Yeasts

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 that ORFs YJR041c, YJR046w and YJR065c are essential genes . Complementation tests by corresponding cognate gene clones confirmed this observation . The non-growing haploid segregants were observed under the microscope . The yjr041c delta haploid cells gave rise to microcolonies comprising about 20 to 50 cells . Most yjr046w delta cells were blocked after one or two cell cycles with heterogeneous bud sizes . The yjr065c delta cells displayed an unbudded spore or were arrested before completion of the first cell division cycle with a bud of variable size . The deduced protein of ORF YJR065c, that we named Act4, belongs to the Arp3 family of actin-related proteins . Three other ORFs, YJR039w, YJR043c and YJR053w are non-essential genes . The yjr043c delta cells hardly grew at 15 degrees C, indicating that this gene is required for growth at low temperature . Complementation tests confirmed that the disruption of YJR043c is responsible for this growth defect . In addition, the mating efficiency of yjr043c delta and yjr053w delta cells appear to be moderately affected.

Gene, 1997 Aug 11, 195(1), 1 - 10
Human genetic diseases: a cross-talk between man and yeast; Foury F; A sequence similarity search has been carried out against the complete Saccharomyces cerevisiae genome to identify the yeast homologues of human disease-associated genes . Using the BLAST algorithm (Basic Local Alignment Search Tool), it was found that 52 out of the 170 disease genes identified without reference to chromosomal map position and 22 of the 80 (27.5%) positionally cloned genes match yeast genes with a P-value of <e(-40) . The percentage of the disease genes identified by positional cloning which bear homology to yeast is similar to that of a random collection of human cDNAs . The biochemical and physiological functions of the large majority of these human genes remain poorly understood and, even though a strict conservation of function cannot safely be assessed from structural homology analysis without the support of experimental and three-dimensional data, functional analogies can often be established between the human and yeast genes.

Appl Microbiol Biotechnol, 1997 Aug, 48(2), 232 - 5
One-step transformation of the dimorphic yeast Yarrowia lipolytica; Chen DC et al.; An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described . Using cells grown overnight on agar plates, the whole process is carried out within 1 h . The transformant clones could be recovered on selective plates as early as 36-48 h after plating . The efficiency was better than 10(5) transformants/micrograms replicative plasmid DNA . Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000 concentration and the wetness of selective plates were investigated.

J Mol Biol, 1997 Sep 5, 271(5), 838 - 45
Investigation of the enzymatic mechanism of the yeast chorismate mutase by docking a transition state analog; Lin SL et al.; The structure of the complex of the chorismate mutase from the yeast Saccharomyces cerevisiae with a transition state analog is constructed using a suite of docking tools . The construction finds the best location for the active site in the enzyme, and the best orientation of the analog compound in the active site . The resulting complex shows extensive salt links and hydrogen bonds between the enzyme and the compound, including those mediated by water molecules . A network of polar interactions between amino acid residues is found to solidify the active site of the enzyme . The enzymatic mechanism suggested for a bacterial chorismate mutase, that the active site is by design capable of selecting an active conformer of the substrate, and of stabilizing the transition state, is apparently intact in the yeast enzyme . No direct evidence is found to support an alternative mechanism which involves specific catalytic groups, although the possibility is not eliminated . This finding reinforces the notion of a function being evolutionarily conserved via a common mechanism, rather than via sequential or structural homology .

Biochemistry, 1997 Sep 23, 36(38), 11472 - 8
Mechanism-based inactivation of a yeast methylamine oxidase mutant: implications for the functional role of the consensus sequence surrounding topaquinone; Cai D et al.; The copper-containing yeast methylamine oxidase E406N mutant has an altered consensus sequence surrounding the topaquinone cofactor (residue 405) . The mutation has no effect on the final yield of the active-site topaquinone cofactor during biogenesis but causes the enzyme to be inactivated by substrate methylamine {Cai, D., and Klinman, J . P . (1994) Biochemistry 33, 7674-7653} . In this study we show that the inactivation leads to the formation of a covalent adduct, which has a UV/vis spectrum very similar to that of a product Schiff base, an intermediate of topaquinone-catalyzed amine oxidation reactions . The kinetic isotope effects on the second-order rate constant for the inactivation and catalytic turnover are identical, indicating that the two processes share a common intermediate that follows C_H bond cleavage . Resonance Raman spectroscopy provides direct evidence for the accumulation of a neutral product Schiff base species . Removal of excess methylamine leads to recovery of both activity and the native absorption spectrum for E406N, indicating that the cofactor in the inactivated enzyme is chemically competent for hydrolysis . The rate of the reactivation is slow, however; the shortest half-life of the inhibited E406N at 25 degrees C is 5.9 min at pH 6.15 . pH effect experiments show that the inactivation and reactivation steps are controlled by a single ionizable group with a pKa of 6.9-7.1; under basic conditions, when this residue is deprotonated, the inactivation is the fastest and the half-life of the inhibited enzyme is the longest . On the basis of the available crystal structures of copper amine oxidases, we propose that a histidine residue in the dimer interface is responsible for the observed ionization . In the wild-type enzyme this histidine is kept protonated by virtue of Glu at position 406 . Unlike methylamine, the larger substrates ethylamine and benzylamine give normal turnover with E406N . Disruption of structure at the subunit interface in E406N may allow a rotation of the relatively small topa-product Schiff base complex (formed from methylamine) away from the active-site base to a conformation that is incompetent toward hydrolysis.

Ukr Biokhim Zh, 1996 Jul-Aug, 68(4), 64 - 75
{Comparative analysis of primary structures of yeast dolicholphosphomannosyl- and dolichophosphoglucosyl synthetases and other dolichol-conjugated enzymes}; Shpakov AO et al.; Primary structure of yeast dolicholphosphomannosyl- (DPMS) and dolicholphosphoglucosylsynthetases (DPGS) have been compared both between themselves and DPMS and DPGS with yeast beta- and alpha-1, 3-mannosyltransferases, glucosyltransferases and rat mannosyl-binding proteins . The long homological segments were revealed . The homological segments of beta- and alpha-1, 3-mannosyltransferases were located in regions having potency to form coiled-coil structures . These structures are known to be included in carbohydrate-binding protein domains . The previous data and the results presented now have made it possible to conclude that the dolichol-coupled enzymes have evolutionary relationship between themselves and common evolutionary roots with carbohydrate-binding proteins (lectins).

J Cell Sci, 1997 Aug, 110 ( Pt 16), 1879 - 91
A yeast heat shock transcription factor (Hsf1) mutant is defective in both Hsc82/Hsp82 synthesis and spindle pole body duplication; Zarzov P et al.; Cdc28 is a cyclin-dependent protein kinase of Saccharomyces cerevisiae that is required for the G1/S and G2/M transitions of the cell division cycle . All previously described cdc28 mutants aside from cdc28-1N arrest division specifically in the G1 phase . cdc28-1N arrests division in G2/mitosis . We show here that the cdc28-109 mutant exhibits a mixed cell division arrest at 37 degrees C with cells in both the G1 and G2 phases . In order to identify proteins that functionally interact with Cdc28, we isolated mutants that are colethal with cdc28-109 at its permissive temperature . We describe here our phenotypic analysis of two such mutants, hsf1-82 and ydj1-10, that affect the heat shock transcription factor and a yeast dnaj-like protein chaperone, respectively . hsf1-82 and ydj1-10 temperature-sensitive mutants arrest the cell division cycle at several stages . However, one predominant class of cells in both mutants was arrested with a large bud and a single vertex of microtubules . Electron microscopic analysis of such hsf1-82 cells showed that they contained an unduplicated spindle pole body with an enlarged half-bridge . Two-dimensional gel electrophoresis of total cell proteins revealed that the hsf1-82 cells were specifically defective in the expression of the Hsc82 and Hsp82 proteins . Furthermore, the hsf1-82 mutation was suppressed by the HSC82 gene on a multicopy plasmid that restored Hsc82 protein to high levels in these cells . These results show that Hsf1 is required for spindle pole body duplication at 37 degrees C.

Environ Health Perspect, 1997 Jul, 105(7), 734 - 42
Evaluation of a recombinant yeast cell estrogen screening assay; Coldham NG et al.; A wide range of chemicals with diverse structures derived from plant and environmental origins are reported to have hormonal activity . The potential for appreciable exposure of humans to such substances prompts the need to develop sensitive screening methods to quantitate and evaluate the risk to the public . Yeast cells transformed with plasmids encoding the human estrogen receptor and an estrogen responsive promoter linked to a reporter gene were evaluated for screening compounds for estrogenic activity . Relative sensitivity to estrogens was evaluated by reference to 17 beta-estradiol (E2) calibration curves derived using the recombinant yeast cells, MCF-7 human breast cancer cells, and a prepubertal mouse uterotrophic bioassay . The recombinant yeast cell bioassay (RCBA) was approximately two and five orders of magnitude more sensitive to E2 than MCF-7 cells and the uterotrophic assay, respectively . The estrogenic potency of 53 chemicals, including steroid hormones, synthetic estrogens, environmental pollutants, and phytoestrogens, was measured using the RCBA . Potency values produced with the RCBA relative to E2 (100) included estrone (9.6), diethylstilbestrol (74.3), tamoxifen (0.0047), alpha-zearalanol (1.3), equol (0.085), 4-nonylphenol (0.005), and butylbenzyl phathalate (0.0004), which were similar to literature values but generally higher than those produced by the uterotrophic assay . Exquisite sensitivity, absence of test compound biotransformation, ease of use, and the possibility of measuring antiestrogenic activity are important attributes that argue for the suitability of the RCBA in screening for potential xenoestrogens to evaluate risk to humans, wildlife, and the environment.

J Bacteriol, 1997 Sep, 179(18), 5843 - 8
Regulation of yeast phospholipid biosynthetic genes in phosphatidylserine decarboxylase mutants; Griac P; In the yeast Saccharomyces cerevisiae, the products of two genes (PSD1 and PSD2) are able to catalyze the decarboxylation of phosphatidylserine (PS) to produce phosphatidylethanolamine (PE) (C . J . Clancey, S . Chang, and W . Dowhan, J . Biol . Chem . 268:24580-24590, 1993; P . J . Trotter, J . Pedretti, and D . R . Voelker, J . Biol . Chem . 268:21416-21424, 1993; P.J . Trotter, and D . R . Voelker, J . Biol . Chem . 270:6062-6070, 1995) . I report that the major mitochondrial PS decarboxylase gene (PSD1) is transcriptionally regulated by inositol in a manner similar to that reported for other coregulated phospholipid biosynthetic genes . The second PS decarboxylase gene (PSD2) is not regulated on a transcriptional level by inositol and/or ethanolamine . In yeast, phosphatidylcholine (PC) biosynthesis is required for the repression of the phospholipid biosynthetic genes, including the INO1 gene, in response to inositol . I show that the presence of a functional major mitochondrial PS decarboxylase encoded by the PSD1 gene is necessary for proper regulation of INO1 in response to inositol in the absence of ethanolamine . Disruption of the second PS decarboxylase gene (PSD2) does not affect the INO1 regulation . Analysis of phospholipid content of PS decarboxylase mutants suggests that the proportion of PC on total cellular phospholipids is not correlated to the cell's ability to repress INO1 in response to inositol . Rather, yeast cells are apparently able to monitor the flux through the phospholipid biosynthetic pathway and modify the transcription of phospholipid biosynthetic genes accordingly.

Curr Genet, 1997 Aug, 32(2), 100 - 7
Cold-sensitive rad52 alleles of yeast; Nguyen MM et al.; We have characterized two rad52 mutations that are cold-sensitive for growth on MMS agar . The mutations change residues 61 and 69, respectively, in the 504 amino-acid open reading frame . Neither mutation has a profound effect on mitotic crossing-over or on gene conversion . One has a severe deficiency in sporulation at all temperatures, while the other has a partial deficiency and reduced spore viability . Both mutants are retarded in growth on MMS agar by a high-copy plasmid expressing RAD51.

Curr Genet, 1997 Aug, 32(2), 83 - 92
Yeast bcy1 mutants with stationary phase-specific defects; Peck VM et al.; Entry into the stationary phase requires the yeast BCY1 gene, which encodes the regulatory subunit of the cAMP-dependent protein kinase (cAPK) . New bcy1 mutants, constructed by in vitro mutagenesis of the 3'-region encoding the cAMP-binding domains, were classified as early or late-acting mutants based on viability studies . The late-acting bcy1 mutants accumulated fewer stationary phase-specific Bcy1p isoforms and had decreased cAPK activity . This late-acting class is novel and dies after 7 days in culture, later than two previously reported stationary phase mutants, ubi4 and ard1.

Proc Natl Acad Sci U S A, 1997 Sep 16, 94(19), 10373 - 8
Direct isolation of human transcribed sequences from yeast artificial chromosomes through the application of RNA fingerprinting; Still IH et al.; The identification of cDNA clones from genomic regions known to contain human genes is usually the rate-limiting factor in positional cloning strategies . We demonstrate here that human genes present on yeast artificial chromosomes (YACs) are transcribed in yeast host cells . We have used the arbitrarily primed RNA (RAP) fingerprinting method to identify human-specific, transcribed sequences from YACs located in the 13q12 chromosome region . By comparing the RAP fingerprints generated using defined, arbitrary primers from various fragmented YACs, megaYACs, and host yeast, we were able to identify and map 20 products transcribed from the human YAC inserts . This method, therefore, permits the simultaneous isolation and mapping of novel expressed sequences directly from whole YACs.

Mol Gen Genet, 1997 Aug, 255(5), 543 - 7
Budding yeast Rad50, Mre11, Xrs2, and Hdf1, but not Rad52, are involved in the formation of deletions on a dicentric plasmid; Tsukamoto Y et al.; We have previously shown that the RAD50, RAD52, MRE11, XRS2, and HDF1 genes of Saccharomyces cervisiae are involved in the formation of deletions by illegitimate recombination on a monocentric plasmid . In this study, we investigated the effects of mutations of these genes on formation of deletions of a dicentric plasmid, in which DNA double-strand breaks are expected to occur frequently because the two centromeres are pulled to opposite poles in mitosis . We transformed yeast cells with a dicentric plasmid, and after incubation for a few division cycles, cells carrying deleted plasmids were detected using negative selection markers . Deletions occurred at a higher frequency than on the monocentric plasmid and there were short regions of homology at the recombination junctions as observed on the monocentric plasmid . In rad50, mre11, xrs2, and hdf1 mutants, the frequency of occurrence of deletions was reduced by about 50-fold, while in the rad52 mutant, it was comparable to that in the wild-type strain . The end-joining functions of Rad50, Mre11, Xrs2, and Hdf1, suggest that these proteins play important roles in the joining of DNA ends produced on the dicentric plasmid during mitosis.

Mol Gen Genet, 1997 Aug, 255(5), 460 - 6
Rig2, a RING finger protein that interacts with the Kin28/Ccl1 CTD kinase in yeast; Faye G et al.; Kin28/Cell, a cyclin-dependent kinase, is essential for the in vivo phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II in Saccharomyces cerevisiae . In a search for mutations co-lethal with a thermosensitive kin28 mutation, we have identified genes whose products interact functionally with Kin28 . In the present work, we have studied a new complementation group of synthetic lethal mutations . The corresponding gene, RIG2, encodes a predicted RING finger protein . Rig2 is likely to be a homolog of MAT1 of higher eukaryotes which forms a ternary complex with MO15(cdk7) and cyclin H . Our genetic data suggest that Rig2 is a component of transcription factor TFIIH . Transcription activity in a rig2-ts mutant is impeded at restrictive temperature . However, none of the rig2-ts mutants obtained was UV sensitive, suggesting that Rig2 is dispensable for nucleotide excision repair.

RNA, 1997 Sep, 3(9), 961 - 73
Kinetics of hairpin ribozyme cleavage in yeast; Donahue CP et al.; Hairpin ribozymes catalyze a self-cleavage reaction that provides a simple model for quantitative analyses of intracellular mechanisms of RNA catalysis . Decay rates of chimeric mRNAs containing self-cleaving ribozymes give a direct measure of intracellular cleavage kinetics in yeast . Intracellular ribozyme-mediated cleavage occurs at similar rates and shows similar inhibition by ribozyme mutations as ribozyme-mediated reactions in vitro, but only when ribozymes are located in a favorable mRNA sequence context . The impact of cleavage on mRNA abundance is shown to depend directly on intrinsic mRNA stability . Surprisingly, cleavage products are no more labile than uncleaved mRNAs despite the loss of terminal cap structures or poly (A).

Biochem J, 1997 Sep 1, 326 ( Pt 2), 339 - 44
Mechanism and ion-dependence of in vitro autoactivation of yeast proteinase A: possible implications for compartmentalized activation in vivo; Van Den Hazel H et al.; Yeast proteinase A is synthesized as a zymogen which transits through the endoplasmic reticulum, the Golgi complex and the endosome to the vacuole . On arrival in the vacuole, activation takes place . It has previously been found that proteinase A can activate autocatalytically; however, the propeptide of proteinase A shows essentially no similarity to other known aspartic proteinase propeptides . To understand why proteinase A activation occurs rapidly in the vacuole but not at all in earlier compartments, we have purified the zymogen and investigated the conditions that trigger autoactivation and the mechanism of autoactivation . Autoactivation was triggered by acidic pH and its rate increased with increasing ionic strength . Kinetic evidence indicates that autoactivation mainly occurs via a bimolecular product-catalysed mechanism in which an active proteinase A molecule activates a zymogen molecule . Both the pH- and ionic-strength-dependence and the predominance of a product-catalysed mechanism are well adapted to the situation in vivo, since slow activation in the absence of active proteinase A helps to prevent activation in prevacuolar compartments, whereas, on delivery to the vacuole, lower pH, higher ionic strength and the presence of already active proteinases ensure rapid activation . Product-catalysed autoactivation may be a general mechanism by which cells ensure autoactivation of intracellular enzymes to be both rapid and compartmentalized.

Mol Carcinog, 1997 Aug, 19(4), 243 - 53
Screening the p53 status of human cell lines using a yeast functional assay; Jia LQ et al.; We have screened the p53 status of 156 human cell lines, including 142 tumor cell lines from 27 different tumor types and 14 cell lines from normal tissues by using functional analysis of separated alleles in yeast . This assay enables us to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS3 via the p53-responsive GAL1 promotor in Saccharomyces cerevisiae . Of 142 tumor cell lines, at least 104 lines (73.2%) were found to express the mutated p53 gene: 94 lines (66.2%) were mutated in both alleles, three lines (2.1%) were heterozygous, and no p53 cDNA was amplified from seven lines (4.9%) . Of the 14 cell lines originating from normal tissues, all the transformed or immortalized cell lines expressed mutant p53 only . Yeast cells expressing mutant p53 derived from 94 cell lines were analyzed for temperature-sensitive growth . p53 cDNA from eight cell lines showed p53-dependent temperature-sensitive growth, growing at 30 degrees C but not at 37 degrees C . Four temperature-sensitive p53 mutations were isolated: CAT-->CGT at codon 214 (H214R), TAC-->TGC at codon 234 (Y234C), GTG-->ATG at codon 272 (V272M), and GAG-->AAG (E285K) . Functionally wild-type p53 was detected in 38 tumor cell lines (26.8%) and all of the diploid fibroblasts at early and late population doubling levels . These results strongly support the previous findings that p53 inactivation is one of the most frequent genetic events that occurs during carcinogenesis and immortalization.

Yeast, 1997 Sep 15, 13(11), 1009 - 20
Accumulation of misfolded protein aggregates leads to the formation of russell body-like dilated endoplasmic reticulum in yeast; Umebayashi K et al.; RNAP-1, an aspartic proteinase from a filamentous fungus Rhizopus niveus, is secreted very efficiently in Saccharomyces cerevisiae . It is synthesized first as a precursor form with signal sequence and prosequence in its amino-terminus . Our previous study indicated that the prosequence of RNAP-I had important roles in its correct folding and secretion in yeast, and that a prosequence-deleted derivative of RNAP-I, delta pro, was not secreted but was retained and degraded in the yeast endoplasmic reticulum (ER) . In the present study, we show that the accumulation of delta pro in the yeast ER caused elevated synthesis of ER resident chaperones, indicating that delta pro is recognized as an unfolded protein species in the ER . Our biochemical data demonstrated that delta pro formed aggregates which contained BiP, but not protein disulfide isomerase (PDI), in the ER . Immunoelectron microscopical analysis revealed that the delta pro aggregates were indeed visible as electron-dense regions in the ER and nuclear envelope . Such 'chaperone-associated misfolded protein bodies' were observed for the first time in yeast . Morphologies of the ER and nucleus were drastically altered by the accumulation of the delta pro aggregates . The ER lost its flat cisternal shape; the ER lumen extended aberrantly and the ER membrane irregularly proliferated . The misfolded delta pro proteins are probably sorted from the ordinary ER lumen to form the aggregates so that the ER function would not be grossly impaired, and the dilated ER may represent an ER subcompartment where the delta pro aggregates are degraded.

Biochemistry, 1997 Sep 16, 36(37), 11273 - 81
Real-time measurement of multiple intramolecular distances during protein folding reactions: a multisite stopped-flow fluorescence energy-transfer study of yeast phosphoglycerate kinase; Lillo MP et al.; Understanding the set of rules which dictate how the primary amino acid sequence determines tertiary structure is an unsolved problem in biophysics . If it were possible to simultaneously measure all of the intramolecular distances in a protein (in real time) during a folding reaction, the "second" genetic code problem would be solved . Regrettably, no such technique currently exists . As a first step toward this goal, an optical distance assay system has been developed for a two-domain protein, yeast phosphoglycerate kinase (PGK), using Forster resonance energy transfer {Lillo, M . P., et al . (1997) Biochemistry 36, 11261-11272} . In this study, real-time stopped-flow distance changes are measured using six unique pairs of donor/acceptor fluorescent labels strategically placed throughout the tertiary structure of PGK . These multiple donor/acceptor sites were genetically engineered into PGK by cysteine substitution mutagenesis followed by extrinsic labeling with fluorescent probes, 5-{{{(2-iodoacetyl)amino}ethyl}amino}naphthalenesulfonic acid (as a donor) and 5-iodoacetamidofluorescein (acceptor) . The unfolding of PGK is found to be a sequential multistep process (native --> I1 --> I2 --> unfolded) with rate constants of 0.30, 0.16, and 0.052 s-1, respectively (from native to unfolded) . Unique to this unfolding study, six intramolecular distance vectors have been resolved for both the I1 and I2 states . With this distance information, it is shown that the transition from the native to I1 state can be modeled as a large hinge-bending motion, in which both domains "swing away" from each other by about 15 A . As the domains move apart, the carboxyl-terminal domain rotates almost 90 degrees about the hinge region connecting the two domains . It is also shown that the amino-terminal domain remains intact during the native --> I1 transition, consistent with our previous site-specific tryptophan fluorescence anisotropy stopped-flow study {Beechem, J . M., et al . (1995) Biochemistry 34, 13943-13948} . Future experiments are proposed which will attempt to resolve in detail the unfolding/refolding transitions in this protein with a resolution of approximately 5-10 A.

Biochemistry, 1997 Sep 16, 36(37), 11261 - 72
Design and characterization of a multisite fluorescence energy-transfer system for protein folding studies: a steady-state and time-resolved study of yeast phosphoglycerate kinase; Lillo MP et al.; A multisite distance-based fluorescence resonance energy-transfer assay system was developed for the study of protein folding reactions . Single- and double-cysteine substitution mutagenesis was utilized to place sulfhydryl residues throughout the tertiary structure of the bidomain enzyme yeast phosphoglycerate kinase (PGK) . These reactive cysteines were covalently modified with extrinsic donor {5-{{2-(2-iodoacetamido)ethyl}amino}-1-naphthalenesulfonic acid} and acceptor (5-iodoacetamidofluorescein) fluorescent labels . A detailed experimental strategy was followed, which revealed that, when these relatively large extrinsic fluorescent labels are covalently attached to properly selected solvent-exposed residues, they do not affect the intrinsic stability of the protein . The PGK crystal structure was combined with molecular dynamics simulations of the dyes built into the protein and time-resolved anisotropy experiments, in order to estimate a more realistic orientation factor, <kappa2>*, for each donor/acceptor pair . Time-resolved and steady-state fluorescence energy-transfer experiments revealed that this distance assay, spanning six different donor-acceptor distances, is linear and accurate (to within 10-20%) over the range of 30-70 A . This distance assay system for PGK allows for the measurement of long-range changes in intra- and interdomain spatial organization during protein folding reactions . The approach which we have developed can be applied to any protein system in which unique one- and two-site cysteine residues can be engineered into a protein . In the following paper {Lillo, M . P., et al . (1997) Biochemistry 36, 11273-11281}, these multisite energy-transfer pairs are utilized for stopped-flow unfolding studies.

