a qualitatively similar manner . Our mapping experiments identify hTFIIB interaction domains within the amino-terminal activation domain of BPV-1 E2 . Finally, we demonstrate in vitro interaction between Saccharomyces cerevisiae TFIIB and BPV-1 E2, an observation that is consistent with the importance of the E2-TFIIB interaction for BPV-1 E2 transactivation in both systems. J Virol, 1997 Oct, 71(10), 7526 - 32 Analysis of the interaction of viral RNA replication proteins by using the yeast two-hybrid assay; O'Reilly EK et al.; The yeast two-hybrid system has been a useful tool in the genetic evaluation of protein-protein interactions . However, the biological relevance of these two-hybrid interactions to viral positive-strand RNA replication has not been demonstrated . The brome mosaic virus (BMV) system has been characterized extensively both genetically and biochemically, providing numerous mutations in the BMV 1a helicase-like and 2a polymerase-like proteins . We have tested wild-type 1a and 18 insertion mutations of 1a and found a perfect correlation between the in planta phenotypes and their ability to interact with 2a in the two-hybrid system . This finding allowed further characterization of the interaction between and among the BMV viral proteins . Using the two-hybrid assay, we have found that the interaction between the helicase-like region of 1a and the N terminus of 2a is stabilized by the presence of the centrally conserved polymerase-like domain of 2a . We have also identified a novel interaction between the 1a helicase-like protein and itself . Additionally, we have found this interaction in two related tripartite RNA viruses, cowpea chlorotic mottle virus and cucumber mosaic virus . We have demonstrated that this protein-protein interaction is specific to homologous pairings of the protein. FEMS Microbiol Lett, 1997 Sep 15, 154(2), 347 - 53 Purification and characterization of a cytosolic cytochrome P450 from yeast Trichosporon cutaneum; Yang Y et al.; A soluble cytochrome P450 from the yeast Trichosporon cutaneum was purified to homogeneity, using ammonium sulfate fractionation followed by fast protein liquid chromatography (FPLC) with DEAE-cellulose and phenyl-Sepharose columns . This procedure resulted in a 45-fold increase in specific activity with an activity yield of 6.8% . One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified enzyme was homogeneous and had a molecular mass of 45 kDa . The purified enzyme contained a heme group and had a characteristic absorption peak at 448 nm in the reduced carbon monoxide difference spectrum . This enzyme was a monomeric protein and catalyzed the conversion of salicylic acid to catechol in the presence of NADH or NADPH . The N-terminal amino acid sequence indicated that the Trichosporon cutaneum cytochrome P450 did not show homology to most eukaryotic cytochromes P450, but had a high degree of homology to one cytochrome P450, the nitric oxide reductase, of Fusarium oxysporum. Curr Genet, 1997 Jul, 32(1), 27 - 31 An approach to identify functional homologues and suppressors of genes in fission yeast; Grallert B et al.; We have developed a procedure using a bank of temperature-sensitive (ts) mutants of fission yeast to identify mutants which can be rescued by expression of a plasmid-borne gene of interest . The procedure has been used to identify new ts alleles of cdc2 and swi7/poll, a ts mutant rescued by actin, and to identify a ts allele of cdc11 which can be rescued by combined mammalian Myc and Max expression . The procedure should also be useful as an alternative approach to identify genes in fission yeast which are functionally homologous to genes of interest from other organisms. Microbiology, 1997 Sep, 143 ( Pt 9), 3063 - 8 Alterations in cellular lipids may be responsible for the transient nature of the yeast heat shock response; Chatterjee MT et al.; The stress-sensing systems leading to the cellular heat shock response (HSR) and the mechanism responsible for the desensitizing of this response in stress-acclimated cells are largely unknown . Here it is demonstrated that there is a close correlation between a 3 degrees C increase in the temperature required for maximal activation of a heat-shock (HS)-inducible gene in Saccharomyces cerevisiae and an increase in the percentage of cellular unsaturated fatty acids when cells are subjected to extended periods of growth at 37 degrees C . The latter occurs with the same kinetics as HS gene down-regulation during a prolonged HS and is reversed by reacclimation to growth at 25 degrees C . The transient nature of the HS may therefore be due to a lipid-mediated decrease in cellular heat sensitivity . Further evidence that unsaturated fatty acids desensitize cells to heat, with a resultant down-regulation of the HSR, is provided by demonstrating a 9 degrees C increase in the temperature required for maximal induction of this HS-inducible gene in cells containing high levels of unsaturated fatty acids assimilated during anaerobic growth at 25 degrees C. EMBO J, 1997 Aug 15, 16(16), 4973 - 82 A human homolog of the yeast Ssk2/Ssk22 MAP kinase kinase kinases, MTK1, mediates stress-induced activation of the p38 and JNK pathways; Takekawa M et al.; A human homolog of the yeast Ssk2 and Ssk22 mitogen-activated protein kinase kinase kinases (MAPKKK) was cloned by functional complementation of the osmosensitivity of the yeast ssk2delta ssk22delta sho1delta triple mutant . This kinase, termed MTK1 (MAP Three Kinase 1), is 1607 amino acids long and is structurally highly similar to the yeast Ssk2 and Ssk22 MAPKKKs . In mammalian cells (COS-7 and HeLa), MTK1 overexpression stimulated both the p38 and JNK MAP kinase pathways, but not the ERK pathway . MTK1 overexpression also activated the MKK3, MKK6 and SEK1 MAPKKs, but not the MEK1 MAPKK . Furthermore, MTK1 phosphorylated and activated MKK6 and SEK1 in vitro . Overexpression of a dominant-negative MTK1 mutant {MTK1(K/R)} strongly inhibited the activation of the p38 pathway by environmental stresses (osmotic shock, UV and anisomycin), but not the p38 activation by the cytokine TNF-alpha . The dominant-negative MTK1(K/R) had no effect on the activation of the JNK pathway or the ERK pathway . These results indicate that MTK1 is a major mediator of environmental stresses that activate the p38 MAPK pathway, and is also a minor mediator of the JNK pathway. EMBO J, 1997 Aug 15, 16(16), 4826 - 38 In vivo disassembly of free polyubiquitin chains by yeast Ubp14 modulates rates of protein degradation by the proteasome; Amerik AYu et al.; Degradation of many eukaryotic proteins requires their prior ligation to polyubiquitin chains, which target substrates to the 26S proteasome, an abundant cellular protease . We describe a yeast deubiquitinating enzyme, Ubp14, that specifically disassembles unanchored ('free') ubiquitin chains in vitro, a specificity shared by mammalian isopeptidase T . Correspondingly, deletion of the UBP14 gene from yeast cells results in a striking accumulation of free ubiquitin chains, which correlates with defects in ubiquitin-dependent proteolysis . Increasing the steady-state levels of ubiquitin chains in wild-type cells (by expressing a derivative of ubiquitin with an altered C-terminus) inhibits protein degradation to a degree comparable with that observed in ubp14delta cells . Inhibition of degradation is also seen when an active site mutant of Ubp14 is overproduced in vivo . Surprisingly, overproduction of wild-type Ubp14 can inhibit degradation of some proteins as well . Finally, Ubp14 and human isopeptidase T are shown to be functional homologs by complementation analysis . We propose that Ubp14 and isopeptidase T facilitate proteolysis in vivo by preventing unanchored ubiquitin chains from competitively inhibiting polyubiquitin-substrate binding to the 26S proteasome. Can J Microbiol, 1997 Aug, 43(8), 792 - 5 A conserved cysteine residue in yeast uroporphyrinogen decarboxylase is not essential for enzymatic activity; Di Flumeri C et al.; Uroporphyrinogen decarboxylase catalyzes the fifth step of heme biosynthesis in Saccharomyces cerevisiae . Studies utilizing sulfhydryl-specific reagents suggest that the enzyme requires a cysteine residue within the catalytic site . This hypothesis was tested directly by site-directed mutagenesis of highly conserved cysteine-52 to serine or alanine . Plasmids containing these mutations were able to complement a hem6 mutant strain . In addition, properties associated with