Biochemistry, 1997 Sep 16, 36(37), 11234 - 40
Role of deprotonation events in ubihydroquinone:cytochrome c oxidoreductase from bovine heart and yeast mitochondria; Brandt U et al.; The pH dependence of bovine and yeast cytochrome bc1 complex catalyzing electron transfer from ubi- and plastohydroquinone to cytochrome c have been analyzed . The pH dependence of the steady-state rate was found to be governed by two protonable groups, one of which (pK approximately 6.6) has to be deprotonated while the other (pK approximately 9.2) has to be protonated to allow catalysis . Using ubideuteroquinone instead of ubihydroquinone as a substrate resulted in 1.4- and 1.7-fold lower steady-state rates for the bovine and yeast enzymes, respectively . The activation energy at pH 8.0 was 33 kJ/mol for the bovine and 44 kJ/mol for the yeast enzyme and exhibited a linear decrease between pH 5.4 and 9.2 . For ubihydroquinone the slope was very close to a value of -5.7 kJ/mol expected if the activation energy depended on a single deprotonation event . When plastohydroquinone was used instead, the slope more than doubled, indicating that a second deprotonation contributed to the activation barrier with this nonphysiological substrate . In contrast to previous kinetic models for the cytochrome bc1 complex, which propose that the activation barrier is associated with the formation of ubisemiquinone at the ubihydroquinone oxidation center, our results strongly suggest that the best approximation of the transition state is the singly deprotonated form of ubihydroquinone . This supports the recently proposed proton-gated charge transfer mechanism, which has control of catalysis by the first deprotonation of ubihydroquinone as one of its key features {Brandt, U . (1996) FEBS Lett . 387, 1-6} . All results reported here can be rationalized in a straightforward way based on other aspects of the same hypothesis.

J Biol Chem, 1997 Sep 12, 272(37), 23360 - 5
Yeast DNA repair proteins Rad6 and Rad18 form a heterodimer that has ubiquitin conjugating, DNA binding, and ATP hydrolytic activities; Bailly V et al.; The RAD6 and RAD18 genes of Saccharomyces cerevisiae are required for postreplicative bypass of ultraviolet (UV)-damaged DNA and for UV mutagenesis . The RAD6 encoded protein is a ubiquitin conjugating enzyme, and RAD18 encodes a protein containing a RING finger motif and a nucleotide binding motif . Rad18 can be co-immunoprecipitated with Rad6, indicating that the two proteins exist in a complex in vivo . Here, we co-overproduce the two proteins using a yeast multicopy plasmid, purify the Rad6-Rad18 complex to near homogeneity, and show that the complex is heterodimeric . The Rad6-Rad18 heterodimer has ubiquitin conjugating activity, binds single-stranded DNA, and possesses single-stranded DNA-dependent ATPase activity . The Rad6-Rad18 complex provides the first example wherein a ubiquitin conjugating activity is physically associated with DNA binding and ATPase activities provided by an associated protein factor . The co-existence of these activities should provide the complex with the ability to recognize single-stranded DNA resulting from stalling of the replication machinery at DNA damage sites and to recognize the components of the DNA replication machinery for ubiquitination by Rad6.

J Biol Chem, 1997 Sep 5, 272(36), 22832 - 9
Substrate specificity of the RNase activity of yeast RNA polymerase III; Bobkova EV et al.; Using yeast RNA polymerase III ternary complexes stalled at various positions on the template, we have analyzed the cleavage products that are retained and released by the transcription complexes . The retained 5' products result from cleavage at uridine residues during retraction, whereas the yield of mononucleotides and dinucleotides released indicates that multiple cuts occur near the 3' end . Comparison of the cleavage patterns of uridine-containing and 5-bromouridine-containing transcripts suggests that RNA within an RNA-DNA hybrid duplex is the substrate for the 3'-5' exonuclease . During transcription of the SUP4 tRNATyr gene, RNA polymerase III produces not only full-length pre-tRNATyr but also short oligonucleotides, indicating that exonuclease digestion and transcription are concurrent processes . To explore the possibility that these oligonucleotides are released by the action of the RNA polymerase III nuclease at previously observed uridine-rich pause sites, we tested modified templates lacking the arrest sites present in the SUP4 tRNATyr gene . Comparative studies of cleavage during transcription for these templates show a direct correlation between the number of natural pause sites and the yield of 3' products made . At the natural arrest sites and the terminator, RNA polymerase III carries out multiple cleavage resynthesis steps, producing short oligoribonucleotides with uridine residues at the 3' terminus.

J Biol Chem, 1997 Sep 5, 272(36), 22495 - 501
Glucose-6-P control of glycogen synthase phosphorylation in yeast; Huang D et al.; The SNF1 gene encodes a protein kinase necessary for expression of glucose-repressible genes and for the synthesis of the storage polysaccharide glycogen . From a genetic screen, we have found that mutation of the PFK2 gene, which encodes the beta-subunit of 6-phosphofructo-1-kinase, restores glycogen accumulation in snf1 cells . Loss of PFK2 causes elevated levels of metabolites such as glucose-6-P, hyperaccumulation of glycogen, and activation of glycogen synthase, whereas glucose-6-P is reduced in snf1 cells . Other mutations that increase glucose-6-P, deletion of PFK1, which codes for the alpha-subunit of 6-phosphofructo-1-kinase, or of PGI1, the phosphoglucoisomerase gene, had similar effects on glycogen metabolism as did pfk2 mutants . We propose that elevated glucose-6-P mediates the effects of these mutations on glycogen storage . Glycogen synthase kinase activity was reduced in extracts from pfk2 cells but was restored to that of wild type if the extract was gel-filtered to remove small molecules . Also, added glucose-6-P inhibited the glycogen synthase kinase activity in extracts from wild-type cells, half-maximally at approximately 2 mM . We suggest that glucose-6-P controls glycogen synthase activity by two separate mechanisms . First, glucose-6-P is a direct activator of glycogen synthase, and second, it controls the phosphorylation state of glycogen synthase by inhibiting a glycogen synthase kinase.

Nature, 1997 Sep 4, 389(6646), 90 - 3
Actin-dependent localization of an RNA encoding a cell-fate determinant in yeast; Takizawa PA et al.; The cytoplasmic localization of messenger RNA creates an asymmetric distribution of proteins that specify cell fate during development in multicellular eukaryotes . The protein Ash1 is a cell-fate determinant in budding yeast which localizes preferentially to the presumptive daughter nucleus, where it inhibits mating-type switching . Here we show that Ash1 mRNA is localized to the distal tip of daughter buds in post-anaphase cells . Three-dimensional imaging reveals that Ash1 mRNA is assembled into particles that associate with the cell cortex . To achieve this localization, Ash1 mRNA must have its 3' untranslated region and the actin cytoskeleton must be intact . Ash1 mRNA is not localized correctly in the absence of a myosin (Myo4) and is mislocalized to the mother-bud neck in the absence of a regulator of the actin cytoskeleton known as Bnil . We propose that Ash1 mRNA particles are transported into the daughter bud along actin filaments and are anchored at the distal tip . Thus, as in higher eukaryotes, Saccharomyces cerevisiae employs RNA localization to generate an asymmetric distribution of proteins and hence to determine cell fate.

Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9757 - 62
Dual roles for DNA sequence identity and the mismatch repair system in the regulation of mitotic crossing-over in yeast; Datta A et al.; Sequence divergence acts as a potent barrier to homologous recombination; much of this barrier derives from an antirecombination activity exerted by mismatch repair proteins . An inverted repeat assay system with recombination substrates ranging in identity from 74% to 100% has been used to define the relationship between sequence divergence and the rate of mitotic crossing-over in yeast . To elucidate the role of the mismatch repair machinery in regulating recombination between mismatched substrates, we performed experiments in both wild-type and mismatch repair defective strains . We find that a single mismatch is sufficient to inhibit recombination between otherwise identical sequences, and that this inhibition is dependent on the mismatch repair system . Additional mismatches have a cumulative negative effect on the recombination rate . With sequence divergence of up to approximately 10%, the inhibitory effect of mismatches results mainly from antirecombination activity of the mismatch repair system . With greater levels of divergence, recombination is inefficient even in the absence of mismatch repair activity . In both wild-type and mismatch repair defective strains, an approximate log-linear relationship is observed between the recombination rate and the level of sequence divergence.

J Bacteriol, 1997 Sep, 179(17), 5614 - 7
The transmission disadvantage of yeast mitochondrial intergenic mutants is eliminated in the mgt1 (cce1) background; Piskur J; A trans-acting element, MGT1 (also called CCE1), has previously been shown to be required in Saccharomyces cerevisiae for the preferential transmission of petite mitochondrial DNA (mtDNA) molecules over wild-type mtDNA molecules . In the present study a possible role of this nuclear gene in the transmission of mtDNA from various respiration-competent mutants was studied . Several of these mutants, lacking one or the other of two biologically active mitochondrial intergenic sequences, were employed in genetic crosses . When these deletion mutants were crossed to the parental wild-type strain in the MGT1/CCE1 background, the progeny contained predominantly wild-type mtDNA molecules . When crosses were performed in the mgt1/cce1 background, the parental molecules interacted in zygotes and underwent homologous recombination but wild-type and intergenic-deletion alleles were transmitted with equal frequencies.

Genetics, 1997 Sep, 147(1), 73 - 85
Probing novel elements for protein splicing in the yeast Vma1 protozyme: a study of replacement mutagenesis and intragenic suppression; Nogami S et al.; Protein splicing is a compelling chemical reaction in which two proteins are produced posttranslationally from a single precursor polypeptide by excision of the internal protein segment and ligation of the flanking regions . This unique autocatalytic reaction was first discovered in the yeast Vma1p protozyme where the 50-kD site-specific endonuclease (VDE) is excised from the 120-kD precursor containing the N- and G-terminal regions of the catalytic subunit of the vacuolar H(+)-ATPase . In this work, we randomized the conserved valine triplet residues three amino acids upstream of the C-terminal splicing junction in the Vma1 protozyme and found that these site-specific random mutations interfere with normal protein splicing to different extents . Intragenic suppressor analysis has revealed that this particular hydrophobic triplet preceding the C-terminal splicing junction genetically interacts with three hydrophobic residues preceding the N-terminal splicing junction . This is the first evidence showing that the N-terminal portion of the V-ATPase subunit is involved in protein splicing . Our genetic evidence is consistent with a structural model that correctly aligns two parallel beta-strands ascribed to the triplets . This model delineates spatial interactions between the two conserved regions both residing upstream of the splicing junctions.

Genetics, 1997 Sep, 147(1), 57 - 71
The Cak1p protein kinase is required at G1/S and G2/M in the budding yeast cell cycle; Sutton A et al.; The CAK1 gene encodes the major CDK-activating kinase (CAK) in budding yeast and is required for activation of Cdc28p for cell cycle progression from G2 to M phase . Here we describe the isolation of a mutant allele of CAK1 in a synthetic lethal screen with the Sit4 protein phosphatase . Analysis of several different cak1 mutants shows that although the G2 to M transition appears most sensitive to loss of Cak1p function, Cak1p is also required for activation of Cdc28p for progression from G1 into S phase . Further characterization of these mutants suggests that, unlike the CAK identified from higher eukaryotes, Cak1p of budding yeast may not play a role in general transcription . Finally, although Cak1 protein levels and in vitro protein kinase activity do not fluctuate during the cell cycle, at least a fraction of Cak1p associates with higher molecular weight proteins, which may be important for its in vivo function.

Hum Mol Genet, 1997 Sep, 6(9), 1535 - 41
Altered metabolism of familial Alzheimer's disease-linked amyloid precursor protein variants in yeast artificial chromosome transgenic mice; Lamb BT et al.; Missense mutations in the beta-amyloid precursor protein gene (APP) co-segregate with a small subset of autosomal dominant familial Alzheimer's disease (FAD) cases wherein deposition of the 39-43 amino acid beta-amyloid (A beta) peptide and neurodegeneration are principal neuropathological hallmarks . To accurately examine the effect of missense mutations on APP metabolism and A beta production in vivo, we have introduced yeast artificial chromosomes (YACs) containing the entire approximately 400 kbp human APP gene encoding APP harboring either the asparagine for lysine and leucine for methionine FAD substitution at codons 670 and 671 (APP(K670N/M671L)), the isoleucine for valine FAD substitution at codon 717 (APP(V7171)) or a combination of both substitutions into transgenic mice . We demonstrate that, relative to YAC transgenic mice expressing wild-type APP, high levels of A beta peptides are detected in the brains of YAC transgenic mice expressing human APP(K670N/M671L) that is associated with a concomitant diminution in the levels of apha-secretase-generated soluble APP derivatives . Moreover, the levels of longer A beta peptides (species terminating at amino acids 42/43) are elevated in YAC transgenic mice expressing human APP(V7171) . These mice should prove valuable for detailed analysis of the in vivo effects of the APP FAD mutations in a variety of tissues and throughout aging and for testing therapeutic agents that specifically alter APP metabolism and A beta production.

Mol Cell Biol, 1997 Sep, 17(9), 5473 - 84
Functional equivalency and diversity of cis-acting elements among yeast replication origins; Lin S et al.; The DNA replication origins of the yeast Saccharomyces cerevisiae require several short functional elements, most of which are not conserved in sequence . To better characterize ARS305, a replicator from a chromosomal origin, we swapped functional DNA elements of ARS305 with defined elements of ARS1 . ARS305 contains elements that are functionally exchangeable with ARS1 A and B1 elements, which are known to bind the origin recognition complex; however, the ARS1 A element differs in that it does not require a 3' box adjacent to the essential autonomously replicating sequence consensus . At the position corresponding to ARS1 B3, ARS305 has a novel element, B4, that can functionally substitute for every type of short element (B1, B2, and B3) in the B domain . Unexpectedly, the replacement of element B4 by ARS1 B3, which binds ABF1p and is known as a replication enhancer, inhibited ARS305 function . ARS305 has no short functional element at or near positions corresponding to the B2 elements in ARS1 and ARS307 but contains an easily unwound region whose functional importance was supported by a broad G+C-rich substitution mutation . Surprisingly, the easily unwound region can functionally substitute for the ARS1 B2 element, even though ARS1 B2 was found to possess a distinct DNA sequence requirement . The functionally conserved B2 element in ARS307 contains a known sequence requirement, and helical stability analysis of linker and minilinker mutations suggested that B2 also contains a DNA unwinding element (DUE) . Our findings suggest that yeast replication origins employ a B2 element or a DUE to mediate a common function, DNA unwinding during initiation, although not necessarily through a common mechanism.

Mol Cell Biol, 1997 Sep, 17(9), 5288 - 98
Genetic analysis of the large subunit of yeast transcription factor IIE reveals two regions with distinct functions; Kuldell NH et al.; Biochemical analysis of proteins necessary for transcription initiation by eukaryotic RNA polymerase II (pol II) has identified transcription factor IIE (TFIIE) as an essential component of the reaction . To better understand the role of TFIIE in transcription, we isolated conditional alleles of TFA1, the gene encoding the large subunit of TFIIE in the yeast Saccharomyces cerevisiae . The mutant Tfa1 proteins fall into two classes . The first class causes thermosensitive growth due to single amino acid substitutions of the cysteines comprising the Zn-binding motif . The second mutant class is made up of proteins that are C-terminally truncated and that cause a cold-sensitive growth phenotype . The behavior of these mutants suggests that Tfa1p possesses at least two domains with genetically distinct functions . The mutations in the Zn-binding motif do not affect the mutant protein's stability at the nonpermissive temperature or its ability to associate with the small subunit of TFIIE . Our studies further determined that wild-type TFIIE can bind to single-stranded DNA in vitro . However, this property is unaffected in the mutant TFIIE complexes . Finally, we have demonstrated the biological importance of TFIIE in pol II-mediated transcription by depleting the Tfa1 protein from the cells and observing a concomitant decrease in total poly(A)+ mRNA.

Mol Cell Biol, 1997 Sep, 17(9), 5210 - 26
Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica; Titorenko VI et al.; We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis . Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis . The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form . Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane . Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion . Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis . At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum.

Mol Cell Biol, 1997 Sep, 17(9), 5023 - 32
Functional analysis of Rrp7p, an essential yeast protein involved in pre-rRNA processing and ribosome assembly; Baudin-Baillieu A et al.; During the functional analysis of open reading frames (ORFs) identified during the sequencing of chromosome III of Saccharomyces cerevisiae, the previously uncharacterized ORF YCL031C (now designated RRP7) was deleted . RRP7 is essential for cell viability, and a conditional null allele was therefore constructed, by placing its expression under the control of a regulated GAL promoter . Genetic depletion of Rrp7p inhibited the pre-rRNA processing steps that lead to the production of the 20S pre-rRNA, resulting in reduced synthesis of the 18S rRNA and a reduced ratio of 40S to 60S ribosomal subunits . A screen for multicopy suppressors of the lethality of the GAL::rrp7 allele isolated the two genes encoding a previously unidentified ribosomal protein (r-protein) that is highly homologous to the rat r-protein S27 . When present in multiple copies, either gene can suppress the lethality of an RRP7 deletion mutation and can partially restore the ribosomal subunit ratio in Rrp7p-depleted cells . Deletion of both r-protein genes is lethal; deletion of either single gene has an effect on pre-rRNA processing similar to that of Rrp7p depletion . We believe that Rrp7p is required for correct assembly of rpS27 into the preribosomal particle, with the inhibition of pre-rRNA processing appearing as a consequence of this defect.

J Virol, 1997 Sep, 71(9), 6863 - 8
Cryo-electron microscopy structure of yeast Ty retrotransposon virus-like particles; Palmer KJ et al.; The virus-like particles (VLPs) produced by the yeast retrotransposon Ty1 are functionally related to retroviral cores . These particles are unusual in that they have variable radif . A paired mass-radius analysis of VLPs by scanning transmission electron microscopy showed that many of these particles form an icosahedral T-number series . Three-dimensional reconstruction to 38-A resolution from cryo-electron micrographs of T = 3 and T = 4 shells revealed that the single structural protein encoded by the TYA gene assembles into spiky shells from trimeric units.

Comp Biochem Physiol A Physiol, 1997 Sep, 118(1), 153 - 8
Calcium homeostasis and yeast phagocytosis in hemocytes of the colonial ascidian Botryllus schlosseri; Ballarin L et al.; This paper reports the effects of drugs affecting the homeostasis of cytosolic-free calcium on in vitro yeast phagocytosis by hemocytes of the colonial ascidian Botryllus schlosseri . Significant inhibition of phagocytosis is observed after exposure of hemocytes to 10 microM or higher concentrations of thimerosal, which is known to deplete intracellular calcium stores in mammalian cells . The two calcium channel blockers nifedipine and verapamil significantly decrease the phagocytic index, the minimum effective concentrations being 10 and 50 microM, respectively . As these substances have no effects at lower concentrations, they probably act through the inhibition of Ca(2+)-ATPase activity, required to restock intracellular calcium stores, due to their interaction with calmodulin . Analogously, pimozide, which suppresses ATPase activity by interacting with calmodulin, and thapsigargin, which inhibits Ca(2+)-ATPase activity, significantly reduce the phagocytic index . Moreover, nifedipine, by altering cytosolic calcium homeostasis, also lowers the production of superoxide anion associated with phagocytosis . Results indicate that in ascidians, as in mammals, a rise in intracellular calcium is required for phagocyte activation and induction of the respiratory burst.

Science, 1997 Aug 29, 277(5330), 1313 - 6
Accelerated aging and nucleolar fragmentation in yeast sgs1 mutants; Sinclair DA et al.; The SGS1 gene of yeast encodes a DNA helicase with homology to the human WRN gene . Mutations in WRN result in Werner's syndrome, a disease with symptoms resembling premature aging . Mutation of SGS1 is shown to cause premature aging in yeast mother cells on the basis of a shortened life-span and the aging-induced phenotypes of sterility and redistribution of the Sir3 silencing protein from telomeres to the nucleolus . Further, in old sgs1 cells the nucleolus is enlarged and fragmented-changes that also occur in old wild-type cells . These findings suggest a conserved mechanism of cellular aging that may be related to nucleolar structure.

J Biol Chem, 1997 Aug 29, 272(35), 22163 - 72
Relationship between yeast polyribosomes and Upf proteins required for nonsense mRNA decay; Atkin AL et al.; In yeast, the accelerated rate of decay of nonsense mutant mRNAs, called nonsense-mediated mRNA decay, requires three proteins, Upf1p, Upf2p, and Upf3p . Single, double, and triple disruptions of the UPF genes had nearly identical effects on nonsense mRNA accumulation, suggesting that the encoded proteins function in a common pathway . We examined the distribution of epitope-tagged versions of Upf proteins by sucrose density gradient fractionation of soluble lysates and found that all three proteins co-distributed with 80 S ribosomal particles and polyribosomes . Treatment of lysates with RNase A caused a coincident collapse of polyribosomes and each Upf protein into fractions containing 80 S ribosomal particles, as expected for proteins that are associated with polyribosomes . Mutations in the cysteine-rich (zinc finger) and RNA helicase domains of Upf1p caused loss of function, but the mutant proteins remained polyribosome-associated . Density gradient profiles for Upf1p were unchanged in the absence of Upf3p, and although similar, were modestly shifted to fractions lighter than those containing polyribosomes in the absence of Upf2p . Upf2p shifted toward heavier polyribosome fractions in the absence of Upf1p and into fractions containing 80 S particles and lighter fractions in the absence of Upf3p . Our results suggest that the association of Upf2p with polyribosomes typically found in a wild-type strain depends on the presence and opposing effects of Upf1p and Upf3p.

J Biol Chem, 1997 Aug 29, 272(35), 21811 - 7
Affinity purification and kinetic analysis of mutant forms of yeast NAD+-specific isocitrate dehydrogenase; Zhao WN et al.; Polyhistidine tags were added to the carboxyl termini of the two homologous subunits of yeast NAD+-specific isocitrate dehydrogenase (IDH) . The tag in either the IDH1 or IDH2 subunit permits one-step affinity purification from yeast cellular extracts of catalytically active and allosterically responsive holoenzyme . This expression system was used to investigate subunit-specific contributions of residues with putative functions in adenine nucleotide binding . The primary effect of simultaneous replacement of the adjacent Asp-279 and Ile-280 residues in IDH1 with alanines is a dramatic loss of activation by AMP . In contrast, alanine replacement of the homologous Asp-286 and Ile-287 residues in IDH2 does not alter the allosteric response to AMP, but produces a 160-fold reduction in Vmax due to a 70-fold increase in the S0.5 value for NAD+ . These results suggest that the targeted aspartate/isoleucine residues may contribute to regulator binding in IDH1 and to cofactor binding in IDH2, i.e . that these homologous residues are located in regions that have evolved for binding the adenine nucleotide components of different ligands . In other mutant enzymes, an alanine replacement of Asp-191 in IDH1 eliminates measurable catalytic activity, and a similar substitution of the homologous Asp-197 in IDH2 produces pleiotropic catalytic effects . A model is presented for the primary function of IDH2 in catalysis and of IDH1 in regulation, with crucial roles for these single aspartate residues in the communication and functional interdependence of the two subunits.

J Biol Chem, 1997 Aug 29, 272(35), 21665 - 8
Yeast Rad7-Rad16 complex, specific for the nucleotide excision repair of the nontranscribed DNA strand, is an ATP-dependent DNA damage sensor; Guzder SN et al.; In eukaryotes, nucleotide excision repair of ultraviolet light-damaged DNA is a highly intricate process that requires a large number of evolutionarily conserved protein factors . Genetic studies in the yeast Saccharomyces cerevisiae have indicated a specific role of the RAD7 and RAD16 genes in the repair of transcriptionally inactive DNA . Here we show that the RAD7- and RAD16-encoded products exist as a complex of 1:1 stoichiometry, exhibiting an apparent dissociation constant (Kd) of <4 x 10(-10) M . The Rad7-Rad16 complex has been purified to near homogeneity in this study and is shown to bind, in an ATP-dependent manner and with high specificity, to DNA damaged by ultraviolet light . Importantly, inclusion of the Rad7-Rad16 complex in the in vitro nucleotide excision repair system that consists entirely of purified components results in a marked stimulation of damage specific incision . Thus, Rad7-Rad16 complex is the ATP-dependent DNA damage sensor that specifically functions with the ensemble of nucleotide excision repair factor (NEF) 1, NEF2, NEF3, and replication protein A in the repair of transcriptionally inactive DNA . We name this novel complex of Rad7 and Rad16 proteins NEF4.

J Cell Biol, 1997 Aug 25, 138(4), 731 - 46
Novel genes involved in endosomal traffic in yeast revealed by suppression of a targeting-defective plasma membrane ATPase mutant; Luo W et al.; A novel genetic selection was used to identify genes regulating traffic in the yeast endosomal system . We took advantage of a temperature-sensitive mutant in PMA1, encoding the plasma membrane ATPase, in which newly synthesized Pma1 is mislocalized to the vacuole via the endosome . Diversion of mutant Pma1 from vacuolar delivery and rerouting to the plasma membrane is a major mechanism of suppression of pma1(ts) . 16 independent suppressor of pma1 (sop) mutants were isolated . Identification of the corresponding genes reveals eight that are identical with VPS genes required for delivery of newly synthesized vacuolar proteins . A second group of SOP genes participates in vacuolar delivery of mutant Pma1 but is not essential for delivery of the vacuolar protease carboxypeptidase Y . Because the biosynthetic pathway to the vacuole intersects with the endocytic pathway, internalization of a bulk membrane endocytic marker FM 4-64 was assayed in the sop mutants . By this means, defective endosome-to-vacuole trafficking was revealed in a subset of sop mutants . Another subset of sop mutants displays perturbed trafficking between endosome and Golgi: impaired pro-alpha factor processing in these strains was found to be due to defective recycling of the trans-Golgi protease Kex2 . One of these strains defective in Kex2 trafficking carries a mutation in SOP2, encoding a homologue of mammalian synaptojanin (implicated in synaptic vesicle endocytosis and recycling) . Thus, cell surface delivery of mutant Pma1 can occur as a consequence of disturbances at several different sites in the endosomal system.