decreased uroporphyrinogen decarboxylase activity were not detected in the mutant strain transformed with these mutant plasmids . These results suggest that the conserved cysteine-52 by itself is not essential for enzymatic activity. Genes Dev, 1997 Sep 1, 11(17), 2272 - 90 mre11S--a yeast mutation that blocks double-strand-break processing and permits nonhomologous synapsis in meiosis; Nairz K et al.; During meiotic prophase the repair of self-inflicted DNA double-strand break (DSB) damage leads to meiotic recombination in yeast . We employed a genetic screen to specifically characterize cellular functions that become essential after this DSB formation . As a result a new allele of MRE11, termed mre11S (for Separation of functions) was isolated that allows initiation but not processing and repair of meiotic DSBs similar to the well-characterized rad50S allele . In contrast, the mre11-1 allele blocks initiation of meiotic DSBs as reported previously by others . The mre11S allele, which is mutated in the 5' part of the gene, can partially complement mre11 alleles disrupted close to the 3' end that cannot initiate DSBs when homozygous . This suggests homodimerization of the Mre11 protein and the presence of separate domains for DSB initiation and 5' resection . The fact that two genes, RAD50 and MRE11, required for DSB processing are also essential for DSB initiation dictates a model in which a bifunctional initiation/repair complex is required to initiate meiotic recombination . A subset of mre11S nuclei was shown to perform extensive but partially nonhomologous synapsis . We propose that the unprocessed DSBs present in mre11S allow for synapsis, but that homologous synapsis is only ensured at a later stage of recombination. EMBO J, 1997 Aug 1, 16(15), 4727 - 37 A multisubunit 3' end processing factor from yeast containing poly(A) polymerase and homologues of the subunits of mammalian cleavage and polyadenylation specificity factor; Preker PJ et al.; Polyadenylation is the second step in 3' end formation of most eukaryotic mRNAs . In Saccharomyces cerevisiae, this step requires three trans-acting factors: poly(A) polymerase (Pap1p), cleavage factor I (CF I) and polyadenylation factor I (PF I) . Here, we describe the purification and subunit composition of a multiprotein complex containing Pap1p and PF I activities . PF I-Pap1p was purified to homogeneity by complementation of extracts mutant in the Fip1p subunit of PF I . In addition to Fip1p and Pap1p, the factor comprises homologues of all four subunits of mammalian cleavage and polyadenylation specificity factor (CPSF), as well as Ptalp, which previously has been implicated in pre-tRNA processing, and several as yet uncharacterized proteins . As expected for a PF I subunit, pta1-1 mutant extracts are deficient for polyadenylation in vitro . PF I also appears to be functionally related to CPSF, as it polyadenylates a substrate RNA more efficiently than Pap1p alone . Possibly, the observed interaction of the complex with RNA tethers Pap1p to its substrate. EMBO J, 1997 Aug 1, 16(15), 4708 - 16 Functional interchangeability of TFIIIB components from yeast and human cells in vitro; Teichmann M et al.; In eukaryotes, TFIIIB is required for proper initiation by RNA polymerase III . In the yeast Saccharomyces cerevisiae a single form of TFIIIB (gammaTFIIIB) is sufficient for transcription of all pol III genes, whereas in extracts derived from human cells two different hTFIIIB complexes exist which we have previously designated as hTFIIIB-alpha and hTFIIIB-beta . Human TFIIIB-alpha is a TBP-free entity and must be complemented by TBP for transcription of pol III genes driven by gene external promoters, whereas hTFIIIB-beta is a TBP-TAF complex which governs transcription from internal pol III promoters . We show that hTFIIIB-beta cannot be replaced by yeast TFIIIB for transcription of tRNA genes, but that the B" component of gammaTFIIIB can substitute for hTFIIIB-alpha activity in transcription of the human U6 gene . Moreover, hTFIIIB-alpha can be chromatographically divided into activities which are functionally related to gammaTFIIIE and recombinant yB"90, suggesting that hTFIIIB-alpha is a human homolog of yeast TFIIIB" . In addition, we show that