Cell, 1997 Aug 22, 90(4), 683 - 93
Yeast Hct1 is a regulator of Clb2 cyclin proteolysis; Schwab M et al.; Stage-specific proteolysis of mitotic cyclins is fundamental to eukaryotic cell cycle regulation . We found that yeast Hct1, a conserved protein of eukaryotes, is a necessary and rate-limiting component of this proteolysis pathway . In hct1 mutants, the mitotic cyclin Clb2 is highly stabilized and inappropriately induces DNA replication, while G1 cyclins and other proteolytic substrates remain short-lived . Viability of hct1 mutants depends on SIC1 . This and further results suggest that inhibition of cyclin-dependent kinases may compensate for defects in cyclin proteolysis . Remarkably, elevated levels of Hct1 ectopically activate destruction box- and Cdc23-dependent degradation of Clb2 and cause phenotypic effects characteristic for a depletion of M-phase cyclins . Hct1 and the related Cdc20 may function as substrate-specific regulators of proteolysis during mitosis.

Cell, 1997 Aug 22, 90(4), 615 - 24
Yeast TAF(II)145 functions as a core promoter selectivity factor, not a general coactivator; Shen WC et al.; In yeast, TATA box binding protein associated factors (TAF(II)s) are dispensable for transcription of most genes . Here we use differential display to identify a small subset of yeast genes whose transcription in vivo requires yTAF(II)145 . Promoter-mapping studies reveal, unexpectedly, that the region of a gene that renders it yTAF(II)145-dependent is not the upstream activating sequence, which contains the activator-binding sites, but rather the core promoter . In fact, a core promoter requiring yTAF(II)145 retained that requirement when its transcription was directed by several unrelated upstream activating sequences and even in the absence of an activator . Taken together, our results indicate that yTAF(II)145 functions in recognition and selection of core promoters by a mechanism not involving upstream activators.

Cell, 1997 Aug 22, 90(4), 607 - 14
Yeast TAF(II)145 required for transcription of G1/S cyclin genes and regulated by the cellular growth state; Walker SS et al.; TFIID comprises the TATA box-binding protein and a set of highly conserved associated factors (TAF(II)s) . yTAF(II)145, the core subunit of the yeast TAF(II) complex, is dispensable for transcription of most yeast genes but specifically required for progression through G1/S . Here we show that transcription of G1 and certain B-type cyclin genes is dependent upon yTAF(II)145 . At high cell density or following nutrient deprivation, yeast cells cease division, enter a G0-like state, and terminate transcription of most genes . In this stationary phase, we find that the levels of yTAF(II)145, several other yTAF(II)s, and TBP are drastically reduced . Collectively, our results indicate that yTAF(II)145 and other TFIID components have a specialized role in transcriptional regulation of cell cycle progression and growth control.

J Biol Chem, 1997 Aug 22, 272(34), 21104 - 12
Properties of a cyclosporin-insensitive permeability transition pore in yeast mitochondria; Jung DW et al.; Yeast mitochondria (Saccharomyces cerevisiae) contain a permeability transition pore which is regulated differently than the pore in mammalian mitochondria . In a mannitol medium containing 10 mM Pi and ethanol (oxidizable substrate), yeast mitochondria accumulate large amounts of Ca2+ (>400 nmol/mg of protein) upon the addition of an electrophoretic Ca2+ ionophore (ETH129) . Pore opening does not occur following Ca2+ uptake, even though ruthenium red-inhibited rat liver mitochondria undergo rapid pore opening under analogous conditions . However, a pore does arise in yeast mitochondria when Ca2+ and Pi are not present, as monitored by swelling, ultrastructure, and matrix solute release . Pore opening is slow unless a respiratory substrate is provided (ethanol or NADH) but also occurs rapidly in response to ATP (2 mM) when oligomycin is present . Pi and ADP inhibit pore opening (EC50 approximately 1 and 4 mM, respectively), however, cyclosporin A (7 microg/ml), oligomycin (20 microg/ml), or carboxyatractyloside (25 microM) have no effect . The pore arising during respiration is also inhibited by nigericin or uncoupler, indicating that an acidic matrix pH antagonizes the process . Pi also inhibits pore opening by lowering the matrix pH (Pi/OH- antiport) . However, inhibition of the ATP-induced pore by Pi is seen in the presence of mersalyl, suggesting a second mechanism of action . Since pore induction by ATP is not sensitive to carboxyatractyloside, ATP appears to act at an external site and Pi may antagonize the interaction . Isoosmotic polyethylene glycol-induced contraction of yeast mitochondria swollen during respiration, or in the presence of ATP, is 50% effective at a solute size of 1.0-1.1 kDa . This suggests that the same pore is induced in both cases and is comparable in size with the permeability transition pore of heart and liver mitochondria.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9202 - 7
The catalytic subunit of yeast telomerase; Counter CM et al.; Telomerase is an RNA-directed DNA polymerase, composed of RNA and protein subunits, that replicates the telomere ends of linear eukaryotic chromosomes . Using a genetic strategy described here, we identify the product of the EST2 gene, Est2p, as a subunit of telomerase in the yeast Saccharomyces cerevisiae . Est2p is required for enzyme catalysis, as mutations in EST2 were found to result in the absence of telomerase activity . Immunochemical experiments show that Est2p is an integral subunit of the telomerase enzyme . Critical catalytic residues present in RNA-directed DNA polymerases are conserved in Est2p; mutation of one such residue abolishes telomerase activity, suggesting a direct catalytic role for Est2p.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9034 - 9
Chromatin assembly in a yeast whole-cell extract; Schultz MC et al.; A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae . The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes . We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast . This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes . Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly . These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.

Cancer Res, 1997 Aug 15, 57(16), 3478 - 85
A functional assay in yeast for the human estrogen receptor displays wild-type and variant estrogen receptor messenger RNAs present in breast carcinoma; van Dijk MA et al.; Human estrogen receptor (hER) variants or mutants with altered functional activity may be responsible for resistance to the antiestrogen tamoxifen in breast cancer . The method presented in this report is a screening method for functional activity of hER in yeast Saccharomyces cerevisiae . hER mRNA isolated from breast cancer tissue is subjected to reverse transcription-PCR, directly cloned into a yeast expression vector in vivo, and subsequently tested for functional activity in a simple yeast growth assay . This technique, functional analysis of separated alleles in yeast of the human estrogen receptor (hER-FASAY), gives a display of the prevalence and functional activity of all of the variant hER mRNAs among normal, wild-type receptors in a breast tumor sample . The hER-FASAY can discriminate among wild-type hER, constitutively active hER, and inactive hER . In contrast to standard immunohistochemistry, this assay gives insight into the functional activity of hER . The hER-FASAY was optimized and validated using breast cancer cell lines MCF-7 and T47D and seven breast cancer biopsies . Phenotypes detected with the hER-FASAY were validated by DNA sequencing . In both cell lines and tumor biopsies, hER variants are highly common and mainly caused by alternative RNA splicing, whereas point mutations and deletions occur only at low frequency.

J Mol Biol, 1997 Aug 15, 271(2), 235 - 43
Modulation of yeast F-actin structure by a mutation in the nucleotide-binding cleft; Orlova A et al.; Although the actin sequence is very highly conserved across evolution, tissue-specific expression of different isoforms in high eukaryotes suggests that different isoforms carry out different functions . However, little information exists about either the differences in filaments made from different actins or the effects on filament structure caused by the various mutations in actin that have been introduced to gain insight into actin function . Using electron microscopy and three-dimensional reconstruction, we have studied the differences in the filaments made by yeast and rabbit skeletal muscle actin, two proteins with 88% homologous sequences, and we have assessed the changes in filament structure caused by the introduction of the S14A mutation into yeast actin . Elimination of the S14 hydroxyl group, assumed to bind to the gamma-phosphate of actin-bound ATP, results in a 40 to 60-fold decrease in actin's affinity for ATP . We show that yeast actin displays less extensive contacts between the two long-pitch helical strands than does muscle actin, and displays the large cooperativity within filaments previously observed for muscle actin . Finally, we demonstrate that the S14A mutation narrows the cleft between the two lobes of the actin subunit and strengthens the inter-strand connections in F-actin.

Arch Biochem Biophys, 1997 Aug 15, 344(2), 309 - 15
Probing energy coupling in the yeast plasma membrane H+-ATPase with acetyl phosphate; Wang G et al.; The energy-rich compound acetyl phosphate (ACP) was examined as a substrate for energy-linked reactions by the yeast plasma membrane H+-ATPase . The hydrolysis of ACP was sensitive to inhibition by vanadate with an IC50 approximately 1 microM, which is comparable to the level obtained in the presence of ATP . A Km of 8.29 +/- 0.65 mM for the hydrolysis of ACP was approximately 10-fold higher than that obtained for ATP, while Vmax values of 8.66 +/- 0.29 and 7.23 +/- 0.34 micromol Pi mg(-1) min(-1) were obtained with ATP and ACP, respectively . ACP formed a phosphorylated intermediate that was efficiently chased with hydroxylamine . Both ACP and ATP effectively protected the enzyme from trypsin-induced inactivation and formed identical tryptic digestion patterns, suggesting that ACP mimics the formation of conformational intermediates induced by ATP . However, unlike ATP, ACP was unable to drive proton transport by H+-ATPase . In addition, a pma1-S368F mutant enzyme that is highly insensitive to inhibition by vanadate in the presence of ATP was largely sensitive to vanadate in the presence of ACP . These results are interpreted in terms of a reverse, short-circuit pathway of the normal P-type ATPase kinetic pathway, in which the formation of E2P by-passes the E1P high-energy intermediate . In this pathway, ACP favors the formation of an E2P conformational state, which can interact with classical inhibitors like vanadate, but possesses insufficient free energy to drive proton transport by the H+-ATPase.

J Biol Chem, 1997 Aug 15, 272(33), 20873 - 83
Role of the yeast phosphatidylinositol/phosphatidylcholine transfer protein (Sec14p) in phosphatidylcholine turnover and INO1 regulation; Patton-Vogt JL et al.; In yeast, mutations in the CDP-choline pathway for phosphatidylcholine biosynthesis permit the cell to grow even when the SEC14 gene is completely deleted (Cleves, A., McGee, T., Whitters, E., Champion, K., Aitken, J., Dowhan, W., Goebl, M., and Bankaitis, V . (1991) Cell 64, 789-800) . We report that strains carrying mutations in the CDP-choline pathway, such as cki1, exhibit a choline excretion phenotype due to production of choline during normal turnover of phosphatidylcholine . Cells carrying cki1 in combination with sec14(ts), a temperature-sensitive allele in the gene encoding the phosphatidylinositol/phosphatidylcholine transporter, have a dramatically increased choline excretion phenotype when grown at the sec14(ts)-restrictive temperature . We show that the increased choline excretion in sec14(ts) cki1 cells is due to increased turnover of phosphatidylcholine via a mechanism consistent with phospholipase D-mediated turnover . We propose that the elevated rate of phosphatidylcholine turnover in sec14(ts) cki1 cells provides the metabolic condition that permits the secretory pathway to function when Sec14p is inactivated . As phosphatidylcholine turnover increases in sec14(ts) cki1 cells shifted to the restrictive temperature, the INO1 gene (encoding inositol-1-phosphate synthase) is also derepressed, leading to an inositol excretion phenotype (Opi-) . Misregulation of the INO1 gene has been observed in many strains with altered phospholipid metabolism, and the relationship between phosphatidylcholine turnover and regulation of INO1 and other co-regulated genes of phospholipid biosynthesis is discussed.

J Biol Chem, 1997 Aug 15, 272(33), 20408 - 13
Phosphate transport in yeast vacuoles; Booth JW et al.; The vacuole of the yeast Saccharomyces cerevisiae is a major storage compartment for phosphate . We have measured phosphate transport across the vacuolar membrane . Isolated intact vacuoles take up large amounts of added {32P}phosphate by counterflow exchange with phosphate present in the vacuoles at the time of their isolation . The bidirectional phosphate transporter has an intrinsic dissociation constant for phosphate of 0.4 mM . Exchange mediated by this carrier is faster than unidirectional efflux of phosphate from the vacuoles . The transporter is highly selective for phosphate; of other anions tested, only arsenate is also a substrate . Transport is strongly pH-dependent with increasing activity at lower pH . Similar phosphate transport behavior was observed in right-side-out vacuolar membrane vesicles.

Science, 1997 Aug 15, 277(5328), 938 - 41
Protein transport by purified yeast Sec complex and Kar2p without membranes; Matlack KE et al.; Posttranslational protein translocation across the endoplasmic reticulum membrane of yeast requires a seven-component transmembrane complex (the Sec complex) in collaboration with the lumenal Kar2 protein (Kar2p) . A translocation substrate was initially bound to the cytosolic face of the purified Sec complex in a signal-sequence-dependent but Kar2p- and nucleotide-independent manner . In a subsequent reaction, in which Kar2p interacted with the lumenal face of the Sec complex and hydrolyzed adenosine triphosphate, the substrate moved through a channel formed by the Sec complex and was released at the lumenal end . Movement through the channel occurred in detergent solution in the absence of a lipid bilayer.

J Cell Biol, 1997 Aug 11, 138(3), 643 - 56
A novel mammalian, mitotic spindle-associated kinase is related to yeast and fly chromosome segregation regulators; Gopalan G et al.; We describe a novel mammalian protein kinase related to two newly identified yeast and fly kinases-Ipl1 and aurora, respectively-mutations in which cause disruption of chromosome segregation . We have designated this kinase as Ipl1- and aurora-related kinase 1 (IAK1) . IAK1 expression in mouse fibroblasts is tightly regulated temporally and spatially during the cell cycle . Transcripts first appear at G1/S boundary, are elevated at M-phase, and disappear rapidly after completion of mitosis . The protein levels and kinase activity of IAK1 are also cell cycle regulated with a peak at M-phase . IAK1 protein has a distinct subcellular and temporal pattern of localization . It is first identified on the centrosomes immediately after the duplicated centrosomes have separated . The protein remains on the centrosome and the centrosome-proximal part of the spindle throughout mitosis and is detected weakly on midbody microtubules at telophase and cytokinesis . In cells recovering from nocodazole treatment and in taxol-treated mitotic cells, IAK1 is associated with microtubule organizing centers . A wild-type and a mutant form of IAK1 cause mitotic spindle defects and lethality in ipl1 mutant yeast cells but not in wild-type cells, suggesting that IAK1 interferes with Ipl1p function in yeast . Taken together, these data strongly suggest that IAK1 may have an important role in centrosome and/ or spindle function during chromosome segregation in mammalian cells . We suggest that IAK1 is a new member of an emerging subfamily of the serine/threonine kinase superfamily . The members of this subfamily may be important regulators of chromosome segregation.

J Cell Biol, 1997 Aug 11, 138(3), 629 - 41
Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex; Carminati JL et al.; Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast . To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)-tubulin fusion protein to observe microtubules in living yeast cells . GFP-tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function . Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells . The dynamic properties of yeast microtubules are modulated during the cell cycle . The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation . Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles . In addition, microtubule dynamics were altered in the absence of dynein . These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8579 - 84
ECA1 complements yeast mutants defective in Ca2+ pumps and encodes an endoplasmic reticulum-type Ca2+-ATPase in Arabidopsis thaliana; Liang F et al.; To understand the structure, role, and regulation of individual Ca2+ pumps in plants, we have used yeast as a heterologous expression system to test the function of a gene from Arabidopsis thaliana (ECA1) . ECA1 encoded a 116-kDa polypeptide that has all the conserved domains common to P-type Ca2+ pumps (EC 3.6.1.38) . The amino acid sequence shared more identity with sarcoplasmic/endoplasmic reticulum (53%) than with plasma membrane (32%) Ca2+ pumps . Yeast mutants defective in a Golgi Ca2+ pump (pmr1) or both Golgi and vacuolar Ca2+ pumps (pmr1 pmc1 cnb1) were sensitive to growth on medium containing 10 mM EGTA or 3 mM Mn2+ . Expression of ECA1 restored growth of either mutant on EGTA . Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with ECA1 to determine if the ECA1 polypeptide (ECA1p) could be phosphorylated as intermediates of the reaction cycle of Ca2+-pumping ATPases . In the presence of {gamma-32P}ATP, ECA1p formed a Ca2+-dependent {32P}phosphoprotein of 106 kDa that was sensitive to hydroxylamine . Cyclopiazonic acid, a blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+ pumps, inhibited the formation of the phosphoprotein, whereas thapsigargin did not . Immunoblotting with an antibody against the carboxyl tail showed that ECA1p was associated mainly with the endoplasmic reticulum membranes isolated from Arabidopsis plants . The results support the model that ECA1 encodes an endoplasmic reticulum-type Ca2+ pump in Arabidopsis . The ability of ECA1p to restore growth of mutant pmr1 on medium containing Mn2+, and the formation of a Mn2+-dependent phosphoprotein suggested that ECA1p may also regulate Mn2+ homeostasis by pumping Mn2+ into endomembrane compartments of plants.

Biochemistry, 1997 Aug 5, 36(31), 9625 - 35
The exchangeable yeast ribosomal acidic protein YP2beta shows characteristics of a partly folded state under physiological conditions; Zurdo J et al.; The eukaryotic acidic ribosomal P proteins, contrary to the standard r-proteins which are rapidly degraded in the cytoplasm, are found forming a large cytoplasmic pool that exchanges with the ribosome-bound proteins during translation . The native structure of the P proteins in solution is therefore an essential determinant of the protein-protein interactions that take place in the exchange process . In this work, the structure of the ribosomal acidic protein YP2beta from Saccharomyces cerevisiae has been investigated by fluorescence spectroscopy, circular dichroism (CD), nuclear magnetic resonance (NMR), and sedimentation equilibrium techniques . We have established the fact that YP2beta bears a 22% alpha-helical secondary structure and a noncompact tertiary structure under physiological conditions (pH 7.0 and 25 degrees C); the hydrophobic core of the protein appears to be solvent-exposed, and very low cooperativity is observed for heat- or urea-induced denaturation . Moreover, the 1H-NMR spectra show a small signal dispersion, and virtually all the amide protons exchange with the solvent on a very short time scale, which is characteristic of an open structure . At low pH, YP2beta maintains its secondary structure content, but there is no evidence for tertiary structure . 2,2,2-Trifluoroethanol (TFE) induces a higher amount of alpha-helical structure but also disrupts any trace of the remaining tertiary fold . These results indicate that YP2beta may have a flexible structure in the cytoplasmic pool, with some of the characteristics of a "molten globule", and also point out the physiological relevance of such flexible protein states in processes other than protein folding.

Biochemistry, 1997 Aug 5, 36(31), 9493 - 500
Antisense inhibition of CAS, the human homologue of the yeast chromosome segregation gene CSE1, interferes with mitosis in HeLa cells; Ogryzko VV et al.; We have analyzed the effects on HeLa cells of reduction of the CAS protein, the human homologue to yeast chromosome segregation protein CSE1 . Expression of CAS antisense cDNA decreases the amount of CAS protein in HeLa cells and perturbs progression from G2 (retards transition from G2) to G1 in the cell cycle . Increased levels of cyclin B in CAS antisense transfected cells correlated with an arrest in G2 phase or mitosis . This arrest upon CAS attenuation is consistent with observations that yeast with CSE1 mutations are defective in mitosis and cyclin B degradation.

FEBS Lett, 1997 Aug 4, 412(3), 518 - 20
Protein splicing in the yeast Vma1 protozyme: evidence for an intramolecular reaction; Kawasaki M et al.; Protein splicing is an autocatalytic reaction of a single polypeptide in which a spliced intervening sequence is excised out and the two external regions are ligated with the peptide bond to yield two mature proteins . We examined the reaction mechanism using a folding-dependent in vitro protein splicing system . Protein splicing proceeds at an optimal pH of 7 and is an intramolecular reaction . The reaction is not inhibited by potential protease inhibitors, suggesting that its mechanism is different from those catalyzed by known proteases.

FEBS Lett, 1997 Aug 4, 412(3), 410 - 4
Submitochondrial distributions and stabilities of subunits 4, 5, and 6 of yeast cytochrome oxidase in assembly defective mutants; Glerum DM et al.; The concentration and submitochondrial distribution of the subunit polypeptides of cytochrome oxidase have been studied in wild type yeast and in different mutants impaired in assembly of this respiratory complex . All the subunit polypeptides of the enzyme are associated with mitochondrial membranes of wild type cells, except for a small fraction of subunits 4 and 6 that is recovered in the soluble protein fraction of mitochondria . Cytochrome oxidase mutants consistently display a severe reduction in the steady-state concentration of subunit 1 due to its increased turnover . As a consequence, most of subunit 4, which normally is associated with subunit 1, is found in the soluble fraction . A similar shift from membrane-bound to soluble subunit 6 is seen in mutants blocked in expression of subunit 5a . In contrast, null mutations in COX6 coding for subunit 6 promote loss of subunit 5a . The absence of subunit 5a in the cox6 mutant is the result of proteolytic degradation rather than regulation of its expression by subunit 6 . The possible role of the ATP-dependent proteases Rca1p and Afg3p in proteolysis of subunits 1 and 5a has been assessed in strains with combined mutations in COX6, RCA1, and/or AFG3 . Immunochemical assays indicate that another protease(s) must be responsible for most of the proteolytic loss of these proteins.

Chromosoma, 1997 Aug, 106(3), 136 - 48
Yeast silencers create domains of nuclease-resistant chromatin in an SIR4-dependent manner; Reimer SK et al.; Previous analysis of the repression of the silent mating type loci in Saccharomyces cerevisiae has linked the mechanism of silencing to the formation of a chromatin domain at the silenced loci . In this study, a TRP1 reporter gene was used to examine changes in chromatin structure in a neutral environment . This enabled the chromatin structure organized by yeast silencers to be compared directly with changes effected by the yeast alpha2 repressor . It was found that silencers mediate the formation of lengthy nuclease-resistant domains on the DNA, rather than specifically positioning nucleosomes over promoter regions as the alpha2 repressor does . Silencing at the TRP1 reporter gene closely resembled silencing at the HMR and HML loci . Repression of the test gene was optimal when two silencers flanking the reporter gene were used, mimicking the situation at the silent loci . In addition, both repression of the reporter gene and the formation of nuclease-resistant chromatin domains was SIR4 dependent.

Mol Biol Cell, 1997 Aug, 8(8), 1481 - 99
Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle; Mulholland J et al.; Many yeast actin cytoskeleton mutants accumulate large secretory vesicles and exhibit phenotypes consistent with defects in polarized growth . This, together with actin's polarized organization, has suggested a role for the actin cytoskeleton in the vectorial transport of late secretory vesicles to the plasma membrane . By using ultrastructural and biochemical analysis, we have characterized defects manifested by mutations in the SLA2 gene (also known as the END4 gene), previously found to affect both the organization of the actin cytoskeleton and endocytosis in yeast . Defects in cell wall morphology, accumulated vesicles, and protein secretion kinetics were found in sla2 mutants similar to defects found in act1 mutants . Vesicles that accumulate in the sla2 and act1 mutants are immunoreactive with antibodies directed against the small GTPase Ypt1p but not with antibodies directed against the homologous Sec4p found on classical "late" secretory vesicles . In contrast, the late-acting secretory mutants sec1-1 and sec6-4 are shown to accumulate anti-Sec4p-positive secretory vesicles as well as vesicles that are immunoreactive with antibodies directed against Ypt1p . The late sec mutant sec4-8 is also shown to accumulate Ypt1p-containing vesicles and to exhibit defects in actin cytoskeleton organization . These results indicate the existence of at least two classes of morphologically similar, late secretory vesicles (associated with Ypt1p+ and Sec4p+, respectively), one of which appears to accumulate when the actin cytoskeleton is disorganized.

Biochem Mol Biol Int, 1997 Aug, 42(5), 1063 - 9
Protection by histidine against oxidative inactivation of AMP deaminase in yeast; Murakami K et al.; Oxidative inactivation of AMP deaminase and its protection were analyzed under the in situ conditions of yeast cells . AMP deaminase was readily inactivated by an exposure to hydrogen peroxide plus copper in permeabilized yeast cells . Addition of ascorbic acid further enhanced the inactivation of the enzyme, suggesting the hydroxyl radical produced by the Fenton reaction is responsible for the inactivation of the enzyme . Addition of histidine caused an effective protection against the inactivation of AMP deaminase by hydrogen peroxide-induced hydroxyl radical . The concentration of histidine required for half-maximal effect was within physiological range . Cysteine showed less effective protection against oxidative inactivation . Other amino acids as potent copper-chelating agents as well as trolox and taurine showed little or no effect . Histidine can act as a physiological "antioxidant" in yeast cells.

Lett Appl Microbiol, 1997 Aug, 25(2), 113 - 6
Utilization of an auxotrophic strain of the yeast Yarrowia lipolytica to improve gamma-decalactone production yields; Pagot Y et al.; gamma-Decalactone is an aroma compound with a pleasant peachy odour . Most industrial processes use the bioconversion of ricinoleic acid by yeasts to produce gamma-decalactone . Peroxisomal beta-oxidation activity is responsible for the bioconversion . Some yeasts, Yarrowia lipolytica in particular, grow during the bioconversion, yielding a low bioconversion rate . Auxotrophy for uracil of a genetically engineered Y . lipolytica strain was used to prevent growth in the bioconversion medium . beta-Oxidation activities and gamma-decalactone production of the auxotrophic strain were measured and compared with a wild-type strain in media supplemented or not . Induction of beta-oxidation was observed in the non-supplemented medium, although to a lesser extent than in supplemented medium . Aroma productivity of the auxotrophic strain in the supplemented medium was similar to that observed for the wild-type strain in both media . However, in the non-supplemented medium the productivity of the auxotrophic strain was 10-20-fold higher.

Photochem Photobiol, 1997 Aug, 66(2), 253 - 9
Diamino acid derivatives of porphyrins penetrate into yeast cells, induce photodamage, but have no mutagenic effect; Zoladek T et al.; The yeast Saccharomyces cerevisiae was used as a model eukaryotic organism to study the uptake of diamino acid derivatives of porphyrins and their phototoxicity with particular emphasis on possible mutagenic effects . The water-soluble hematoporphyrin derivatives diarginate (HpD{Arg}2) and 1-arginin di(N-amino acid)-protoporphyrinate used in this study are effective photosensitizers in tumor photodynamic therapy . Depending on the amino acid substituent, the porphyrin derivatives differ in their affinity for yeast cells . It is shown that HpD(Arg)2 and PP(Met)2 (Arg)2 penetrate into the yeast cell and are metabolized . Both compounds sensitize yeast cells to photodamage but have no mutagenic effect on nuclear or mitochondrial genomes.

Genes Dev, 1997 Aug 1, 11(15), 1899 - 911
Alpha2p controls donor preference during mating type interconversion in yeast by inactivating a recombinational enhancer of chromosome III; Szeto L et al.; Homothallic strains of Saccharomyces cerevisiae can change mating type as often as every generation by replacing the allele at the MAT locus with a copy of mating type information present at one of two storage loci, HML and HMR, located on either end of chromosome III . Selection of the appropriate donor locus is dictated by a mating type-specific repressor protein, alpha2p: Cells containing alpha2p select HMR, whereas those lacking alpha2p select HML . As a repressor protein, alpha2p binds to DNA cooperatively with the transcriptional activator Mcm1p . Here we show that two alpha2p/Mcm1p-binding sites, DPS1 and DPS2, control donor selection . DPS1 and DPS2 are located approximately 30 kb from the left arm of chromosome III, well removed from HML, HMR, and MAT . Precise deletion of only DPS1 and DPS2 results in random selection of donor loci and in a cells without affecting selection in alpha cells . Reciprocally, deletion of only the alpha2p binding segments in each of these two sites results in selection of the wrong donor loci in alpha cells without affecting preference in a cells . These results suggest that Mcm1p, bound to these two sites in the absence of alpha2p, activates HML as donor . Binding of alpha2p blocks the ability of Mcm1p bound to DPS1 and DPS2 to activate HML, resulting in default selection of HMR as donor . DPS1 and DPS2 also regulate expression of several noncoding RNAs, although deletion of at least one of these RNA loci does not affect donor preference . This suggests that transcriptional activation, rather than transcription of a specific product, is the initiating event in activating the left arm of chromosome III for donor selection.

Yeast, 1997 Aug, 13(10), 917 - 30
GCR1-dependent transcriptional activation of yeast retrotransposon Ty2-917; Turkel S et al.; Transcription of Saccharomyces cerevisiae Ty2-917 retrotransposon depends on regulatory elements both upstream and downstream of the transcription initiation site . An upstream activation sequence (UAS) and a downstream enhancer stimulate transcription synergistically . Here we show that activation by both of these sites depends on the GCR1 product, a transcription factor which also regulates the genes encoding yeast glycolytic enzymes . Eliminating GCR1 causes a 100-fold decrease in transcription of Ty2-917 . Activation by the isolated Ty2-917 UAS also strongly depends on GCR1 . Unexpectedly, GCR1-dependent activation by the Ty2-917 enhancer is strongly position-dependent . Activation by the enhancer in its normal position within the transcription unit depended strongly on GCR1, but eliminating GCR1 reduced activation only three-fold when the enhancer was moved upstream of the transcribed region . Gel mobility shift and DNaseI protection assays indicated that GCR1 binds specifically to multiple sites within the Ty2-917 UAS and enhancer regions.

J Cell Sci, 1997 Aug, 110 ( Pt 15), 1793 - 804
Distinct subunit functions and cell cycle regulated phosphorylation of 20S APC/cyclosome required for anaphase in fission yeast; Yamada H et al.; We show here that the fission yeast gene products Cut9 and Nuc2 are the subunits of the 20S complex, the putative APC (anaphase promoting complex)/cyclosome which contains ubiquitin ligase activity required for cyclin and Cut2 destruction . The assembly of Cut9 into the 20S complex requires functional Nuc2, and vice versa . The size of fission yeast APC/cyclosome is similar to that of higher eukaryotes, but differs greatly from that (36S) of budding yeast . The 20S complex is present in cells arrested at different stages of the cell cycle, and becomes slightly heavier in mitosis than interphase . Cut9 in the 20S complex is hyperphosphorylated specifically at the time of metaphase . The truncated forms of Cut9 block entry into mitosis, however . The 20S assembly impaired in the cut9 mutant can be restored by elevating the level of a novel gene product Hcnl, similar to budding yeast Cdc26 . Furthermore, deletion of protein kinase PKA (Pkal) suppresses the phenotype of the cut9 mutation and reduces phosphorylation of Cut9 . In contrast, PP1 (Dis2) phosphatase mutation shows the reverse effect on the phenotype of cut9 . The Cut9 subunit is likely to be a target for regulating APC/ cyclosome function through protein-protein interactions and phosphorylation.

Hum Mol Genet, 1997 Aug, 6(8), 1305 - 13
Sequence comparison of human and yeast telomeres identifies structurally distinct subtelomeric domains; Flint J et al.; We have sequenced and compared DNA from the ends of three human chromosomes: 4p, 16p and 22q . In all cases the pro-terminal regions are subdivided by degenerate (TTAGGG)n repeats into distal and proximal sub-domains with entirely different patterns of homology to other chromosome ends . The distal regions contain numerous, short (<2 kb) segments of interrupted homology to many other human telomeric regions . The proximal regions show much longer (approximately 10-40 kb) uninterrupted homology to a few chromosome ends . A comparison of all yeast subtelomeric regions indicates that they too are subdivided by degenerate TTAGGG repeats into distal and proximal sub-domains with similarly different patterns of identity to other non-homologous chromosome ends . Sequence comparisons indicate that the distal and proximal sub-domains do not interact with each other and that they interact quite differently with the corresponding regions on other, non-homologous, chromosomes . These findings suggest that the degenerate TTAGGG repeats identify a previously unrecognized, evolutionarily conserved boundary between remarkably different subtelomeric domains.

Genetics, 1997 Aug, 146(4), 1265 - 74
Molecular and genetic analysis of REC103, an early meiotic recombination gene in yeast; Gardiner JM et al.; In the yeast Saccharomyces cerevisiae at least 10 genes are required to begin meiotic recombination . A new early recombination gene REC103 is described in this paper . It was initially defined by the rec103-1 mutation found in a selection for mutations overcoming the spore inviability of a rad52 spo13 haploid strain . Mutations in REC103 also rescue rad52 in spo13 diploids . rec103 spo13 strains produce viable spores; these spores show no evidence of meiotic recombination . rec103 SPO13 diploids produce no viable spores, consistent with loss of recombination . Mutations in REC103 do not affect mitotic recombination, growth, or repair . These phenotypes are identical to those conferred by mutations in several other early meiotic recombination genes (e.g., REC102, REC104, REC114, ME14, MER2, and SPO11) . REC103 maps to chromosome VII between ADE5 and RAD54 . Cloning and sequencing of REC103 reveals that REC103 is identical to SK18, a gene that depresses the expression of yeast double-stranded ("killer") (ds)RNA viruses . REC103/SK18 is transcribed in mitotic cells and is induced approximately 15-fold in meiosis . REC103 has 26% amino acid identity to the Schizasaccharomyces pombe rec14+ gene; mutations in both genes confer similar meiotic phenotypes, suggesting that they may play similar roles in meiotic recombination.

Nat Biotechnol, 1997 Aug, 15(8), 794 - 7
Increased potato tuber size resulting from apoplastic expression of a yeast invertase; Sonnewald U et al.; The role of sucrose cleavage in determining sink strength in potato was investigated by generating transgenic potato plants that expressed a yeast invertase in either the cytosol or apoplast of tubers . Cytosolic localization gave rise to a reduction in tuber size and an increase in tuber number per plant whereas apoplastic targeting led to an increase in tuber size and a decrease in tuber number per plant . Sink organ size can be manipulated through modification of sucrose metabolism.

Biophys J, 1997 Aug, 73(2), 1103 - 9
Electrorotation of single yeast cells at frequencies between 100 Hz and 1.6 GHz; Holzel R; The determination of complete electrorotation spectra of living cells has been made possible by the development of a quadrature generator and an electrode assembly that span the frequency range between 100 Hz and 1.6 GHz . Multiple spectra of single cells of the yeast Saccharomyces cerevisiae have been measured at different medium conductivities ranging from 0.7 to 550 microS cm-1 . A spherical four-shell model was applied that simulated the experimental data well and disclosed the four-layer structure of the cell envelope attributed to the plasma membrane, the periplasmic space, and a thick inner and a thin outer wall region . Below 10 kHz an additional rotation effect was found, which changed its direction depending on the ionic strength of the medium . This is supposed to be connected with properties of the cell surface and its close vicinity . From the four-shell simulation the following physical properties of cell compartments could be derived: specific capacitance of plasma membrane (0.76 microF cm-2), periplasmic space (0.5 microF cm-2), and outer wall region (0.1 microF cm-2) . The conductivity of cytoplasm, plasma membrane, and inner wall region were found to vary with medium ionic strength from 9 to 12 mS cm-1, 5.8 nS cm-1 to approximately 50 nS cm-1, and 6 microS cm-1 to 240 microS cm-1, respectively.

Arch Biochem Biophys, 1997 Aug 1, 344(1), 139 - 49
Mutations in the IDH2 gene encoding the catalytic subunit of the yeast NAD+-dependent isocitrate dehydrogenase can be suppressed by mutations in the CIT1 gene encoding citrate synthase and other genes of oxidative metabolism; Gadde DM et al.; During a screen for respiration competent yeast mutants that were unable to grow with acetate as a carbon source, two idh2 cit1 double mutants were identified . These strains were defective in the catalytic subunit of the NAD(+)-dependent isocitrate dehydrogenase and citrate synthase of the tricarboxylic acid (TCA) cycle . The strains harboring the idh2 alleles from these strains had two unusual phenotypes . First, their growth on many nonfermentable carbon sources was much poorer than strains containing other idh2 mutations . Second, the poor growth phenotype could be suppressed by the presence of mutations in CIT1 and other genes encoding oxidative functions . Spontaneous suppressor mutants that restore fast growth on glycerol medium to strains harboring two idh2 alleles were isolated, and a large percentage of the suppressor mutations have been identified within the CIT1 gene and at several other loci . Elevated levels of several TCA cycle proteins were observed in these idh2 mutants that were not observed in the presence of suppressing cit1 mutations . Citrate and isocitrate concentrations were also elevated in the idh2 mutants, but probably not to toxic levels . Five idh2 alleles were sequenced to understand the defects of the two classes of mutations . Sequence analysis indicated that the poor growth phenotype was caused by the loss of Idh2p protein . Similarly, eight cit1 alleles were sequenced to understand their characteristics as glycerol suppressors of idh2 . These and other studies indicate that any mutation within CIT1 was capable of suppressing the idh2 mutations . Several models to explain these interactions are discussed.

Arch Biochem Biophys, 1997 Aug 1, 344(1), 53 - 60
Metabolic effects of altering redundant targeting signals for yeast mitochondrial malate dehydrogenase; Small WC et al.; Eukaryotic cells contain highly homologous isozymes of malate dehydrogenase which catalyze the same reaction in different cellular compartments . To examine whether the metabolic functions of these isozymes are interchangeable, we have altered the cellular localization of mitochondrial malate dehydrogenase (MDH1) in yeast . Since a previous study showed that removal of the targeting presequence from MDH1 does not prevent mitochondrial import in vivo, we tested the role of a putative cryptic targeting sequence near the amino terminus of the mature polypeptide . Three residues in this region were changed to residues present in analogous positions in the other two yeast MDH isozymes . Alone, these replacements did not affect activity or localization of MDH1 but, in combination with deletion of the presequence, prevented mitochondrial import in vivo . Measurable levels of the resulting cytosolic form of MDH1 were low with expression from a centromere-based plasmid but were comparable to normal cellular levels with expression from a multicopy plasmid . The cytosolic form of MDH1 restored the ability of a deltaMDH1 disruption strain to grow on ethanol or acetate, suggesting that mitochondrial localization of MDH1 is not essential for its function in the TCA cycle . This TCA cycle function observed for the cytosolic form of MDH1 is unique to that isozyme since overexpression of MDH2 and of a cytosolic form of MDH3 in a deltaMDH1 strain failed to restore growth . Finally, only partial restoration of growth of a deltaMDH2 disruption mutant was attained with the cytosolic form of MDH1, suggesting that MDH2 may also have unique metabolic functions.

Blood, 1997 Aug 1, 90(3), 1233 - 40
A chromosome 14q11/TCR alpha/delta specific yeast artificial chromosome improves the detection rate and characterization of chromosome abnormalities in T-lymphoproliferative disorders; Rack KA et al.; The rate of detection of chromosome abnormalities in T-cell proliferations is lower than that observed in B-cell malignancies . The former frequently involve the TCR alpha/delta locus at chromosome band 14q11 . We have identified a YAC encompassing 70% of the TCR alpha/delta locus, which has been used as a fluorescence in situ hybridization probe to detect chromosome rearrangements involving 14q11, both at metaphase and within interphase nuclei, in patients with a variety of T-lymphoproliferative disorders . Its use allowed detection of previously unsuspected TCR alpha/delta rearrangements in 4/13 (30%) immature T-lineage acute leukemias, including two t(10;14) and 2 minor inversion 14s . It also clarified interpretation of complex chromosome 14 abnormalities in mature T-cell proliferations (T-prolymphocytic leukemia and ataxia telangiectasia) . Use of this probe will aid the detection and characterization of abnormalities involving the TCR alpha/delta locus, particularly in cases with normal or complex karyotypes and in those proliferations for which mitoses are difficult to obtain.

Nat Genet, 1997 Aug, 16(4), 352 - 7
Respiratory deficiency due to loss of mitochondrial DNA in yeast lacking the frataxin homologue; Wilson RB et al.; Friedreich's ataxia (FRDA) is an autosomal recessive degenerative disorder that primarily affects the nervous system and heart . Patients with FRDA have point mutations or trinucleotide repeat expansions in both alleles of FRDA, which encodes a protein termed frataxin . We show that the yeast frataxin homologue, which we have named YFH1, localizes to mitochondria and is required to maintain mitochondrial DNA . The YFH1-homologous domain of frataxin functions in yeast and a disease-associated missense mutation of this domain, or the corresponding domain in YFH1, reduces function . Our data suggest that mitochondrial dysfunction contributes to FRDA pathophysiology.

Nat Genet, 1997 Aug, 16(4), 345 - 51
Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin; Koutnikova H et al.; Friedreich's ataxia is due to loss of function mutations in the gene encoding frataxin (FRDA) . Frataxin is a protein of unknown function . In situ hybridization analyses revealed that mouse frataxin expression correlates well with the main site of neurodegeneration, but the expression pattern is broader than expected from the pathology of the disease . Frataxin mRNA is predominantly expressed in tissues with a high metabolic rate, including liver, kidney, brown fat and heart . We found that mouse and yeast frataxin homologues contain a potential mitochondrial targeting sequence in their N-terminal domains and that disruption of the yeast gene results in mitochondrial dysfunction . Finally, tagging experiments demonstrate that human frataxin co-localizes with a mitochondrial protein . Friedreich's ataxia is therefore a mitochondrial disease caused by a mutation in the nuclear genome.

J Biol Chem, 1997 Aug 1, 272(31), 19343 - 50
Structural and functional roles of tyrosine 78 of yeast guanylate kinase; Zhang Y et al.; The hydroxyl group of Tyr-78 of yeast guanylate kinase (GK) is hydrogen-bonded to the phosphate of the bound GMP as revealed by x-ray crystallography . The structural and functional roles of Tyr-78 were evaluated by site-directed mutagenesis, kinetics, guanidine hydrochloride-induced denaturation, and nuclear magnetic resonance spectroscopy (NMR) . Substitution of Tyr-78 with a phenylalanine resulted in a decrease in kcat by a factor of 131, an increase in Km(GMP) by a factor of 20 and an increase in Ki(GMP) by a factor of 18 . Km(MgATP) and Ki(MgATP) were very similar to those of the wild-type (WT) GK . The conformational stability of the mutant was lower than that of the WT by 1.0 kcal/mol as measured by guanidine hydrochloride-induced denaturation . Detailed comparison of the TOCSY and NOESY spectra of the WT GK and the mutant indicated that the conformation of Y78F is little perturbed relative to that of the WT GK at the free state and the conformation of Y78F.GMP complex is also very similar to that of the WT.GMP complex . The results taken together showed that the hydrogen bond between Tyr-78 and GMP stabilizes the GK.GMP complex by 1.7 kcal/mol, the ternary complex by 1.8 kcal/mol, and the transition state by 4.6 kcal/mol . Tyr-78 is not essential for proper folding of the enzyme but it may contribute to the conformational stability . Solvent-accessible aromatic residues were identified by using the paramagnetic probe 4-hydroxy-2, 2,6,6-tetramethylpiperidine-1-oxyl . Comparison of the free and GMP-bound forms of the WT GK by NMR indicated that there are changes in conformation and dynamics upon binding of GMP.

J Biol Chem, 1997 Aug 1, 272(31), 19319 - 27
Genes for Tfb2, Tfb3, and Tfb4 subunits of yeast transcription/repair factor IIH . Homology to human cyclin-dependent kinase activating kinase and IIH subunits; Feaver WJ et al.; Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase transcription factor IIH (TFIIH) are described . All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit transcription in vitro . A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH) . An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the factor . The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH . Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast factor . These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.

J Biol Chem, 1997 Aug 1, 272(31), 19176 - 86
Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-cytochrome P450 reductases with P450 CYP73A5; Urban P et al.; Two NADPH-cytochrome P450 reductase-encoding cDNAs were isolated from an Arabidopsis cDNA library by metabolic interference in a Saccharomyces cerevisiae mutant disrupted for its endogenous cpr1 gene . ATR1 encodes a protein of 692 amino acids, while ATR2 encodes either a 712-residue protein (ATR2-1), or a 702-residue protein (ATR2-2) depending on the choice of the initiation codon . Comparative analysis of ATR1 and ATR2-1 indicates 64% amino acid sequence identity and the absence of conservation in the third base of conserved amino acid codons . The two Arabidopsis reductases are encoded by distinct genes whose divergence is expected an early event in angiosperms evolution . A poly(Ser/Thr) stretch reminiscent of a plant chloroplastic targeting signal is present at the ATR2-1 N-terminal end but absent in ATR1 . The cDNA open reading frames were expressed in yeast . The recombinant polypeptides were found present in the yeast endoplasmic reticulum membrane and exhibited a high specific NADPH-cytochrome c reductase activity . To gain more insight into the respective functions of the two reductases, the Arabidopsis cDNA encoding cinnamate 4-hydroxylase (CYP73A5) was cloned and co-expressed with ATR1 or ATR2 in yeast . Biochemical characterization of the Arabidopsis ATR1/CYP73A5 and ATR2-1/CYP73A5 systems demonstrates that the two distantly related Arabidopsis reductases similarly support the first oxidative step of the phenylpropanoid general pathway.

Mol Cell Biol, 1997 Aug, 17(8), 4811 - 9
The yeast SWI-SNF complex facilitates binding of a transcriptional activator to nucleosomal sites in vivo; Burns LG et al.; The Saccharomyces cerevisiae SWI-SNF complex is a 2-MDa protein assembly that is required for the function of many transcriptional activators . Here we describe experiments on the role of the SWI-SNF complex in activation of transcription by the yeast activator GAL4 . We find that while SWI-SNF activity is not required for the GAL4 activator to bind to and activate transcription from nucleosome-free binding sites, the complex is required for GAL4 to bind to and function at low-affinity, nucleosomal binding sites in vivo . This SWI-SNF dependence can be overcome by (i) replacing the low-affinity sites with higher-affinity, consensus GAL4 binding sequences or (ii) placing the low-affinity sites into a nucleosome-free region . These results define the criteria for the SWI-SNF dependence of gene expression and provide the first in vivo evidence that the SWI-SNF complex can regulate gene expression by modulating the DNA binding of an upstream activator protein.

Mol Cell Biol, 1997 Aug, 17(8), 4622 - 32
A transcriptional mediator protein that is required for activation of many RNA polymerase II promoters and is conserved from yeast to humans; Lee YC et al.; A temperature-sensitive mutation was obtained in Med6p, a component of the mediator complex from the yeast Saccharomyces cerevisiae . The mediator complex has been shown to enable transcriptional activation in vitro . This mutation in Med6p abolished activation of transcription from four of five inducible promoters tested in vivo . There was no effect, however, on uninduced transcription, transcription of constitutively expressed genes, or transcription by RNA polymerases I and III . Mediator-RNA polymerase II complex isolated from the mutant yeast strain was temperature sensitive for transcriptional activation in a reconstituted in vitro system due to a defect in initiation complex formation . A database search revealed the existence of MED6-related genes in humans and Caenorhabditis elegans, suggesting that the role of mediator in transcriptional activation is conserved throughout the evolution.

Mol Cell Biol, 1997 Aug, 17(8), 4536 - 43
Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein; Bailly V et al.; The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme required for postreplicational repair of UV-damaged DNA and for damage-induced mutagenesis . In addition, Rad6 functions in the N end rule pathway of protein degradation . Rad6 mediates its DNA repair role via its association with Rad18, whose DNA binding activity may target the Rad6-Rad18 complex to damaged sites in DNA . In its role in N end-dependent protein degradation, Rad6 interacts with the UBR1-encoded ubiquitin protein ligase (E3) enzyme . Previous studies have indicated the involvement of N-terminal and C-terminal regions of Rad6 in interactions with Ubr1 . Here, we identify the regions of Rad6 and Rad18 that are involved in the dimerization of these two proteins . We show that a region of 40 amino acids towards the C terminus of Rad18 (residues 371 to 410) is sufficient for interaction with Rad6 . This region of Rad18 contains a number of nonpolar residues that have been conserved in helix-loop-helix motifs of other proteins . Our studies indicate the requirement for residues 141 to 149 at the C terminus, and suggest the involvement of residues 10 to 22 at the N terminus of Rad6, in the interaction with Rad18 . Each of these regions of Rad6 is indicated to form an amphipathic helix.

Endocrinology, 1997 Aug, 138(8), 3459 - 75
Phosphoinositolglycan-peptides from yeast potently induce metabolic insulin actions in isolated rat adipocytes, cardiomyocytes, and diaphragms; Muller G et al.; Polar headgroups of free glycosyl-phosphatidylinositol (GPI) lipids or protein-bound GPI membrane anchors have been shown to exhibit insulin-mimetic activity in different cell types . However, elucidation of the molecular mode of action of these phospho-inositolglycan (PIG) molecules has been hampered by 1) lack of knowledge of their exact structure; 2) variable action profiles; and 3) rather modest effects . In the present study, these problems were circumvented by preparation of PIG-peptides (PIG-P) in sufficient quantity by sequential proteolytic (V8 protease) and lipolytic (phosphatidylinositol-specific phospholipase C) cleavage of the GPI-anchored plasma membrane protein, Gce1p, from the yeast Saccharomyces cerevisiae . The structure of the resulting PIG-P, NH2-Tyr-Cys-Asn-ethanolamine-PO4-6(Man1-2)Man1-2Man1-+ ++6Man1-4GlcNH(2)1-6myo-inositol-1,2-cyclicPO4, was revealed by amino acid analysis and Dionex exchange chromatography of fragments generated enzymatically or chemically from the neutral glycan core and is in accordance with the known consensus structures of yeast GPI anchors . PIG-P stimulated glucose transport and lipogenesis in normal, desensitized and receptor-depleted isolated rat adipocytes, increased glycerol-3-phosphate acyltransferase activity and translocation of the glucose transporter isoform 4, and inhibited isoproterenol-induced lipolysis and protein kinase A activation in adipocytes . Furthermore, PIG-P was found to stimulate glucose transport in isolated rat cardiomyocytes and glycogenesis and glycogen synthase in isolated rat diaphragms . The concentration-dependent effects of the PIG-P reached 70-90% of the maximal insulin activity with EC50-values of 0.5-5 microM . Chemical or enzymic cleavages within the glycan or peptide portion of the PIG-P led to decrease or loss of activity . The data demonstrate that PIG-P exhibits a potent insulin-mimetic activity which covers a broad spectrum of metabolic insulin actions on glucose transport and metabolism.