yeast TBP can only be exchanged against human TBP for in vitro transcription of the human and yeast U6 gene but virtually not for that of the yeast tRNA4Sup gene . This deficiency can be counteracted by a mutant of human TBP (R231K) which is able to replace yeast TBP for transcription of yeast tRNA genes in vitro. EMBO J, 1997 Aug 1, 16(15), 4676 - 88 Control of S-phase periodic transcription in the fission yeast mitotic cycle; Baum B et al.; In fission yeast, passage through START and into S-phase requires cyclin-dependent kinase (CDK) activity and the periodic transcription of genes essential for S-phase ('S-phase transcription') . Here we investigate the control of this transcription in the mitotic cell cycle . We demonstrate that the periodicity of S-phase transcription is likely to be controlled independently of CDK activity . This contrasts with the equivalent system in budding yeast . Furthermore, the CDK function required for S-phase acts after the onset of S-phase transcription and after the accumulation of cdc18p, a critical target of this transcriptional machinery . We investigate the role of individual components of the S-phase transcriptional machinery, cdc10p, res1p, res2p and rep2p, and define a new role for res2p, previously demonstrated to be important in the meiotic cycle, in switching off S-phase transcription during G2 of the mitotic cycle . We show that the presence of the in vitro bandshift activity DSC1, conventionally thought to represent the active complex, requires res2p and correlates with inactive transcription . We suggest that S-phase transcription is controlled by both activation and repression, and that res2p represses transcription in G2 of the cell cycle as a part of the DSC1 complex. EMBO J, 1997 Aug 1, 16(15), 4665 - 75 Stress and developmental regulation of the yeast C-type cyclin Ume3p (Srb11p/Ssn8p); Cooper KF et al.; The ume3-1 allele was identified as a mutation that allowed the aberrant expression of several meiotic genes (e.g . SPO11, SPO13) during mitotic cell division in Saccharomyces cerevisiae . Here we report that UME3 is also required for the full repression of the HSP70 family member SSA1 . UME3 encodes a non-essential C-type cyclin (Ume3p) whose levels do not vary through the mitotic cell cycle . However, Ume3p is destroyed during meiosis or when cultures are subjected to heat shock . Ume3p mutants resistant to degradation resulted in a 2-fold reduction in SPO13 mRNA levels during meiosis, indicating that the down-regulation of this cyclin is important for normal meiotic gene expression . Mutational analysis identified two regions (PEST-rich and RXXL) that mediate Ume3p degradation . A third destruction signal lies within the highly conserved cyclin box, a region that mediates cyclin-cyclin-dependent kinase (Cdk) interactions . However, the Cdk activated by Ume3p (Ume5p) is not required for the rapid destruction of this cyclin . Finally, Ume3p destruction was not affected in mutants defective for ubiquitin-dependent proteolysis . These results support a model in which Ume3p, when exposed to heat shock or sporulation conditions, is targeted for destruction to allow the expression of genes necessary for the cell to respond correctly to these environmental cues. Yeast, 1997 Sep 30, 13(12), 1181 - 94 Disruption of six novel yeast genes reveals three genes essential for vegetative growth and one required for growth at low temperature; Huang ME et al.; We describe here the construction of six deletion mutants and their basic phenotypic analysis . Six open reading frames (ORFs) from chromosome X, YJR039w, YJR041c, YJR043c, YJR046w, YJR053w and YJR065c, were disrupted by deletion cassettes with long (LFH) or short (SFH) flanking regions homologous to the target locus . The LFH deletion cassette was made by introducing into the kanMX4 marker module two polymerase chain reaction (PCR) fragments several hundred base pairs (bp) in size homologous to the promoter and terminator regions of a given ORF . The SFH gene disruption construct was obtained by PCR amplification of the kanMX4 marker with primers providing homology to the target gene . The region of homology to mediate homologous recombination was about 70 bp . Sporulation and tetrad analysis revealed