J Virol, 1997 Aug, 71(8), 6075 - 82
The tobacco mosaic virus RNA polymerase complex contains a plant protein related to the RNA-binding subunit of yeast eIF-3; Osman TA et al.; A sucrose density gradient-purified, membrane-bound tobacco mosaic virus (tomato strain L) (TMV-L) RNA polymerase containing endogenous RNA template was efficiently solubilized with sodium taurodeoxycholate . Solubilization resulted in an increase in the synthesis of positive-strand, 6.4-kb genome-length single-stranded RNA (ssRNA) and a decrease in the production of 6.4-kbp double-stranded RNA (dsRNA) to levels close to the limits of detection . The solubilized TMV-L RNA polymerase was purified by chromatography on columns of DEAE-Bio-Gel and High Q . Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed that purified RNA polymerase preparations consistently contained proteins with molecular masses of 183, 126, 56, 54, and 50 kDa, which were not found in equivalent material from healthy plants . Western blotting showed that the two largest of these proteins are the TMV-L-encoded 183- and 126-kDa replication proteins and that the 56-kDa protein is related to the 54.6-kDa GCD10 protein, the RNA-binding subunit of yeast eIF-3 . The 126-, 183-, and 56-kDa proteins were coimmunoaffinity selected by antibodies against the TMV-L 126-kDa protein and by antibodies against the GCD10 protein . Antibody-linked polymerase assays showed that active TMV-L RNA polymerase bound to antibodies against the TMV-L 126-kDa protein and to antibodies against the GCD10 protein . Synthesis of genome-length ssRNA and dsRNA by a template-dependent, membrane-bound RNA polymerase was inhibited by antibodies against the GCD10 protein, and this inhibition was reversed by prior addition of GCD10 protein.

Gene, 1997 Jul 31, 194(2), 227 - 33
Integration of the yeast retrovirus-like element Ty3 upstream of a human tRNA gene expressed in yeast; Dildine SL et al.; The retrovirus-like element Ty3 of Saccharomyces cerevisae integrates into the yeast genomic DNA in a position specific manner . Ty3 integrates within 1-2 base pairs of the site of transcription initiation by RNA polymerase III . The human tRNA(Lys)3 gene was used as a target for transposition in a plasmid-based assay to determine whether Ty3 integration can be targeted to a human tRNA gene . Each transposition event observed was adjacent to the site of initiation of transcription of the human tRNA gene . Therefore, heterologous tRNA genes can serve as targets for Ty3 in yeast . This is a first step toward development of a system for targeted integrations in heterologous organisms.

Nature, 1997 Jul 31, 388(6641), 465 - 8
The advantage of sex in evolving yeast populations; Zeyl C et al.; Sex is a general feature of the life cycle of eukaryotes . It is not universal, however, as many organisms seem to lack sex entirely . The widespread occurrence of sex is puzzling, both because meiotic recombination can disrupt co-adapted combinations of genes, and because it halves the potential rate of reproduction in organisms with strongly differentiated male and female gametes . Most attempts to explain the maintenance of sexuality invoke differences between parents and sexual offspring . These differences may be advantageous in novel or changing environments if new gene combinations are favoured from time to time . Sex would then serve to concentrate beneficial mutations that have arisen independently into the same line of descent . But in a stable environment sex might serve to concentrate deleterious mutations, so that they will be more effectively purged from the population by selection . We have studied the effect of sex on mean fitness in experimental populations of the budding yeast Saccharomyces cerevisiae . Our results show that sex increases mean fitness in an environment to which the populations were well adapted, but not in an environment to which new adaptation occurred, supporting the hypothesis that the advantage of sexuality lay in the removal of deleterious mutations.

Biochemistry, 1997 Jul 29, 36(30), 9246 - 52
Yeast protein farnesyltransferase . Site-directed mutagenesis of conserved residues in the beta-subunit; Dolence JM et al.; Protein prenyltransferases catalyze the posttranslational modification of cysteines by isoprenoid hydrocarbon chains . A protein farnesyltransferase (PFTase) and a protein geranylgeranyltransferase (PGGTase-I) alkylate cysteines in a CaaX C-terminal tetrapeptide sequence, where a is usually an aliphatic amino acid and X is an amino acid that specifies whether a C15 farnesyl or C20 geranylgeranyl moiety is added . A third enzyme, PGGTase-II, adds geranylgeranyl groups to both cysteines at the C-terminus of Rab proteins . All three enzymes are Zn2+ metalloproteins and also require Mg2+ for activity . The protein prenyltransferases are heterodimers . PFTase and PGGTase I contain identical alpha-subunits and distinctive beta-subunits, which are responsible for the differences in substrate selectivity seen for the two enzymes . The subunits in PGGTase-II are similar, but not identical, to their counterparts in the other two enzymes . An alignment of amino acid sequences for the beta-subunits of all three enzymes shows five regions of high similarity . Thirteen of the conserved polar and charged residues in yeast PFTase were selected for substitution by site-directed mutagenesis . Kinetic studies revealed a subset of five enzymes, R211Q, D307A, C309A, Y310F, and H363A, with substantially reduced catalytic constants (kcat) . Metal analyses of wild-type enzyme and the five least reactive mutants showed that the substitutions had compromised Zn2+ binding in the D307A, C309A, and H363A enzymes.

FEBS Lett, 1997 Jul 28, 412(2), 355 - 8
The presence of a transcription activation function in the hormone-binding domain of androgen receptor is revealed by studies in yeast cells; Moilanen A et al.; To assess the importance of various regions of the androgen receptor (AR) in transcriptional regulation, we have compared its activation functions (AFs) in yeast and mammalian cells . The receptor's amino-terminal region contains a major transcriptional activator (AF-1) in both cell types, whereas AF-2 in the ligand-binding domain (LBD) is very weak in mammalian cells but clearly functional in the yeast . Hormone-binding ability of LBD is mandatory for AF-2 to operate, as illustrated by mutated LBD constructs . The activity of AF-2 in yeast is severely attenuated when the hinge region is attached to LBD, suggesting that the former region modulates AF-2 in vivo, probably by presenting an interface for interacting proteins.

J Biotechnol, 1997 Jul 23, 56(1), 41 - 7
PolI-driven integrative expression vectors for yeast; Blancafort P et al.; A novel expression vector for yeast has been constructed from the regulatory elements present in the polI promoter and the enhancer/termination region (E/T) of rDNA . Under some conditions, this promoter/vector combination produces small RNAs such as the hammerhead RNA sequence at levels comparable to polII- and polIII-dependent systems . No stable transcription product can be demonstrated with this vector when the enhancer/termination sequence is less than 100 nucleotides downstream from the promoter . On the other hand, high expression of a stable, hammerhead RNA molecule can be obtained from this vector by inserting a 400-bp fragment containing the ADH1 transcription termination region upstream of the E/T . RNAs produced by this vector are polyadenylated and multiple copies of this plasmid can be stably integrated into the yeast chromosome.

Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 7897 - 902
The major yeast poly(A)-binding protein is associated with cleavage factor IA and functions in premessenger RNA 3'-end formation; Minvielle-Sebastia L et al.; Polyadenylation of premessenger RNAs occurs posttranscriptionally in the nucleus of eukaryotic cells by cleavage of the precursor and polymerization of adenosine residues . In the yeast Saccharomyces cerevisiae, the mature poly(A) tail ranges from 60 to 70 nucleotides . 3'-end processing can be reproduced in vitro with purified factors . The cleavage reaction requires cleavage factors I and II (CF I and CF II), whereas polyadenylation involves CF I, polyadenylation factor I (PFI), and poly(A) polymerase (Pap1p) . CF I has recently been separated into two factors, CF IA and CF IB . We have independently purified CF IA and found that five polypeptides cofractionate with the activity . They include Rna14p, Rna15p, Pcf11p, a new protein called Clp1p, and remarkably, the major poly(A)-binding protein Pab1p . Extracts from strains where the PAB1 gene is mutated or deleted are active for cleavage but generate transcripts bearing abnormally long poly(A) tracts . Complementation with recombinant Pab1p not only restores the length of the poly(A) tails to normal, but also triggers a poly(A) shortening activity . In addition, a monoclonal Pab1p antibody prevents the formation of poly(A) tails in extracts or in a reconstituted system . Our data support the notion that Pab1p is involved in the length control of the poly(A) tails of yeast mRNAs and define a new essential function for Pab1p in the formation of mature mRNAs.

Virology, 1997 Jul 21, 234(1), 84 - 92
Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system; Wittmann S et al.; The yeast LexA interaction trap was used to screen a cDNA library from Arabidopsis thaliana in order to identify proteins that interact with the viral protein genome linked (VPg)-proteinase of turnip mosaic potyvirus . The screen allowed the isolation of four candidate cDNA clones . Clones pHC4, pHC21, and pHC40 were partially sequenced but no homologies to known proteins were found . However, the amino acid sequence deduced from the complete nucleotide sequence of pSW56 revealed that it was the eukaryotic initiation factor (iso) 4E {eIF(iso)4E} . Deletion analysis indicated that the VPg domain was involved in the interaction with the plant protein . Interaction between the viral protein and the cellular protein was confirmed by ELISA-based binding experiments . eIF(iso)4E plays an essential role in the initiation of the translation of capped mRNAs and its association with VPg would point to a role of the viral protein in the translation of the virus.

Science, 1997 Jul 18, 277(5324), 383 - 7
Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA; Long RM et al.; Cell divisions that produce progeny differing in their patterns of gene expression are key to the development of multicellular organisms . In the budding yeast Saccharomyces cerevisiae, mother cells but not daughter cells can switch mating type because they selectively express the HO endonuclease gene . This asymmetry is due to the preferential accumulation of an unstable transcriptional repressor protein, Ash1p, in daughter cell nuclei . Here it is shown that ASH1 messenger RNA (mRNA) preferentially accumulates in daughter cells by a process that is dependent on actin and myosin . A cis-acting element in the 3'-untranslated region of ASH1 mRNA is sufficient to localize a chimeric RNA to daughter cells . These results suggest that localization of mRNA may have been an early property of the eukaryotic lineage.

Science, 1997 Jul 18, 277(5324), 381 - 3
In vitro propagation of the prion-like state of yeast Sup35 protein; Paushkin SV et al.; The yeast cytoplasmically inherited genetic determinant {PSI+} is presumed to be a manifestation of the prion-like properties of the Sup35 protein (Sup35p) . Here, cell-free conversion of Sup35p from {psi-} cells (Sup35ppsi-) to the prion-like {PSI+}-specific form (Sup35pPSI+) was observed . The conversion reaction could be repeated for several consecutive cycles, thus modeling in vitro continuous {PSI+} propagation . Size fractionation of lysates of {PSI+} cells demonstrated that the converting activity was associated solely with Sup35pPSI+ aggregates, which agrees with the nucleation model for {PSI+} propagation . Sup35pPSI+ was purified and showed high conversion activity, thus confirming the prion hypothesis for Sup35p.

J Biol Chem, 1997 Jul 18, 272(29), 18281 - 9
Interactions of nucleotide release factor Dss4p with Sec4p in the post-Golgi secretory pathway of yeast; Collins RN et al.; SEC4 is an essential gene encoding a small GTPase that is involved in Golgi to cell surface transport in Saccharomyces cerevisiae and is a paradigm for studies on the mode of action of Rab proteins . We describe here the features of interaction of Sec4p with the accessory protein Dss4p . Dss4p is found both on membranes and in the cytosol; however, it is the membrane fraction that is complexed to Sec4p . Dss4p, like its mammalian counterpart, Mss4, binds zinc, and disruption of the zinc-binding site disrupts the ability of the protein to interact with Sec4p . DSS4 overexpression can rescue the lethal phenotype of two alleles of SEC4, corresponding to dominant mutations of Ras . We demonstrate that this suppression is due to the ability of Dss4p to form a tight complex with the mutant forms of Sec4p and hence sequester the mutant protein from its inhibitory effect . These results imply an in vivo role for Dss4p as a guanine nucleotide dissociation stimulator . In vitro the protein has the ability to stimulate the dissociation rate of both GDP and GTP from Sec4p . We examined the relationship of GDI1 and DSS4 with SEC4 both genetically and biochemically . These results exclude a role for DSS4 in the recruitment of Sec4p/GDI onto membranes.

EMBO J, 1997 Jul 16, 16(14), 4352 - 60
Cell type-specific chromatin organization of the region that governs directionality of yeast mating type switching; Weiss K et al.; Switching of mating type in Saccharomyces cerevisiae is directional; MAT alpha cells recombine to transfer information from HMRa while MATa cells switch using the silent cassette at HML alpha . Genetic analysis recently has defined a 700 bp recombination enhancer approximately 29 kb from the left end of chromosome III that is necessary for directionality . The chromatin structure of this region differs strikingly in a- and alpha-cells . Mat alpha2p organizes a 3.7 kb chromatin domain that opposes interaction of trans-acting proteins with the enhancer . In a-cells lacking the alpha2 repressor, two footprinted regions flank an approximately 100 bp section having a unique DNA structure . This structural signature probably reflects interactions of proteins that result in directional mating type switching.

EMBO J, 1997 Jul 16, 16(14), 4194 - 204
Suppressors of YCK-encoded yeast casein kinase 1 deficiency define the four subunits of a novel clathrin AP-like complex; Panek HR et al.; In Saccharomyces cerevisiae, the redundant YCK1 and YCK2 genes (Yeast Casein Kinase 1) are required for viability . We describe here the molecular analysis of four mutations that eliminate the requirement for Yck activity . These mutations alter proteins that resemble the four subunits of clathrin adaptors (APs), with highest sequence similarity to those of the recently identified AP-3 complex . The four yeast subunits are associated in a high-molecular-weight complex . These proteins have no essential function and are not redundant for function with other yeast AP-related proteins . Combination of suppressor mutations with a clathrin heavy chain mutation (chc1-ts) confers no synthetic growth defects . However, a yck(ts) mutation shows a strong synthetic growth defect with chc1-ts . Moreover, endocytosis of Ste3p is dramatically decreased in yck(ts) cells and is partially restored by the AP suppressor mutations . These results suggest that vesicle trafficking at the plasma membrane requires the activity of Yck protein kinases, and that the new AP-related complex may participate in this process.

Biochemistry, 1997 Jul 15, 36(28), 8586 - 93
Pressure-induced dissociation of yeast glyceraldehyde-3-phosphate dehydrogenase: heterogeneous kinetics and perturbations of subunit structure; Cioni P et al.; In studies of pressure-induced subunit dissociation of oligomeric proteins, the thermodynamic dissociation constant and the dissociation volume change are derived by assuming that high pressure itself does not significantly perturb the structure of both oligomer and isolated subunit . In this report, the intrinsic phosphorescence emission of Trp reveals that high-pressure dissociation of tetrameric yeast glyceraldehyde-3-phosphate dehydrogenase results in a dramatic shortening of the phosphorescence lifetime, from 300 to less than 2 ms, that is consistent with a profound loosening of the polypeptide structure about the phosphorescence probe . On pressure release, subunit reassociation occurs readily whereas recovery of the native phosphorescence properties is a very slow, thermally activated, process which goes hand in hand with the recovery of the catalytic activity . Further, the comparison between the kinetic traces that describe the degree of dissociation and the change in phosphorescence lifetime, at various applied pressures, has established the following: (1) that high pressure plays a direct role on the structural rearrangement, the extent of which increases with pressure; (2) that the conformational change in the monomer is concomitant with, or follows closely after, the break up of the tetramer, in any case long before an apparent tetramer-monomer equilibrium is established; (3) that native tetramers are highly heterogeneous with regard to their rate of dissociation . The influence of temperature, of protein concentration, of binding of NAD+, and of the addition of 2 M urea on the dissociation/phosphorescence kinetic profiles was also examined . The complications arising from these conformational changes for the derivation of the dissociation free energy change as well as their relevance for understanding the lack of concentration dependence of the degree of dissociation are discussed.

Nucleic Acids Res, 1997 Jul 15, 25(14), 2694 - 701
Pleiotropic effects of intron removal on base modification pattern of yeast tRNAPhe: an in vitro study; Jiang HQ et al.; Cell-free yeast extract has been successfully used to catalyze the enzymatic formation of 11 out of the 14 naturally occurring modified nucleotides in yeast tRNAPhe(anticodon GAA) . They are m2G10, D17, m22G26, Cm32, Gm34,psi39, m5C40, m7G46, m5C49, T54 andpsi55 . Only D16, Y37 and m1A58 were not formed under in vitro conditions . However, m1G37was quantitatively produced instead of Y37 . The naturally occurring intron was absolutely required for m5C40formation while it hindered completely the enzymatic formation of Cm32, Gm34and m1G37 . Enzymatic formation of m22G26,psi39, m7G46, m5C49, T54 andpsi55were not or only slightly affected by the presence of the intron . These results allow us to classify the different tRNA modification enzymes into three groups: intron insensitive, intron dependent, and those requiring the absence of the intron . The fact that truncated tRNAPheconsisting of the anticodon stem and loop prolonged with the 19 nucleotide long intron is a substrate for tRNA: cytosine-40 methylase demonstrates that the enzyme is not only strictly intron dependent, but also does not require fully structured tRNA.

FEBS Lett, 1997 Jul 14, 411(2-3), 308 - 12
Glucose activation of the yeast plasma membrane H+-ATPase requires the ubiquitin-proteasome proteolytic pathway; de la Fuente N et al.; Glucose triggers transcriptional and post-transcriptional mechanisms that increase the level and activity of Saccharomyces cerevisiae plasma membrane H+-ATPase . We have studied the post-transcriptional activation of the enzyme by glucose and have found that Rsp5, a ubiquitin-protein ligase enzyme, Ubc4, a ubiquitin-conjugating enzyme, and the 26S proteasome complex are implicated in this activation . These results suggest that ATPase activation by glucose requires the ubiquitin-proteasome proteolytic pathway . This is supported by the fact that over-expression of the ubiquitin-specific protease Ubp2, which cleaves ubiquitin from its branched conjugates, inhibits this activation . We propose that glucose triggers degradation of an inhibitory protein resulting in enzyme activation.

FEBS Lett, 1997 Jul 14, 411(2-3), 195 - 200
Yeast mitochondrial F1F0-ATPase: the novel subunit e is identical to Tim11; Arnold I et al.; We report here the identification of the novel subunit of the mitochondrial F1F0-ATPase from Saccharomyces cerevisiae, ATPase subunit e . Yeast ATPase subunit e displays significant similarities in both amino acid sequence, properties (hydropathy and predicted coiled-coil structure) and orientation in the inner membrane, with previously identified mammalian ATPase subunit e proteins . Estimation of its native molecular mass and ability to be co-immunoprecipitated with a subunit of the F1-ATPase, demonstrate that subunit e is a subunit of the F1F0-ATPase . Stable expression of subunit e requires the presence of the mitochondrially encoded subunits of the F0-ATPase . Subunit e had been previously identified as Tim11 and was proposed to be involved in the process of sorting of proteins to the mitochondrial inner membrane.

J Cell Biol, 1997 Jul 14, 138(1), 95 - 103
In vitro reconstitution of cortical actin assembly sites in budding yeast; Lechler T et al.; We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro . Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud . After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract . Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors . Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution . This finding, together with the severe defects in actin organization associated with the bee1 null mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex . The factor that acts in the second step of the reconstitution has been identified by conventional chromatography . It is composed of a novel protein, Pca1 . Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.

J Cell Biol, 1997 Jul 14, 138(1), 17 - 36
A small conserved domain in the yeast Spa2p is necessary and sufficient for its polarized localization; Arkowitz RA et al.; SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud . The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions . Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip . Finally, during cytokinesis Spa2p is present as a ring at the mother-daughter bud neck . The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip . Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth . This localization domain and the amino terminus of Spa2p are essential for its function in mating . Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (a2p omolog), with significant homology to the localization domain and amino terminus of Spa2p . This protein also localizes to sites of polarized growth in budding and mating cells . SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation . Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component . The identification of a 150-amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.

J Biol Chem, 1997 Jul 11, 272(28), 17762 - 75
Architecture of the yeast cell wall . Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin; Kollar R et al.; In a previous study (Kollar, R., Petrakova, E., Ashwell, G., Robbins, P . W., and Cabib, E . (1995) J . Biol . Chem . 270, 1170-1178), the linkage region between chitin and beta(1-->3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with beta(1-->3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase . In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material . We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, beta(1-->3)-glucan, beta(1-->6)-glucan, chitin, and mannoprotein are linked together . Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to beta(1-->6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five alpha-linked mannosyl residues . The beta(1-->6)-glucan has some beta(1-->3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a beta(1-->4) or beta(1-->2) linkage . Finally, the reducing end of beta(1-->6)-glucan is connected to the nonreducing terminal glucose of beta(1-->3)-glucan through a linkage that remains to be established . A fraction of the isolated material has three of the main components but lacks mannoprotein . From these results and previous findings on the linkage between mannoproteins and beta(1-->6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall . The possible mechanism of synthesis and physiological significance of the cross-links is discussed.

J Biol Chem, 1997 Jul 11, 272(28), 17749 - 55
Two protein-tyrosine phosphatases inactivate the osmotic stress response pathway in yeast by targeting the mitogen-activated protein kinase, Hog1; Jacoby T et al.; Protein phosphatases inactivate mitogen-activated protein kinase (MAPK) signaling pathways by dephosphorylating components of the MAPK cascade . Two genes encoding protein-tyrosine phosphatases, PTP2, and a new phosphatase, PTP3, have been isolated in a genetic selection for negative regulators of an osmotic stress response pathway called HOG, for high osmolarity glycerol, in budding yeast . PTP2 and PTP3 were isolated as multicopy suppressors of a severe growth defect due to hyperactivation of the HOG pathway . Phosphatase activity is required for suppression since mutation of the catalytic Cys residue in Ptp2 and Ptp3, destroys suppressor function and biochemical activity . The substrate of these phosphatases is likely to be the MAPK, Hog1 . Catalytically inactive Ptp2 and Ptp3 coprecipitate with Hog1 from yeast extracts . In addition, strains lacking PTP2 and PTP3 do not dephosphorylate Hog1-phosphotyrosine as well as wild type . The latter suggests that PTP2 and PTP3 play a role in adaptation . Consistent with this role, osmotic stress induces expression of PTP2 and PTP3 transcripts in a Hog1-dependent manner . Thus Ptp2 and Ptp3 likely act in a negative feedback loop to inactivate Hog1.

J Biol Chem, 1997 Jul 11, 272(28), 17711 - 8
Homeostatic regulation of copper uptake in yeast via direct binding of MAC1 protein to upstream regulatory sequences of FRE1 and CTR1; Yamaguchi-Iwai Y et al.; Copper deprivation of Saccharomyces cerevisiae induces transcription of the FRE1 and CTR1 genes . FRE1 encodes a surface reductase capable of reducing and mobilizing copper chelates outside the cell, and CTR1 encodes a protein mediating copper uptake at the plasma membrane . In this paper, the protein encoded by MAC1 is identified as the factor mediating this homeostatic control . A novel dominant allele of MAC1, MAC1(up2), is mutated in a Cys-rich domain that may function in copper sensing (a G to A change of nucleotide 812 resulting in a Cys-271 to Tyr substitution) . This mutant is functionally similar to the MAC1(up1) allele in which His-279 in the same domain has been replaced by Gln . Both mutations confer constitutive copper-independent expression of FRE1 and CTR1 . A sequence including the palindrome TTTGCTCA .. . TGAGCAAA, appearing within the 5'-flanking region of the CTR1 promoter, is necessary and sufficient for the copper- and MAC1-dependent CTR1 transcriptional regulation . An identical sequence appears as a direct repeat in the FRE1 promoter . The data indicate that the signal resulting from copper deprivation is transduced via the Cys-rich motif of MAC1 encompassing residues 264-279 . MAC1 then binds directly and specifically to the CTR1 and FRE1 promoter elements, inducing transcription of those target genes . This model defines the homeostatic mechanism by which yeast regulates the cell acquisition of copper in response to copper scarcity or excess.

J Biol Chem, 1997 Jul 11, 272(28), 17333 - 41
Dynamics of the U1 small nuclear ribonucleoprotein during yeast spliceosome assembly; Ruby SW; U1 small nuclear ribonucleoprotein (snRNP) may function during several steps of spliceosome assembly . Most spliceosome assembly assays, however, fail to detect the U1 snRNP . Here, I used a new native gel electrophoretic assay to find the yeast U1 snRNP in three pre-splicing complexes (delta, beta1, alpha2) formed in vitro . The order of complex formation is deduced to be delta --> beta1 --> alpha2 --> alpha1 --> beta2, the active spliceosome . The delta complex is formed when U1 snRNP binds to pre-mRNA in the absence of ATP . There are two forms of delta: a major one, deltaun, unstable to competitor RNA; and a minor one, deltacommit, committed to the splicing pathway . The other complexes are formed in the presence of ATP and contain the following snRNPs: beta1, the pre-spliceosome, has both U1 and U2; alpha2 has all five, however, U1 is reduced compared with the others; and alpha1 and beta2 have U2, U5, and U6 . Prior work by others suggests that U1 is "handing off" the 5' splice site region to the U5 and U6 snRNPs before splicing begins . The reduced levels of U1 snRNP in the alpha2 complex suggests that the handoff occurs during formation of this complex.

Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7481 - 6
hCTR1: a human gene for copper uptake identified by complementation in yeast; Zhou B et al.; The molecular mechanisms responsible for the cellular uptake of copper in mammalian cells are unknown . We describe isolation of a human gene involved in this process by complementation of the yeast high-affinity copper uptake mutant, ctr1 . Besides complementing ctr1 growth defect on nonfermentable media, the human gene also rescues iron transport and SOD1 defects in ctr1 yeast . Overexpression of the gene in yeast leads to vulnerability to the toxicity of copper overload . In addition, its expression in ctr1 yeast significantly increases the level of cellular copper, as demonstrated by atomic absorption . We propose this gene as a candidate for high-affinity copper uptake in humans and by analogy have named it hCTR1 . The hCTR1 and yeast CTR1 predicted transmembrane proteins are 29% identical, but the human protein is substantially smaller in both the extracellular metal-binding and intracellular domains . An additional human gene similar to hCTR1, here named hCTR2, was identified in a database search . Both hCTR1 and hCTR2 are expressed in all human tissues examined, and both genes are located in 9q31/32 . These studies, together with the previously recognized functional and sequence similarity between the Menkes/Wilson copper export proteins and CCC2 in yeast, demonstrate that similar copper homeostatic mechanisms are used in these evolutionarily divergent organisms.

Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7384 - 7
Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast; Larionov V et al.; Mutant forms of the BRCA2 gene contribute significantly to hereditary breast cancer . Isolation of the normal and mutant forms of the BRCA2 gene with its natural promoter would greatly facilitate analysis of the gene and its contribution to breast cancer . We have accomplished the direct isolation of the 90-kb gene from total human DNA by transformation-associated recombination in yeast using a small amount of 5' and 3' BRCA2 sequence information . Because the entire isolation procedure of a single chromosomal gene could be accomplished in approximately 2 weeks, the transformation-associated recombination cloning approach is readily applicable to studies of chromosome alterations and human genetic diseases.

Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7378 - 83
Substantial narrowing of the Niemann-Pick C candidate interval by yeast artificial chromosome complementation; Gu JZ et al.; Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses . This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification . The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C . To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype . Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7 . Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome . These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man . This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.

Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7331 - 6
Mso1p: a yeast protein that functions in secretion and interacts physically and genetically with Sec1p; Aalto MK et al.; The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane . We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1-1 mutation . We now describe a third suppressor of sec1-1, which we call MSO1 . Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes . MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction . Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired . Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes . We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system . These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.

Proc Natl Acad Sci U S A, 1997 Jul 8, 94(14), 7156 - 61
Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation; Arendt CS et al.; The proteasome is responsible for degradation of substrates of the ubiquitin pathway . 20S proteasomes are cylindrical particles with subunits arranged in a stack of four heptameric rings . The outer rings are composed of alpha subunits, and the inner rings are composed of beta subunits . A well-characterized archaeal proteasome has a single type of each subunit, and the N-terminal threonine of the beta subunit is the active-site nucleophile . Yeast proteasomes have seven different beta subunits and exhibit several distinct peptidase activities, which were proposed to derive from disparate active sites . We show that mutating the N-terminal threonine in the yeast Pup1 beta subunit eliminates cleavage after basic residues in peptide substrates, and mutating the corresponding threonine of Pre3 prevents cleavage after acidic residues . Surprisingly, neither mutation has a strong effect on cell growth, and they have at most minor effects on ubiquitin-dependent proteolysis . We show that Pup1 interacts with Pup3 in each beta subunit ring . Our data reveal that different proteasome active sites contribute very differently to protein breakdown in vivo, that contacts between particular subunits in each beta subunit ring are critical for active-site formation, and that active sites in archaea and different eukaryotes are highly similar.

FEBS Lett, 1997 Jul 7, 411(1), 133 - 9
Brefeldin A interferes with peroxisomal protein sorting in the yeast Hansenula polymorpha; Salomons FA et al.; We have studied the effect of brefeldin A (BFA), a fungal toxin that interferes with coated vesicle formation, on the biogenesis of peroxisomes in the yeast Hansenula polymorpha . Addition of BFA (20 microg/ml) to cultures of H . polymorpha partially inhibited the development of peroxisomes and resulted in the reversible accumulation of newly synthesized peroxisomal membrane and matrix proteins at the endoplasmic reticulum . In contrast, BFA did not interfere with the selective degradation of peroxisomes . Taken together, our data suggest that the ER plays a crucial role in peroxisome biogenesis in H . polymorpha, possibly in the biosynthesis of the peroxisomal membrane.

FEBS Lett, 1997 Jul 7, 411(1), 48 - 52
High expression of the yeast syntaxin-related Vam3 protein suppresses the protein transport defects of a pep12 null mutant; Gotte M et al.; The Pep12 protein of Saccharomyces cerevisiae is a member of the syntaxin family thought to function as target membrane receptor (t-SNARE) for vesicular intermediates travelling between the Golgi apparatus and the vacuole . Exploiting the temperature-sensitive growth phenotype of pep12 deletion strains, we identified VAM3 as a multicopy suppressor . Vam3p is another syntaxin-related protein which on high expression restored vacuole acidification of pep12 null mutants and effectively suppressed their sorting and maturation defects of vacuolar hydrolases . We conclude that Vam3p acts either as a bypass suppressor or by functionally replacing Pep12p at an endosomal, prevacuolar compartment.

J Biol Chem, 1997 Jul 4, 272(27), 16972 - 7
The yeast Rab escort protein binds intracellular membranes in vivo and in vitro; Miaczynska M et al.; In both mammals and yeast, intracellular vesicular transport depends on the correct shuttling between membrane and cytosol of the Rab/Ypt small G proteins . Membrane association of these proteins requires prenylation by the Rab geranylgeranyl transferase that recognizes a complex formed by the Rab/Ypt protein and the Rab escort protein (REP) . After prenylation the Rab/Ypt protein is delivered to the target membranes by REP . Little is known about the early steps of the Rab-REP complex formation and where this association occurs in the cell . Although prenylation is believed to take place in the cytosol, we show that the yeast Rab escort protein Mrs6 is present in both soluble and particulate fractions of cell extracts . Mrs6p is associated with the heavy microsomal fraction that contains endoplasmic reticulum-Golgi membranes but is absent in the plasma membrane, vacuoles, mitochondria, and microsomal subfraction associated with mitochondria . The solubilization pattern of the particulate pool of Mrs6p implies that this protein is peripherally but tightly associated with membranes via hydrophobic interactions and metal ions . We also report that the C terminus of Mrs6p is important for maintaining the solubility of the protein because its deletion or replacement with the C terminus of RabGDI results in a protein that localizes only to membranes.

J Biol Chem, 1997 Jul 4, 272(27), 16829 - 37
Fluorescence probing of yeast actin subdomain 3/4 hydrophobic loop 262-274 . Actin-actin and actin-myosin interactions in actin filaments; Feng L et al.; Residues 262-274 form a loop between subdomains 3 and 4 of actin . This loop may play an important role in actin filament formation and stabilization . To assess directly the behavior of this loop, we mutated Ser265 of yeast actin to cysteine (S265C) and created another mutant (S265C/C374A) by changing Cys374 of S265C actin to alanine . These changes allowed us to attach a pyrene maleimide stoichiometrically to either Cys374 or Cys265 . These mutations had no detectable effects on the protease susceptibility, intrinsic ATPase activity, and thermal stability of labeled or unlabeled G-actin . The presence of the loop cysteine, either labeled or unlabeled, did not affect the actin-activated S1 ATPase activity or the in vitro motility of the actin . Both mutant actins, either labeled or unlabeled, nucleated filament formation considerably faster than wild-type (WT) actin, although the critical concentration was not affected . Whereas the fluorescence of the C-terminal (WT) probe increased during polymerization, that of the loop (S265C/C374A) probe decreased, and the fluorescence of the doubly labeled actin (S265C) was approximately 50% less than the sum of the fluorescence of the individual fluorophores . Quenching was also observed in copolymers of labeled WT and S265C/C374A actins . An excimer peak was present in the emission spectrum of labeled S265C F-actin and in the labeled S265C/C374A-WT actin copolymers . These results show that in the filaments, the C-terminal pyrene of a substantial fraction of monomers directly interacts with the loop pyrene of neighboring monomers, bringing the two cysteine sulfurs to within 18 A of one another . Finally, when bound to labeled S265C/C374A F-actin, myosin S1, but not tropomyosin, caused an increase in fluorescence of the loop probe . Both proteins had no effect on excimer fluorescence . These results help establish the orientation of monomers in F-actin and show that the binding of S1 to actin subdomains 1 and 2 affects the environment of the loop between subdomains 3 and 4.

J Biol Chem, 1997 Jul 4, 272(27), 16753 - 60
Differential inhibition of the yeast bc1 complex by phenanthrolines and ferroin . Implications for structure and catalytic mechanism; Boumans H et al.; o-Phenanthroline and m-phenanthroline both inhibit the electron transfer activity of lauryl maltoside-solubilized yeast bc1 complex progressively with time . Pre-steady-state kinetics indicate that these compounds bind to the complex on the intermembrane space side, thereby blocking reduction of cytochrome b via the ubiquinol oxidation site . o-Phenanthroline is additionally capable of chelating an iron atom derived from the Rieske Fe-S cluster, thereby distorting the structure of the Rieske protein . EPR analysis shows that the secondary effect of o-phenanthroline occurs after initial inactivation and that m-phenanthroline, which lacks chelating activity, does not affect the Rieske Fe-S cluster . Spectral analysis shows that the b and c1 cytochromes are still dithionite-reducible after inactivation by o-phenanthroline, indicating that they remain intact . Inactivation by o-phenanthroline can be prevented by the addition of Fe2+ . Surprisingly, ferroin, the o-phenanthroline-ferrous sulfate complex, also inhibits the bc1 complex activity . In contrast to o-phenanthroline, this effect is instantaneous . The two types of inhibition are clearly distinguishable by pre-steady-state reduction kinetics . Interestingly, ferroin can only inhibit electron transfer activity by about 50% . This behavior is discussed in relation to the dimeric structure of the bc1 complex, and we conclude that ferroin binds to only one of the two protomers . The rate of inactivation by o-phenanthroline is dependent on the incubation temperature and can be quantitated in terms of the half-life for a certain temperature, the time at which the bc1 activity is reduced to 50% . In contrast to the solubilized form, the bc1 complex in intact mitochondria is insensitive to o-phenanthroline, suggesting that the inactivation rate by o-phenanthroline is dependent on accessibility of the complex to the agent . Reaction with o-phenanthroline is thus a useful technique for study of structural stability of the bc1 complex under different conditions and should provide a sensitive tool for determination of the relative stability of mutant enzymes.

J Biol Chem, 1997 Jul 4, 272(27), 16721 - 4
Amino acid residues responsible for galactose recognition in yeast Gal2 transporter; Kasahara M et al.; A novel, systematic approach was used to identify amino acid residues responsible for substrate recognition in the transmembrane 10 region of the Gal2 galactose transporter of Saccharomyces cerevisiae . A mixture of approximately 25,000 distinct plasmids that encode all the combinations of 12 amino acids in transmembrane 10 that are different in Gal2 and the homologous glucose transporter Hxt2 was synthesized . Selection of galactose transport-positive clones on galactose limited agar plates yielded 19 clones, all of which contained the Tyr446 residue found in Gal2 . 14 of the 19 clones contained Trp455 found in Gal2, whereas the other 5 contained Cys455, a residue not found in either Gal2 or Hxt2 . When Tyr446 of Gal2 was replaced with any of the other 19 amino acids, no galactose transport activity was observed in the resulting transporters, indicating that Tyr446 plays an essential role in the transport of this sugar . Replacement of 2 amino acids of Hxt2 with the corresponding Tyr446 and Trp455 of Gal2 allowed the modified Hxt2 to transport galactose . The Km of galactose transport for the modified transporter was 8-fold higher than that of Gal2 . These results and other evidence unequivocally show that Tyr446 is essential and Trp455 is important for the discrimination of galactose versus glucose.

Mol Cell Biochem, 1997 Jul, 172(1-2), 67 - 79
Identification of proteins that interact with a protein of interest: applications of the yeast two-hybrid system; Gietz RD et al.; The yeast two-hybrid system is a molecular genetic test for protein interaction . Here we describe a step by step procedure to screen for proteins that interact with a protein of interest using the two-hybrid system . This process includes, construction and testing of the bait plasmid, screening a plasmid library for interacting fusion proteins, elimination of false positives and deletion analysis of true positives . This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with your protein of interest.

Plant J, 1997 Jul, 12(1), 169 - 78
Yeast 5-aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco; Zavgorodnyaya A et al.; Synthesis of the tetrapyrrole precursor 5-aminolevulinate (ALA) in plants starts with glutamate and is a tRNA-dependent pathway consisting of three enzymatic steps localized in plastids . In animals and yeast, ALA is formed in a single step from succinyl CoA and glycine by aminolevulinate synthase (ALA-S) in mitochondria . A gene encoding a fusion protein of yeast ALA-S with an aminoterminal transit sequence for the small subunit of ribulose bisphosphate carboxylase was introduced into the genome of wild-type tobacco and a chlorophyll-deficient transgenic line expressing glutamate 1-semi-aldehyde aminotransferase (GSA-AT) antisense RNA . Expression of ALA-S in the GSA-AT antisense transgenic line provided green-pigmented co-transformants similar to wild-type in chlorophyll content, while transformants derived from wild-type plants did not show phenotypical changes . The capacity to synthesize ALA and chlorophyll was increased in transformed plants, indicating a contribution of ALA-S to the ALA supply for chlorophyll synthesis . ALA-S activity was detected in plastids of the transformants . Preliminary evidence is presented that succinyl CoA, the substrate for ALA-S, can be synthesized and metabolized in plastids . The transgenic plants formed chlorophyll in the presence of gabaculine, an inhibitor of GSA-AT . Steady-state RNA and protein levels and consequently, the enzyme activity of GSA-AT were reduced in plants expressing ALA-S . In analogy to the light-dependent ALA synthesis attributed to feedback regulation, a mechanism at the level of intermediates or tetrapyrrole end-products is proposed, which co-ordinates the need for heme and chlorophyll precursors and restricts synthesis of ALA by regulating GSA-AT gene expression . The genetically engineered tobacco plants containing the yeast ALA-S activity demonstrate functional complementation of the catalytic activity of the plant ALA-synthesizing pathway and open strategies for producing tolerance against inhibitors of the C5 pathway.

Biol Pharm Bull, 1997 Jul, 20(7), 770 - 5
Molecular cloning of pea cDNA encoding cycloartenol synthase and its functional expression in yeast; Morita M et al.; The cDNA encoding cycloartenol synthase {EC 5.4.99.8} has been isolated from pea seedling by an efficient PCR using sets of degenerate primers based on the highly conserved sequences of the known 2,3-oxidosqualene cyclase cDNAs . The obtained cDNA contains a 2271-bp open reading frame and is encoding a predicted protein of 757 amino acids with high homology (81%) to Arabidopsis thaliana cycloartenol synthase . The PCR-amplified open reading frame (ORF) has been inserted into pYES2, an expression vector in yeast, under the control of galactose-inducible promoter . Significant cycloartenol synthase activity has been found in the homogenate of the yeast transformed with the plasmid containing PCR-amplified ORF.

Int J Biol Macromol, 1997 Jul, 20(4), 307 - 13
Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by 4,4'-dithiodipyridine; Zheng SY et al.; The course of inactivation of yeast alcohol dehydrogenase (YADH) using 4,4'-dithiodipyridine (DSDP) has been studied in this paper . The results show that the reaction mechanism between DSDP and YADH is a competitive, complexing inhibition . The microscopic constants for the inactivation of the free enzyme and the enzyme-substrate complex were determined . The presence of the substrate NAD+ offers strong protection for this enzyme against inactivation by DSDP . The above results suggest that two Cys residues are essential for activity and are situated at the active site . These essential Cys residues should be Cys-46 and Cys-174 which are ligands to the catalytic zinc ion . Another Cys residue, which can be modified by DSDP, is non-essential for activity of the enzyme.

J Cell Sci, 1997 Jul, 110 ( Pt 14), 1615 - 24
NNF1 is an essential yeast gene required for proper spindle orientation, nucleolar and nuclear envelope structure and mRNA export; Shan X et al.; The nuclear envelope is central to nuclear structure and function . It plays a role in maintaining nuclear shape, allowing the exchange of macromolecules between the nucleus and the cytoplasm (via the nuclear pore complexes), and providing attachment sites for microtubules during chromosome segregation and nuclear migration (via the spindle pole body) . We have isolated an essential yeast gene, NNF1 that is required for a number of nuclear functions . Cells depleted of Nnf1p or containing a temperature-sensitive nnf1 mutation have elongated microtubules and become bi- and multinucleate . They also have a fragmented nucleolous and accumulate poly(A)+ RNA inside the nucleus . A similar constellation of phenotypes has been reported in cells carrying mutations in a number of nuclear pore proteins, components of the Ran GTPase cycle, and the nuclear localization sequence receptor protein . Our results suggest that Nnf1p plays a role in a number of nuclear functions.

Mol Biol Cell, 1997 Jul, 8(7), 1361 - 75
The yeast actin-related protein Arp2p is required for the internalization step of endocytosis; Moreau V et al.; The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton . We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1 . The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role in the endocytic pathway . Inhibition of normal endocytosis as revealed by maintenance of active uracil permease at the plasma membrane and strong protection against subsequent vacuolar degradation of the protein were observed in the mutant at the restrictive temperature . Furthermore, arp2-1 cells accumulated ubiquitin-permease conjugates, formed prior to internalization . These effects were also visible at permissive temperature, whereas the actin cytoskeleton appeared to be normally polarized . The soluble hydrolase carboxypeptidase Y and the lipophilic dye FM 4-64 were targeted normally to the vacuole in arp2-1 cells . Thus, Arp2p is required for internalization but does not play a major role in later steps of endocytosis . Synthetic lethality was demonstrated between arp2-1 and the endocytic mutant end3-1, suggesting participation of Arp2p and End3p in the same process . Finally, no evidence for a major defect in secretion was apparent; invertase secretion and delivery of uracil permease to the plasma membrane were unaffected in arp2-1 cells.

Mol Biol Cell, 1997 Jul, 8(7), 1317 - 27
The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors; Givan SA et al.; The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis . Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation . Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis . We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis . One protein identified was Akr1p, an ankyrin repeat-containing protein . We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p . First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells . Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells . Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells . Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface . akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p) . Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p . In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p . We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins).

Eur J Endocrinol, 1997 Jul, 137(1), 1 - 9
Exocytosis in excitable cells: a conserved molecular machinery from yeast to neuron; Lledo PM; One of the basic cellular functions of nearly every cell type is the exocytotic release of synthesized molecules, stored and packaged into intracellular vesicles or granules . A variety of approaches has been used to identify and characterize the molecules that mediate vesicular trafficking along the secretory pathway . The findings obtained with these approaches suggest that common mechanisms may underlie a wide variety of vesicle-mediated transport steps . This review presents some of the recent findings regarding the study of the cellular mechanisms which control neurotransmitter and hormone release from neurons and endocrine cells respectively, and focuses on regulation of these mechanisms . The similarities between these two cell types can be seen as evidence to support the hypothesis according to which the regulated exocytosis apparatus could have evolved from a constitutive fusion machinery to which some key modulators have been added . Insight into secretory vesicles will be relevant not only to the understanding of vesicular trafficking or cell polarity but also to the understanding of higher nervous functions resulting from synaptic plasticity.

Methods, 1997 Jul, 12(3), 212 - 6
Yeast RNA polymerase II transcription reconstituted with purified proteins; Myers LC et al.; Protocols are presented for the preparation of a fully defined yeast RNA polymerase II transcription system, consisting of essentially pure TFIIB, -E, -F, and -H, TATA-binding protein, RNA polymerase II, and mediator of transcriptional regulation . This system, comprising 44 polypeptides, is able to initiate transcription at any of a dozen yeast and mammalian promoters thus far tested and responds to a variety of transcriptional activator proteins.

EMBO J, 1997 Jul 1, 16(13), 4082 - 91
Identification and characterization of a yeast homolog of U1 snRNP-specific protein C; Tang J et al.; U1C is one of the three human U1 small nuclear ribonucleoprotein (snRNP)-specific proteins and is important for efficient complex formation between U1 snRNP and the pre-mRNA 5' splice site . We identified a hypothetical open reading frame in Saccharomyces cerevisiae as the yeast homolog of the human U1C protein . The gene is essential, and its product, YU1C, is associated with U1 snRNP . YU1C depletion gives rise to normal levels of U1 snRNP and does not have any detectable effect on U1 snRNP assembly . YU1C depletion and YU1C ts mutants affect pre-mRNA splicing in vivo, and extracts from these strains form low levels of commitment complexes and spliceosomes in vitro . These experiments indicate a role for YU1C in snRNP function . Structure probing with RNases shows that only the U1 snRNA 5' arm is hypersensitive to RNase I digestion when YU1C is depleted . Similar results were obtained with YU1C ts mutants, indicating that U1C contributes to a proper 5' arm structure prior to its base pairing interaction with the pre-mRNA 5' splice site.

Photochem Photobiol, 1997 Jul, 66(1), 72 - 5
Interaction between cytochrome b2 core and flavodehydrogenase from the yeast Hansenula anomala; Albani JR; The binding of cytochrome b2 core (a monomer) to flavodehydrogenase (a tetramer), both purified from Hansenula anomala flavocytochrome b2, has been studied in the presence of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) . The association constant of the TNS-flavodehydrogenase complex was found to be equal to 0.64 microM-1 with a stoichiometry of one TNS per tetramer . Binding of cytochrome b2 core to flavodehydrogenase was followed by monitoring changes in the TNS fluorescence . Our results indicated that the binding is cooperative, with a stoichiometry of four cytochrome b2 cores per tetramer of flavodehydrogenase.

Genomics, 1997 Jul 1, 43(1), 95 - 8
Yeast artificial chromosome transfer into human renal carcinoma cells by spheroplast fusion; Julicher K et al.; Successful transfer of yeast artificial chromosomes (YACs) into human cells has been described in only a single study . We here report on the evaluation of YAC transfer strategies into a human renal cell carcinoma cell line by yeast spheroplast fusion and cationic lipids . While the latter approach proved inefficient, significant numbers of clones containing both vector arms were obtained by spheroplast fusion . FISH analyses on such clones revealed the presence of YAC integration and the co-localization of both vector arms with insert sequences . These data demonstrate that under certain experimental conditions efficient YAC transfer into human cells by spheroplast fusion is possible and may be useful for the cloning of human disease-related genes.

J Bacteriol, 1997 Jul, 179(14), 4480 - 5
Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, methylamine, and choline; Sakai Y et al.; The structural gene (FDH1) coding for NAD(+)-dependent formate dehydrogenase (FDH) was cloned from a genomic library of Candida boidinii, and the FDH1 gene was disrupted in the C . boidinii genome (fdh1 delta) by one-step gene disruption . In a batch culture experiment, although the fdh1 delta strain was still able to grow on methanol, its growth was greatly inhibited and a toxic level of formate was detected in the medium . In a methanol-limited chemostat culture at a low dilution rate (0.03 to 0.05 h{-1}), formate was not detected in the culture medium of the fdh1 delta strain; however, the fdh1 delta strain showed only one-fourth of the growth yield of the wild-type strain . Expression of FDH1 was found to be induced by choline or methylamine (used as a nitrogen source), as well as by methanol (used as a carbon source) . Induction of FDH1 was not repressed in the presence of glucose when cells were grown on methylamine, choline, or formate, and expression of FDH1 was shown to be regulated at the mRNA level . Growth on methylamine or choline as a nitrogen source in a batch culture was compared between the wild type and the fdh1 delta mutant . Although the growth of the fdh1 delta mutant was impaired and the level of formate was higher in the fdh1 delta mutant than in the wild-type strain, the growth defect caused by FDH1 gene disruption was small and less severe than that caused by growth on methanol . As judged from these results, the main physiological role of FDH with all of the FDH1-inducing growth substrates seems to be detoxification of formate, and during growth on methanol, FDH seems to contribute significantly to the energy yield.

J Cell Sci, 1997 Jul, 110 ( Pt 13), 1523 - 31
The anaphase-promoting complex is required in G1 arrested yeast cells to inhibit B-type cyclin accumulation and to prevent uncontrolled entry into S-phase; Irniger S et al.; Inactivation of B-type cyclin dependent kinases due to ubiquitin-mediated cyclin proteolysis is necessary for the exit from mitosis . In Saccharomyces cerevisiae, proteolysis is initiated at the onset of anaphase and remains active until Cln1 and Cln2 cyclins appear in late G1 of the subsequent cell cycle . A large particle called the anaphase-promoting complex (APC) which is composed of the TPR proteins Cdc16p/Cdc23p/Cdc27p and other proteins is required for B-type cyclin ubiquitination in both anaphase and during G1 phase . The APC has an essential role for the separation of sister chromatids and for the exit from mitosis, but until now it was unclear whether the persistence of APC activity throughout G1 had any physiological role . We show here that the APC is needed in G1 arrested cells to inhibit premature appearance of B-type cyclins and to prevent unscheduled initiation of DNA replication . When pheromone arrested cells of cdc16 and cdc23 mutants were shifted to the restrictive temperature, they underwent DNA replication in the presence of pheromone . DNA replication also occurred in a G1 arrest induced by G1 cyclin (Cln) depletion, indicating that mutant cells with a defective APC initiate DNA replication without the Cln G1 cyclins, which are normally needed for the onset of S-phase . Degradation of Clb2p, Clb3p and Clb5p depends on the APC . This suggests that accumulation of any one of the six B-type cyclin proteins could account for the precocious replication of cdc16 and cdc23 mutants.

Genes Dev, 1997 Jul 1, 11(13), 1640 - 50
Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex; Grant PA et al.; The transcriptional adaptor protein Gcn5 has been identified as a nuclear histone acetyltransferase (HAT) . Although recombinant yeast Gcn5 efficiently acetylates free histones, it fails to acetylate histones contained in nucleosomes, indicating that additional components are required for acetylation of chromosomal histones . We report here that Gcn5 functions as a catalytic subunit in two high-molecular-mass native HAT complexes, with apparent molecular masses of 0.8 and 1.8 megadalton (MD), respectively, which acetylate nucleosomal histones . Both the 0.8- and 1.8-MD Gcn5-containing complexes cofractionate with Ada2 and are lost in gcn5delta, ada2delta, or ada3delta yeast strains, illustrating that these HAT complexes are bona fide native Ada-transcriptional adaptor complexes . Importantly, the 1.8-MD adaptor/HAT complex also contains Spt gene products that are linked to TATA-binding protein (TBP) function . This complex is lost in spt20/ada5delta and spt7delta strains and Spt3, Spt7, Spt20/Ada5, Ada2, and Gcn5 all copurify with this nucleosomal HAT complex . Therefore, the 1.8-MD adaptor/HAT complex illustrates an interaction between Ada and Spt gene products and confirms the existence of a complex containing the TBP group of Spt proteins as demonstrated by genetic and biochemical studies . We have named this novel transcription regulatory complex SAGA (Spt-Ada-Gcn5-Acetyltransferase) . The function of Gcn5 as a histone acetyltransferase within the Ada and SAGA adaptor complexes indicates the importance of histone acetylation during steps in transcription activation mediated by interactions with transcription activators and general transcription factors (i.e., TBP).

Proteins, 1997 Jul, 28(3), 380 - 7
Picosecond dynamical changes on denaturation of yeast phosphoglycerate kinase revealed by quasielastic neutron scattering; Receveur V et al.; Quasielastic neutron scattering experiments performed on yeast phosphoglycerate kinase in the native form and denatured in 1.5 M guanidinium chloride reveal a change in the fast (picosecond time scale) diffusive internal dynamics of the protein . The momentum and energy transfer dependences of the scattering for both states are fitted by an analytical model in which, on the experimentally accessible picosecond time scale and angstrom length scale, the dynamics of a fraction of the nonexchangeable hydrogens in the protein is described as a superposition of vibrations with uniform diffusion in a sphere, the rest of the hydrogens undergoing only vibrational motion . The fraction diffusing changes, from approximately 60% in the native protein to approximately 82% in the denatured protein . The radius of the sphere also changes slightly, from approximately 1.8 A in the native protein to approximately 2.2 A in the denatured protein . Possible implications of these results for the general protein folding problem are discussed.

Genetics, 1997 Jul, 146(3), 769 - 79
Microsatellite instability in yeast: dependence on the length of the microsatellite; Wierdl M et al.; One of the most common microsatellites in eukaryotes consists of tandem arrays {usually 15-50 base pairs (bp) in length} of the dinucleotide GT . We examined the rates of instability for poly GT tracts of 15, 33, 51, 99 and 105 bp in wild-type and mismatch repair-deficient strains of Saccharomyces cerevisiae . Rates of instability increased more than two orders of magnitude as tracts increased in size from 15 to 99 bp in both wild-type and msh2 strains . The types of alterations observed in long and short tracts in wild-type strains were different in two ways . First, tracts > or = 51 bp had significantly more large deletions than tracts < or = 33 bp . Second, for the 99- and 105-bp tracts, almost all events involving single repeats were additions; for the smaller tracts, both additions and deletions of single repeats were common.

Antimicrob Agents Chemother, 1997 Jul, 41(7), 1537 - 40
Evaluation of a flow cytofluorometric method for rapid determination of amphotericin B susceptibility of yeast isolates; Peyron F et al.; A rapid-flow cytofluorometric susceptibility test for in vitro amphotericin B testing of yeasts was evaluated and compared to the National Committee for Clinical Laboratory Standards (NCCLS) M27-T reference broth macrodilution method . The flow cytofluorometric method is based on the detection of decreased green fluorescence intensity of cells stained with DiOC5(3), a membrane potential-sensitive cationic dye, after drug treatment . Testing was performed on 134 clinical isolates (Candida spp . and Torulopsis glabrata) . From the dose-response curve obtained for each isolate, three endpoints were calculated by computer analysis (the concentrations at which the fluorescence intensity was reduced by 50, 80, and 90%, i.e., 50% inhibitory concentration {IC50}, IC80, and IC90, respectively) . A regression analysis correlating these endpoints with the M27-T MICs showed that the best agreement was obtained with IC80 . The flow cytofluorometric method showed good reproducibility with control strains . These initial results suggest that the flow cytofluorometric method is a valid alternative to the NCCLS reference method.

Mol Cell Biol, 1997 Jul, 17(7), 4124 - 32
The rRNA-processing function of the yeast U14 small nucleolar RNA can be rescued by a conserved RNA helicase-like protein; Liang WQ et al.; The phylogenetically conserved U14 small nucleolar RNA is required for processing of rRNA, and this function involves base pairing with conserved complementary sequences in 18S RNA . With a view to identifying other important U14 interactions, a stem-loop domain required for activity of Saccharomyces cerevisiae U14 RNAs (the Y domain) was first subjected to detailed mutational analysis . The mapping results showed that most nucleotides of the Y domain can be replaced without affecting function, except for loop nucleotides conserved among five different yeast species . Defective variants were then used to identify both intragenic and extragenic suppressor mutations . All of the intragenic mutations mapped within six nucleotides of the primary mutation, suggesting that suppression involves a change in conformation and that the loop element is involved in an essential intermolecular interaction rather than intramolecular base pairing . A high-copy extragenic suppressor gene, designated DBP4 (DEAD box protein 4), encodes an essential, putative RNA helicase of the DEAD-DEXH box family . Suppression by DBP4 (initially CA4 {T.-H . Chang, J . Arenas, and J . Abelson, Proc . Natl . Acad . Sci . USA 87:1571-1575, 1990}) restores the level of 18S rRNA and is specific for the Y domain but is not allele specific . DBP4 is predicted to function either in assembly of the U14 small nucleolar RNP or, more likely, in its interaction with other components of the rRNA processing apparatus . Mediating the interaction of U14 with precursor 18S RNA is an especially attractive possibility.

Mol Cell Biol, 1997 Jul, 17(7), 4033 - 42
Small heat shock protein suppression of Vpr-induced cytoskeletal defects in budding yeast; Gu J et al.; Expression of the auxiliary human immunodeficiency virus type 1 (HIV-1) protein Vpr causes arrest of primate host cells in G2 . Expression of this protein in budding yeast has been previously reported to cause growth arrest and a large-cell phenotype . Investigation of the effect of Vpr expression in budding yeast, reported here, showed that it causes disruption of the actin cytoskeleton . Expression of HSP42, the gene for a small heat shock protein (sHSP), from a high-copy-number plasmid reversed this effect . The sHSPs are induced by exposure of cells to thermal, osmotic, and oxidative stresses and to mitogens . In animal cells, overexpression of sHSPs causes increased resistance to stress and stabilization of actin stress fibers . Yeast cells subjected to mild stress, such as shifting from 23 to 39 degrees C, arrest growth and then resume cell division . Growth arrest is accompanied by transient disorganization of the cytoskeleton . Yeast in which the HSP42 gene was disrupted and which was subjected to moderate thermal stress reorganized the actin cytoskeleton more slowly than did wild-type control cells . These results demonstrate that in yeast, as in metazoan cells, sHSPs promote maintenance of the actin cytoskeleton.

Mol Cell Biol, 1997 Jul, 17(7), 4024 - 32
Analysis of the sorting signals directing NADH-cytochrome b5 reductase to two locations within yeast mitochondria; Haucke V et al.; Mitochondrial NADH-cytochrome b5 reductase (Mcr1p) is encoded by a single nuclear gene and imported into two different submitochondrial compartments: the outer membrane and the intermembrane space . We now show that the amino-terminal 47 amino acids suffice to target the Mcr1 protein to both destinations . The first 12 residues of this sequence function as a weak matrix-targeting signal; the remaining residues are mostly hydrophobic and serve as an intramitochondrial sorting signal for the outer membrane and the intermembrane space . A double point mutation within the hydrophobic region of the targeting sequence virtually abolishes the ability of the precursor to be inserted into the outer membrane but increases the efficiency of transport into the intermembrane space . Import of Mcr1p into the intermembrane space requires an electrochemical potential across the inner membrane, as well as ATP in the matrix, and is strongly impaired in mitochondria lacking Tom7p or Tim11p, two components of the translocation machineries in the outer and inner mitochondrial membranes, respectively . These results indicate that intramitochondrial sorting of the Mcr1 protein is mediated by specific interactions between the bipartite targeting sequence and components of both mitochondrial translocation systems.

Mol Cell Biol, 1997 Jul, 17(7), 3694 - 701
Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro; Amrani N et al.; In Saccharomyces cerevisiae, the single poly(A) binding protein, Pab1, is the major ribonucleoprotein associated with the poly(A) tails of mRNAs in both the nucleus and the cytoplasm . We found that Pab1 interacts with Rna15 in two-hybrid assays and in coimmunoprecipitation experiments . Overexpression of PAB1 partially but specifically suppressed the rna15-2 mutation in vivo . RNA15 codes for a component of the cleavage and polyadenylation factor CF I, one of the four factors needed for pre-mRNA 3'-end processing . We show that Pab1 and CF I copurify in anion-exchange chromatography . These data suggest that Pab1 is physically associated with CF I . Extracts from a thermosensitive pab1 mutant and from a wild-type strain immunoneutralized for Pab1 showed normal cleavage activity but a large increase in poly(A) tail length . A normal tail length was restored by adding recombinant Pab1 to the mutant extract . The longer poly(A) tails were not due to an inhibition of exonuclease activities . Pab1 has previously been implicated in the regulation of translation initiation and in cytoplasmic mRNA stability . Our data indicate that Pab1 is also a part of the 3'-end RNA-processing complex and thus participates in the control of the poly(A) tail lengths during the polyadenylation reaction.

Mol Cell Biol, 1997 Jul, 17(7), 3640 - 8
Met31p and Met32p, two related zinc finger proteins, are involved in transcriptional regulation of yeast sulfur amino acid metabolism; Blaiseau PL et al.; Sulfur amino acid metabolism in Saccharomyces cerevisiae is regulated by the level of intracellular S-adenosylmethionine (AdoMet) . Two cis-acting elements have been previously identified within the 5' upstream regions of the structural genes of the sulfur network . The first contains the CACGTG motif and is the target of the transcription activation complex Cbflp-Met4p-Met28p . We report here the identification of two new factors, Met31p and Met32p, that recognize the second cis-acting element . Met31p was isolated through the use of the one-hybrid method, while Met32p was identified during the analysis of the yeast methionine transport system . Met31p and Met32p are highly related zinc finger-containing proteins . Both LexA-Met31p and LexA-Met32p fusion proteins activate the transcription of a LexAop-containing promoter in a Met4p-dependent manner . Northern blot analyses of cells that do not express either Met31p and/or Met32p suggest that the function of the two proteins during the transcriptional regulation of the sulfur network varies from one gene to the other . While the expression of both the MET3 and MET14 genes was shown to strictly depend upon the presence of either Met31p or Met32p, the transcription of the MET25 gene is constitutive in cells lacking both Met31p and Met32p . These results therefore emphasise the diversity of the mechanisms allowing regulation of the expression of the methionine biosynthetic genes.

Mol Cell Biol, 1997 Jul, 17(7), 3520 - 6
Topoisomerase function during replication-independent chromatin assembly in yeast; Garinther WI et al.; DNA topoisomerases I and II are the two major nuclear enzymes capable of relieving torsional strain in DNA . Of these enzymes, topoisomerase I plays the dominant role in relieving torsional strain during chromatin assembly in cell extracts from oocytes, eggs, and early embryos . We tested if the topoisomerases are used differentially during chromatin assembly in Saccharomyces cerevisiae by a combined biochemical and pharmacological approach . As measured by plasmid supercoiling, nucleosome deposition is severely impaired in assembly extracts from a yeast mutant with no topoisomerase I and a temperature-sensitive form of topoisomerase II (strain top1-top2) . Expression of wild-type topoisomerase II in strain top1-top2 fully restored assembly-driven supercoiling, and assembly was equally efficient in extracts from strains expressing either topoisomerase I or II alone . Supercoiling in top1-top2 extract was rescued by adding back either purified topoisomerase I or II . Using the topoisomerase II poison VP-16, we show that topoisomerase II activity during chromatin assembly is the same in the presence and absence of topoisomerase I . We conclude that both topoisomerases I and II can provide the DNA relaxation activity required for efficient chromatin assembly in mitotically cycling yeast cells.

FEBS Lett, 1997 Jun 30, 410(2-3), 213 - 8
Sequence and characterization of two Arabidopsis thaliana cDNAs isolated by functional complementation of a yeast gln3 gdh1 mutant; Truong HN et al.; We have isolated two Arabidopsis thaliana cDNAs by complementation of a yeast gln3 gdh1 strain that is affected in the regulation of nitrogen metabolism . The two clones (RGA1 and RGA2) are homologous to each other and to the SCARECROW (SCR) gene that is involved in regulating an asymmetric cell division in plants . RGA1, RGA2 and SCR share several structural features and may define a new family of genes . RGA1 and RGA2 have been mapped, respectively, to chromosome II and I, and their expression in plant is constitutive.

Yeast, 1997 Jun 30, 13(8), 747 - 61
Cloning of the multicopy suppressor gene SUR7: evidence for a functional relationship between the yeast actin-binding protein Rvs167 and a putative membranous protein; Sivadon P et al.; The rvs161 and rvs167 mutant cells exhibit several identical phenotypes including sensitivity to several different growth conditions and morphological defects such as alteration of the actin cytoskeleton and budding patterns . The selection of genes that, when overexpressed, are able to suppress the reduced viability upon carbon starvation of the rvs167 mutant strain, has allowed the cloning of the SUR7 gene (Accession Number Z46729x11) . We showed that the suppressive ability of the overexpressed SUR7 gene concerns all the rvs167 phenotypes . However, this suppression is only partial since the rvs167-suppressed strain is not of wild-type phenotype . Moreover, SUR7 is also able to suppress partially the phenotypes exhibited by the rvs161 and rvs167 and rvs161 mutant strains . The SUR7 gene encodes a putative integral membrane protein with four transmembrane domains . Furthermore, sequence comparisons revealed that Sur7p and two other proteins, Yn1194p and Yd1222p, present significant sequence and structural similarities . Taken together, these results strongly suggest that the Rvs161 and Rvs167 proteins act together in relation with Sur7p . Moreover, the putative transmembranous character of Sur7p suggests a membrane localization of the Rvs function, a localization which is consistent with the different rvs phenotypes and the actin-Rvs167p interaction.

Yeast, 1997 Jun 30, 13(8), 691 - 7
BTN1, a yeast gene corresponding to the human gene responsible for Batten's disease, is not essential for viability, mitochondrial function, or degradation of mitochondrial ATP synthase; Pearce DA et al.; The Saccharomyces cerevisiae gene BTN1, encodes a 408 amino acid putative integral membrane protein, which is 39% identical and 59% similar to the human Cln3p, whose mutant forms are responsible for Batten's disease and for a diminished degradation of mitochondrial ATPase synthase subunit c . Disruption experiments established that Btn1p is not essential for viability, mitochondrial function, or degradation of mitochondrial ATP synthase in yeast.

J Cell Biol, 1997 Jun 30, 137(7), 1581 - 8
Human Bcl-2 reverses survival defects in yeast lacking superoxide dismutase and delays death of wild-type yeast; Longo VD et al.; We expressed the human anti-apoptotic protein, Bcl-2, in Saccharomyces cerevisiae to investigate its effects on antioxidant protection and stationary phase survival . Yeast lacking copper-zinc superoxide dismutase (sod1Delta) show a profound defect in entry into and survival during stationary phase even under conditions optimal for survival of wild-type strains (incubation in water after stationary phase is reached) . Expression of Bcl-2 in the sod1Delta strain caused a large improvement in viability at entry into stationary phase, as well as increased resistance to 100% oxygen and increased catalase activity . In addition, Bcl-2 expression reduced mutation frequency in both wild-type and sod1Delta strains . In another set of experiments, wild-type yeast incubated in expired minimal medium instead of water lost viability quickly; expression of Bcl-2 significantly delayed this stationary phase death . Our results demonstrate that Bcl-2 has activities in yeast that are similar to activities it is known to possess in mammalian cells: (a) stimulation of antioxidant protection and (b) delay of processes leading to cell death.

J Cell Biol, 1997 Jun 30, 137(7), 1511 - 24
The yeast v-SNARE Vti1p mediates two vesicle transport pathways through interactions with the t-SNAREs Sed5p and Pep12p; von Mollard GF et al.; Membrane traffic in eukaryotic cells requires that specific v-SNAREs on transport vesicles interact with specific t-SNAREs on target membranes . We identified a novel Saccharomyces cerevisiae v-SNARE (Vti1p) encoded by the essential gene, VTI1 . Vti1p interacts with the prevacuolar t-SNARE Pep12p to direct Golgi to prevacuolar traffic . vti1-1 mutant cells missorted and secreted the soluble vacuolar hydrolase carboxypeptidase Y (CPY) rapidly and reversibly when vti1-1 cells were shifted to the restrictive temperature . However, overexpression of Pep12p suppressed the CPY secretion defect exhibited by vti1-1 cells at 36 degrees C . Characterization of a second vti1 mutant, vti1-11, revealed that Vti1p also plays a role in membrane traffic at a cis-Golgi stage . vti1-11 mutant cells displayed a growth defect and accumulated the ER and early Golgi forms of both CPY and the secreted protein invertase at the nonpermissive temperature . Overexpression of the yeast cis-Golgi t-SNARE Sed5p suppressed the accumulation of the ER form of CPY but did not lead to CPY transport to the vacuole in vti1-11 cells . Overexpression of Sed5p allowed growth in the absence of Vti1p . In vitro binding and coimmunoprecipitation studies revealed that Vti1p interacts directly with the two t-SNAREs, Sed5p and Pep12p . These data suggest that Vti1p plays a role in cis-Golgi membrane traffic, which is essential for yeast viability, and a nonessential role in the fusion of Golgi-derived vesicles with the prevacuolar compartment . Therefore, a single v-SNARE can interact functionally with two different t-SNAREs in directing membrane traffic in yeast.

Cell, 1997 Jun 27, 89(7), 1077 - 86
The yeast spindle pole body is assembled around a central crystal of Spc42p; Bullitt E et al.; The spindle pole body (SPB) is the microtubule organizing center (MTOC) in the yeast Saccharomyces that plays a pivotal role in such diverse processes as mitosis, budding, and mating . We have used cryoelectron microscopy and image processing to study the structure of isolated diploid SPBs . We show that SPBs are present in two lateral-size classes, sharing a similar vertical architecture comprised of six major layers . Tomographic reconstructions of heparin-stripped SPBs reveal a central hexagonally packed layer . Overexpression of Spc42p results in the growth of a similar layer, forming a crystal that encircles the SPB . Hence, the SPB is an MTOC that utilizes crystallographic packing of subunits in its construction.

J Biol Chem, 1997 Jun 27, 272(26), 16618 - 23
Reconstitution in vitro of the V1 complex from the yeast vacuolar proton-translocating ATPase . Assembly recapitulates mechanism; Tomashek JJ et al.; Oligomeric assembly is a fundamental aspect of many complex enzymes . Using our native gel technique for examining subcomplexes of the V-ATPase V1 sector, we have developed an in vitro reconstitution assay for assembly of this complex . Assembly of complex II, the soluble V1 complex observed in native gels, is dependent upon the presence of divalent cations and physiological temperatures . Assembly of soluble V1 can occur in a stepwise fashion from smaller subcomplexes found in some strains deleted for V-ATPase subunits . Specifically, V1 can be assembled directly from complex III (subunits E and G) with complex IV (subunits A, B, D, and F) without prior disassembly of complex IV . The formation of complex III in vivo is also shown to be essential and could not be achieved in vitro . Assembly from simpler precursors is possible and is enhanced by added ATP . Assembly can be blocked by N-ethylmaleimide in a Vma1p (subunit A)-specific manner . From these data, we extend our previous model to consider an assembly pathway whose steps reflect the catalytic mechanism of the Boyer binding-change model.

J Biol Chem, 1997 Jun 27, 272(26), 16610 - 7
Analysis of a yeast SNARE complex reveals remarkable similarity to the neuronal SNARE complex and a novel function for the C terminus of the SNAP-25 homolog, Sec9; Rossi G et al.; SNARE proteins represent a family of related proteins that are thought to have a central role in vesicle targeting and fusion in all eukaryotic cells . The binding properties of the neuronal proteins synaptobrevin 1 (VAMP1), syntaxin 1, SNAP-25, and soluble N-ethylmaleimide-sensitive factor attachment protein (alpha-SNAP), have been extensively studied . We report here the first biochemical characterization of a nonneuronal SNARE complex using recombinant forms of the yeast exocytic SNARE proteins Snc1, Sso1, and Sec9 and the yeast alpha-SNAP homolog, Sec17 . Despite the low level of sequence identity, the association properties of the yeast and neuronal complexes are remarkably similar . The most striking difference we have found between the yeast and neuronal proteins is that individually neither of the target membrane SNAREs (t-SNAREs), Sso1 nor Sec9, show any detectable binding to the synaptobrevin homolog, Snc1 . However, as a hetero-oligomeric complex, Sec9 and Sso1 show strong binding to Snc1 . The clear dependence on the Sso1-Sec9 complex for t-SNARE function suggests that regulating the formation of this complex may be a key step in determining the site of vesicle fusion . In addition, we have used this in vitro assay to examine the biochemical effects of several mutations in Sec9 that result in pronounced growth defects in vivo . As expected, a temperature-sensitive mutation in the region most highly conserved between Sec9 and SNAP-25 is severely diminished in its ability to bind Sso1 and Snc1 in vitro . In contrast, a temperature-sensitive mutation near the C terminus of Sec9 shows no defect in SNARE binding in vitro . Similarly, a deletion of the C-terminal 17 residues, which is lethal in vivo, also binds Sso1 and Snc1 normally in vitro . Interestingly, we find that these same two C-terminal mutants, but not mutants that show SNARE assembly defects in vitro, act as potent dominant negative alleles when expressed behind a strong regulated promoter . Taken together these results suggest that the C-terminal domain of Sec9 is specifically required for a novel interaction that is required at a step following SNARE assembly.

J Biol Chem, 1997 Jun 27, 272(26), 16591 - 8
Conserved alpha-helical segments on yeast homologs of the synaptobrevin/VAMP family of v-SNAREs mediate exocytic function; Gerst JE; We are studying yeast homologs of the synaptobrevin/VAMP family of vesicle-associated membrane proteins, which act as vesicular compartment-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs) in cells having a capacity for stimulus-coupled secretion, as well as in other cell types . The yeast homologs, Snc1 and Snc2, localize to secretory vesicles and are required for normal bulk secretion in Saccharomyces cerevisiae . Here we have used Snc deletion mutants and chimeric Snc-VAMP proteins to demonstrate that these v-SNAREs can be dissected into regions that are either indispensable or dispensable for exocytic function in vivo . We have found that a region encompassing two predicted amphipathic alpha-helices (helix 1 and helix 2) (residues 32-85), which are thought to form coiled-coil structures, is essential for conferring exocytosis in yeast . Deletions in either the helix 1 or helix 2 segments result in a complete loss in the ability of the protein to confer secretion competence to snc cells and to interact genetically with components of the proposed fusion complex: the Sec9 and Sso2 t-SNAREs and the Sec17 alpha-SNAP homolog . In contrast, deletions in either the variable (residues 2-27) or putative intravesicular (residues 115-117) regions have no deleterious effect upon v-SNARE function . This makes it unlikely that sequences in either the amino or carboxyl terminus act in an exocytic capacity . Along with additional studies utilizing chimeric Snc-VAMP proteins, we suggest that although the Snc and synaptobrevin/VAMP proteins have evolved to mediate vastly different exocytic programs, their structural requirements and actions have remained remarkably well-conserved in evolution.

Proc Natl Acad Sci U S A, 1997 Jun 24, 94(13), 7082 - 7
Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein; Yun DJ et al.; Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family . However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plant Atriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell . A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain . We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of all PIR genes in a tolerant strain resulted in sensitivity . Pir proteins have been immunolocalized to the cell wall . The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action . Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins.

Proc Natl Acad Sci U S A, 1997 Jun 24, 94(13), 6618 - 22
Prion-inducing domain 2-114 of yeast Sup35 protein transforms in vitro into amyloid-like filaments; King CY et al.; The yeast non-Mendelian genetic factor {PSI}, which enhances the efficiency of tRNA-mediated nonsense suppression in Saccharomyces cerevisiae, is thought to be an abnormal cellular isoform of the Sup35 protein . Genetic studies have established that the N-terminal part of the Sup35 protein is sufficient for the genesis as well as the maintenance of {PSI} . Here we demonstrate that the N-terminal polypeptide fragment consisting of residues 2-114 of Sup35p, Sup35pN, spontaneously aggregates to form thin filaments in vitro . The filaments show a beta-sheet-type circular dichroism spectrum, exhibit increased protease resistance, and show amyloid-like optical properties . It is further shown that filament growth in freshly prepared Sup35pN solutions can be induced by seeding with a dilute suspension of preformed filaments . These results suggest that the abnormal cellular isoform of Sup35p is an amyloid-like aggregate and further indicate that seeding might be responsible for the maintenance of the {PSI} element in vivo.

Biochim Biophys Acta, 1997 Jun 20, 1340(1), 88 - 96
Effect of site-directed mutagenesis of His373 of yeast enolase on some of its physical and enzymatic properties; Brewer JM et al.; The X-ray structure of yeast enolase shows His373 interacting with a water molecule also held by residues Glu168 and Glu211 . The water molecule is suggested to participate in the catalytic mechanism (Lebioda, L . and Stec, B . (1991) Biochemistry 30, 2817-2822) . Replacement of His373 with asparagine (H373N enolase) or phenylalanine (H373F enolase) reduces enzymatic activity to ca . 10% and 0.0003% of the native enzyme activity, respectively . H373N enolase exhibits a reduced Km for the substrate, 2-phosphoglycerate, and produces the same absorbance changes in the chromophoric substrate analogues TSP1 and AEP1, relative to native enolase . H373F enolase binds AEP less strongly, producing a smaller absorbance change than native enolase, and reacts very little with TSP . H373F enolase dissociates to monomers in the absence of substrate; H373N enolase subunit dissociation is less than H373F enolase but more than native enolase . Substrate and Mg2+ increase subunit association in both mutants . Differential scanning calorimetric experiments indicate that the interaction with substrate that stabilizes enolase to thermal denaturation involves His373 . We suggest that the function of His373 in the enolase reaction may involve hydrogen bonding rather than acid/base catalysis, through interaction with the Glu168/Glu211/H2O system, which produces removal or addition of hydroxyl at carbon-3 of the substrate.

J Biol Chem, 1997 Jun 20, 272(25), 15951 - 8
Copper-specific transcriptional repression of yeast genes encoding critical components in the copper transport pathway; Labbe S et al.; Copper is an essential micronutrient that is toxic in excess . To maintain an adequate yet non-toxic concentration of copper, cells possess several modes of control . One involves copper uptake mediated by genes encoding proteins that play key roles in high affinity copper transport . These include the FRE1-encoded Cu2+/Fe3+ reductase and the CTR1 and CTR3-encoded membrane-associated copper transport proteins . Each of these genes is transcriptionally regulated as a function of copper availability: repressed when cells are grown in the presence of copper and highly activated during copper starvation . Our data demonstrate that repression of CTR3 transcription is exquisitely copper-sensitive and specific . Although copper represses CTR3 gene expression at picomolar metal concentrations, cadmium and mercury down-regulate CTR3 expression only at concentrations 3 orders magnitude greater . Furthermore, copper-starvation rapidly and potently induces CTR3 gene expression . We demonstrate that the CTR1, CTR3, and FRE1 genes involved in high affinity copper uptake share a common promoter element, TTTGCTC, which is necessary for both copper repression and copper-starvation activation of gene expression . Furthermore, the Mac1p is essential for down- or up-regulation of the copper-transport genes . In vivo footprinting studies reveal that the cis-acting element, termed CuRE (copper-response element), is occupied under copper-starvation and accessible to DNA modifying agents in response to copper repression, and that this regulated occupancy requires a functional MAC1 gene . Therefore, yeast cells coordinately express genes involved in high affinity copper transport through the action of a common signaling pathway.

Biochem Biophys Res Commun, 1997 Jun 18, 235(2), 377 - 82
Photoaffinity labeling of yeast farnesyl protein transferase and enzymatic synthesis of a Ras protein incorporating a photoactive isoprenoid; Edelstein RL et al.; Farnesyl protein transferase (FPTase) catalyzes the covalent attachment of a farnesyl (C15) group from farnesyl pyrophosphate (FPP) to a specific cysteine residue of Ras and several other proteins . In this report, photoactive farnesyl and geranylgeranyl pyrophosphate analogs 2-diazo-3,3,3-trifluoropropionyloxy-geranyl pyrophosphate (DATFP-GPP) and 2-diazo-3,3,3-trifluoropropionyloxy-farnesyl pyrophosphate (DATFP-FPP) were used to study the active site of Saccharomyces cerevisiae FPTase . Both analogs are substrates for the enzyme, and upon irradiation, DATFP-GPP inhibits FPTase activity in a time-dependent manner . Photoinactivation by DATFP-GPP is prevented by the presence of the natural substrate FPP . Photolysis of radiolabeled DATFP-GPP results in preferential labeling of the beta subunit of FPTase, suggesting that this subunit is involved in recognition of FPP . Of particular importance, DATFP-GPP and DATFP-FPP were used to enzymatically transfer the photoactive isoprenoid moieties to peptides and to Ras; such molecules should be useful for identifying cellular components which specifically recognize farnesylated Ras and other prenylated proteins.

Biochem Biophys Res Commun, 1997 Jun 18, 235(2), 336 - 42
Synergistic responses of steroidal estrogens in vitro (yeast) and in vivo (turtles); Arnold SF et al.; Many environmental agents exert estrogenic activity . Previous studies from our laboratories demonstrated that certain combinations of environmental estrogens (i) reverse the sex of male turtle embryos in a synergistic manner (Bergeron et al., (1994) Environ . Hlth Perspect . 102, 780-782), and (ii) synergistically transactivate the human estrogen receptor (hER) in yeast and mammalian cells (Arnold et al., (1996) Science 272, 1489-1492) . Because our findings with synthetic estrogens suggested that combinations of naturally-occurring steroidal estrogens might also produce synergistic activity of the ER, we used the same model systems to measure the activity of combinations of steroidal estrogens . The activity of combinations of estrone, estradiol-17beta or estradiol-17alpha in yeast strains expressing hER was synergistic at submaximal concentrations of both estrogenic compounds . However, synergy was not observed with mixtures of estrogens when the concentration of one of the estrogens alone was maximally active in yeast . Ligand-binding assays in yeast performed with various radiolabeled estrogens suggested that multiple estrogens may interact with the receptor . The estrogen-dependent process of sex-reversal of turtle embryos incubated at a male-producing temperature was used to determine whether steroidal estrogens also had synergistic activity in vivo . In this instance, a combination of estriol and estradiol-17beta was effective in reversing the gonadal sex of turtle embryos from males to females in a synergistic manner . Our results suggest that the synergy of some combinations of estrogens, synthetic or steroidal, may play a role in the estrogen-dependent process of sexual development in certain species.

FEMS Microbiol Lett, 1997 Jun 15, 151(2), 191 - 6
Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila; Deegenaars ML et al.; Conditions are described for the heat shock acquisition of thermotolerance, peroxide tolerance and synthesis of heat shock proteins (hsps) in the Antarctic, psychrophilic yeast Candida psychrophila . Cells grown at 15 degrees C and heat shocked at 25 degrees C (3 h) acquired tolerance to heat (35 degrees C) and hydrogen peroxide (100 mM) . Novel heat shock inducible proteins at 80 and 110 kDa were observed as well as the presence of hsp 90, 70 and 60 . The latter hsps were not significantly heat shock inducible . The absence of hsp 104 was intriguing and it was speculated that the 110 kDa protein may play a role in stress tolerance in psychrophilic yeasts, similar to that of hsp 104 in mesophilic species.

Genomics, 1997 Jun 15, 42(3), 467 - 73
Construction and characterization of a Plasmodium vivax genomic library in yeast artificial chromosomes; Camargo AA et al.; Here we describe the construction of a representative YAC library for the human malarial parasite Plasmodium vivax . As P . vivax cannot be maintained continuously under laboratory conditions, the P . vivax DNA necessary for the library construction was isolated from a single human patient presenting himself with vivax malaria to a local hospital in the Brazilian Amazon . Thus, this YAC library is the first of its kind to be generated from patient-derived material . The YAC library consists of 560 clones with an average insert size of 180 kb . Of 9 published P . vivax genes, 8 were found to be present in the library . In addition, 12 P . vivax telomeric YAC clones were identified.

Genes Dev, 1997 Jun 15, 11(12), 1548 - 60
Fission yeast WD-repeat protein pop1 regulates genome ploidy through ubiquitin-proteasome-mediated degradation of the CDK inhibitor Rum1 and the S-phase initiator Cdc18; Kominami K et al.; In fission yeast, maintenance of genome ploidy is controlled by at least two mechanisms . One operates through the Cdc2/Cdc13 kinase, which also involves the CDK inhibitor Rum1, and the other through the S-phase regulator Cdc18 . By screening for sterile mutants that show increased ploidy, we have identified a new gene, pop1+, in mutants that become polyploid . The pop1 mutation shows a synthetic lethal interaction with the temperature-sensitive cdc2 or cdc13 mutation . In a pop1 mutant Rum1 and Cdc18 proteins become accumulated to high levels . The high ploidy phenotype in the pop1 mutant is dependent on the presence of the rum1+ gene, whereas the accumulation of Cdc18 is independent of Rum1 . The predicted sequence of the Pop1 protein indicates that it belongs to a WD-repeat family with highest homology to budding yeast Cdc4, which participates in the ubiquitin-dependent pathway . Consistent with this notion, in a mutant of the 26S proteasome, higher molecular weight forms of Rum1 and Cdc18 are accumulated corresponding to polyubiquitination of these proteins . In the pop1 mutant, however, no ubiquitinated forms of these proteins are detected . Finally we show that Pop1 binds Cdc18 in vivo . We propose that Pop1 functions as a recognition factor for Rum1 and Cdc18, which are subsequently ubiquitinated and targeted to the 26S proteasome for degradation.

Genes Dev, 1997 Jun 15, 11(12), 1504 - 18
ORC-dependent and origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei; Pasero P et al.; We describe an in vitro replication assay from yeast in which the addition of intact nuclei to an S-phase nuclear extract results in the incorporation of deoxynucleotides into genomic DNA at spatially discrete foci . When BrdUTP is substituted for dTTP, part of the newly synthesized DNA shifts to a density on CsCl gradients, indicative of semiconservative replication . Initiation occurs in an origin-specific manner and can be detected in G1- or S-phase nuclei, but not in G2-phase or mitotic nuclei . The S-phase extract contains a heat- and 6-DMAP-sensitive component necessary to promote replication in G1-phase nuclei . Replication of nuclear DNA is blocked at the restrictive temperature in an orc2-1 mutant, and the inactive Orc2p cannot be complemented in trans by an extract containing wild-type ORC . The initiation of DNA replication in cln-deficient nuclei blocked in G1 indicates that the ORC-dependent prereplication complex is formed before Start . This represents the first nonviral and nonembryonic replication system in which DNA replication initiates in an ORC-dependent and origin-specific manner in vitro.

Yeast, 1997 Jun 15, 13(7), 647 - 53
'Marker swap' plasmids: convenient tools for budding yeast molecular genetics; Cross FR; One-step gene disruption constructs for disruption of HIS3, LEU2, TRP1 or URA3 with each of the other three markers have been constructed . All of these constructs have been tested and found to effectively convert markers either in gene disruptions or on plasmids . The 'swapped' strains allow the unambiguous genetic analysis of synthetic phenotypes with multiple genes, even if the original gene disruptions were made with the same marker . They also allow introduction of multiple plasmids in a single transformant, even if the original plasmids had the same marker, and allow transformation of plasmids into strains containing gene disruptions made with the same marker that is on the plasmids . These 'marker-swap' plasmids therefore eliminate the need for much subcloning to change markers . Marker-swapped alleles are acceptably stable mitotically and meiotically for most applications.

Yeast, 1997 Jun 15, 13(7), 639 - 45
Sequence comparison of the Ty1 and Ty2 elements of the yeast genome supports the structural model of the tRNAiMet-Ty1 RNA reverse transcription initiation complex; Friant S et al.; In the reverse transcription initiation complex of the yeast Ty1 retrotransposon, interaction between the template RNA and primer tRNAiMet is not limited to base pairing of the primer binding site (PBS) with ten nucleotides at the 3' end of tRNAiMet, but three regions named boxes O, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet . Sequence comparison of 33 Ty1 elements and 13 closely related Ty2 elements found in the yeast genome shows that the nucleotide sequence of all elements is highly conserved in the region spanning the PBS and the three boxes . Since the domain of the template RNA encodes a portion of protein TyA, we have calculated its amino acid profile and its nucleotide profile to evaluate the role played by nucleotide sequence conservation in the selection for TyA function and in the maintenance of base pairing interactions for the priming function of Ty1 RNA . Our results show that the nucleotide sequence conservation of Ty1 RNA is constrained not only by selection for Ty1 function but also by maintenance of a given nucleotide sequence able to base pair with the tRNAiMet in the primer-template initiation complex.

Nucleic Acids Res, 1997 Jun 15, 25(12), 2464 - 9
Variations of the C2H2 zinc finger motif in the yeast genome and classification of yeast zinc finger proteins; Bohm S et al.; The PROSITE pattern Zinc_Finger_C2H2 was extended to permit the detection of all C2H2 zinc fingers and their parent proteins in the recently completed sequence of the yeast genome . Additionally, a new computer program was written that extracts other zinc binding motifs (non C2H2 'fingers'), overlapping with the classical zinc finger pattern, from the found set of yeast C2H2 fingers . The complete and correct detection of all fingers is a prerequisite for the classification of the yeast zinc finger proteins in functional terms . The detected 53 yeast C2H2 zinc finger proteins do not contain finger clusters with 10 or more repeats, as is frequently found in higher eukaryotes . Only three proteins contain four or more fingers in a cluster . Moreover, nearly all 27 yeast proteins with tandem arrays of two or three finger domains can be classified into nine subgroups with high sequence conservation in their finger clusters, in particular of their DNA recognition helices . These results and application of the recently elaborated finger/DNA recognition rules suggest that the yeast proteins belonging to the same subgroup may recognize identical or very similar DNA sites.

Cell, 1997 Jun 13, 89(6), 939 - 49
tea1 and the microtubular cytoskeleton are important for generating global spatial order within the fission yeast cell; Mata J et al.; Fission yeast cells identify and maintain growing regions exactly opposed at the ends of a cylindrical cell . tea1 mutants disrupt this organization, producing bent and T-shaped cells . We have cloned tea1 and shown that tea1 is located at the cell poles . Microtubules are continuously required to transfer tea1 to the cell ends, and tea1 is located at the ends of microtubules growing toward the cell poles . We suggest that tea1 acts as an end marker, directing the growth machinery to the cell poles . tea1 is down-regulated in cells treated with pheromone that grow toward a mating partner and no longer maintain their ends exactly opposed . tea1 may also influence microtubular organization, affecting the maintenance of a single central axis.

Cell, 1997 Jun 13, 89(6), 849 - 58
The yeast tRNA splicing endonuclease: a tetrameric enzyme with two active site subunits homologous to the archaeal tRNA endonucleases; Trotta CR et al.; The splicing of tRNA precursors is essential for the production of mature tRNA in organisms from all major phyla . In yeast, the tRNA splicing endonuclease is responsible for identification and cleavage of the splice sites in pre-tRNA . We have cloned the genes encoding all four protein subunits of endonuclease . Each gene is essential . Two subunits, Sen2p and Sen34p, contain a homologous domain of approximately 130 amino acids . This domain is found in the gene encoding the archaeal tRNA splicing endonuclease of H . volcanii and in other Archaea . Our results demonstrate that the eucaryal tRNA splicing endonuclease contains two functionally independent active sites for cleavage of the 5' and 3' splice sites, encoded by SEN2 and SEN34, respectively . The presence of endonuclease in Eucarya and Archaea suggests an ancient origin for the tRNA splicing reaction.

J Biol Chem, 1997 Jun 13, 272(24), 15547 - 52
Modulation of cell death in yeast by the Bcl-2 family of proteins; Tao W et al.; Bcl-2 family members are regulators of cell death . The precise biochemical properties of these proteins are unclear although intrafamily protein-protein association is thought to be involved . To elucidate structure-activity relationships among Bcl-2 proteins and identify the pathways in which they act, an inducible death suppressor assay was developed in yeast . Only Bax and Bak killed yeast via a process that did not require interleukin-1beta-converting enzyme-like proteases . Bax/Bak lethality was suppressed by coexpression of Bcl-2 family members that are anti-apoptotic in vertebrates, namely Bcl-xL, Bcl-2, Mcl-1, and A1 . Furthermore, Bcl-xL and Bcl-2 suppressed Bax toxicity by distinct mechanisms in yeast . Bad, Bcl-xS, and Ced-9 lacked suppressor activity . These inactive proteins bound to anti-apoptotic members of the Bcl-2 family but not to Bax or Bak . In contrast, most Bcl-2 family proteins that attenuated death bound to Bax and Bak . However, two mutants of Bcl-xL suppressed Bax-induced cell death while having no Bax binding activity . Therefore, Bcl-xL functions independently of Bax binding, perhaps by interacting with a common target or promoting a pathway that antagonizes Bax . Thus, the pathways downstream of Bax and Bcl-xL may be conserved between vertebrates and yeast . This suppressor assay could be used to isolate components of these pathways.

J Biol Chem, 1997 Jun 13, 272(24), 15527 - 31
The Ktr1p, Ktr3p, and Kre2p/Mnt1p mannosyltransferases participate in the elaboration of yeast O- and N-linked carbohydrate chains; Lussier M et al.; We have determined a role for Ktr1p and Ktr3p as mannosyltransferases in the synthesis of the carbohydrate chains attached to Saccharomyces cerevisiae O- and N-modified proteins . KTR1 and KTR3 encode related proteins that are highly similar to the Kre2p/Mnt1p Golgi alpha1,2-mannosyltransferase (Lussier, M., Camirand, A., Sdicu, A.-M., and Bussey, H . (1993) Yeast 9, 1057-1063; Mallet, L., Bussereau, F., and Jacquet, M . (1994) Yeast 10, 819-831) . Examination of the electrophoretic mobility of a specifically O-linked protein from mutants and an analysis of their total O-linked mannosyl chains demonstrates that Ktr1p, Ktr3p, and Kre2p/Mnt1p have overlapping roles and collectively add most of the second and the third alpha1,2-linked mannose residues on O-linked oligosaccharides . Determination of the mobility of the specifically N-linked glycoprotein invertase in different null strains indicates that Ktr1p, Ktr3p, and Kre2p are also jointly involved in N-linked glycosylation, possibly in establishing some of the outer chain alpha1,2-linkages.

J Biol Chem, 1997 Jun 13, 272(24), 15358 - 65
A yeast ATP-binding cassette-type protein mediating ATP-dependent bile acid transport; Ortiz DF et al.; ATP-dependent transport of bile acids is a key determinant of bile flow in mammalian liver and is associated with cholesterol excretion, gallstone formation, and numerous inherited and acquired hepatobiliary diseases . Secretory vesicles and a vacuole enriched fraction purified from Saccharomyces cerevisiae also exhibit ATP-dependent bile acid transport . ATP-dependent transport of bile acids by the vacuolar fraction was independent of the vacuolar proton ATPase, responded to changes in the osmotically sensitive intravesicular space, and was saturable, exhibiting a Km of 63 microM for taurocholate . The BAT1 (bile acid transporter) gene was isolated from yeast DNA by polymerase chain reaction amplification using degenerate oligonucleotides hybridizing to conserved regions of ABC-type proteins . ATP-dependent bile acid transport was abolished when the BAT1 coding region was deleted from the genome and restored upon reintroduction of the gene . The deduced amino acid sequence predicts that Bat1p is an ABC-type protein 1661 amino acids in length, similar to mammalian cMOAT/cMRP1 and MRP1 transporters, yeast Ycf1p, and two yeast proteins of unknown function . Information obtained from the yeast BAT1 gene may aid identification of the gene encoding the mammalian bile acid transporter.

Proc Natl Acad Sci U S A, 1997 Jun 10, 94(12), 6142 - 7
Interaction of the S phase regulator cdc18 with cyclin-dependent kinase in fission yeast; Brown GW et al.; The fission yeast gene cdc18(+) is required for entry into S phase and for coupling mitosis to the successful completion of S phase . Cdc18 is a highly unstable protein that is expressed only once per cell cycle at the G1/S boundary . Overexpression of Cdc18 causes a mitotic delay and reinitiation of DNA replication, suggesting that the inactivation of Cdc18 plays a role in preventing rereplication within a given cell cycle . In this paper, we present evidence that Cdc18 is associated with active cyclin-dependent kinase in vivo . We have expressed Cdc18 as a glutathione S-transferase fusion in fission yeast and demonstrated that the fusion protein is functional in vivo . We find that the Cdc18 fusion protein copurifies with a kinase activity capable of phosphorylating histone H1 and Cdc18 . The activity was identified by a variety of methods as the cyclin-dependent kinase containing the product of the cdc2(+) gene . The amino terminus of Cdc18 is required for association with cyclin-dependent kinase, but the association does not require the consensus cyclin-dependent kinase phosphorylation sites in this region . Additionally, both G1/S and mitotic forms of cyclin-dependent kinase phosphorylate and interact with Cdc18 . These interactions between Cdc18 and cyclin-dependent kinases suggest mechanisms by which cyclin-dependent kinases could activate the initiation of DNA replication and could prevent rereplication.

Mutat Res, 1997 Jun 9, 384(1), 23 - 32
Preferential incision of interstrand crosslinks induced by 8-methoxypsoralen plus UVA in yeast during the cell cycle; Meniel V et al.; Interstrand crosslink (ICL) induction by 8-methoxypsoralen plus UVA and the incision step of the repair have been investigated during the mitotic cell cycle of haploid Saccharomyces cerevisiae . Cells were synchronised by elutriation and events were examined at the level of the MAT alpha and the HML alpha loci in a SIR strain . The DNA sequence of these two loci is identical, but the MAT alpha locus may be replicated earlier in S phase and is transcriptionally active while the HML alpha locus may be replicated later in S phase and is transcriptionally inactive because of Sir repression that creates a heterochromatin-like structure at this locus . ICL were induced to similar extents in both loci during the stages of the cell cycle examined, and these levels were identical to those reported for asynchronous cultures . Preferential incisions occurred for ICL in the MAT alpha locus compared to those in the HML alpha locus, independently of the cell cycle phase studied . The levels of incision were comparable for events in the early G1 phase (eG1), late G1 phase (lG1), early S phase (eS), middle S phase (mS), late S phase (lS) or G2 phase (G2) . Thus the preferential incision of ICL observed previously in asynchronous cell culture is maintained throughout the cell cycle and, surprisingly, occurs equally well in G1 . Here the opportunities for recombination to further process the incised damaged are substantially limited compared to those in the S and G2 phases.

J Biol Chem, 1997 Jun 6, 272(23), 14705 - 12
Effects of oxygen concentration on the expression of cytochrome c and cytochrome c oxidase genes in yeast; Burke PV et al.; Oxygen is an important environmental regulator for the transcription of several genes in Saccharomyces cerevisiae, but it is not yet clear how this yeast or other eukaryotes actually sense oxygen . To begin to address this we have examined the effects of oxygen concentration on the expression of several nuclear genes (CYC1, CYC7, COX4, COX5a, COX5b, COX6, COX7, COX8, and COX9) for proteins of the terminal portion of the respiratory chain . COX5b and CYC7 are hypoxic genes; the rest are aerobic genes . We have found that the level of expression of these genes is determined by oxygen concentration per se and not merely the presence or absence of oxygen and that each of these genes has a low oxygen threshold (0 . 5-1 microM O2) for expression . For some aerobic genes (COX4, COX5a, COX7, COX8, and COX9) there is a gradual decline in expression between 200 microM O2 (air) and their oxygen threshold . Below this threshold expression drops precipitously . For others (COX5a and CYC1) the level of expression is nearly constant between 200 microM O2 and their threshold and then drops off . The hypoxic genes COX5b and CYC7 are not expressed until the oxygen concentration is below 0.5 microM O2 . These studies have also revealed that COX5a and CYC1, the genes for the aerobic isoforms of cytochrome c oxidase subunit V and cytochrome c, and COX5b and CYC7, the genes for the hypoxic isoforms of cytochrome c oxidase subunit V and cytochrome c, are coexpressed at a variety of oxygen concentrations and switch on or off at extremely low oxygen concentrations . By shifting cells from one oxygen concentration to another we have found that aerobic genes are induced faster than hypoxic genes and that transcripts from both types of gene are turned over quickly . These findings have important implications for cytochrome c oxidase function and biogenesis and for models of oxygen sensing in yeast.

Gene, 1997 Jun 3, 191(2), 173 - 81
The human immunoglobulin kappa locus on yeast artificial chromosomes (YACs); Brensing-Kuppers J et al.; The human immunoglobulin kappa locus is a duplicated structure . Contigs of 600 kb with 40 Vkappa genes and 440 kb with 36 Vkappa genes had been established for the Ckappa proximal (p) and distal (d) copies, respectively . In addition the human genome contains more than 24 dispersed Vkappa genes, called orphons . In the present study, 22 kappa-locus derived YACs were analyzed in detail, while 30 orphon-derived YACs were characterized only with respect to some parameters . The kappa-locus derived YACs allowed three gaps to be closed which previously could not be bridged by cosmid and phage lambda cloning . At the 5' side, the p contig was extended in the YACs by 50 kb and the d contig by 16 kb . At the 3'side, the d contig was extended by 11.5 kb . Beyond the 3' end of the d contig a new Vkappa gene was found, which is located, according to pulsed field gel electrophoresis (PFGE) experiments, at a distance of at least 140 kb from the last Vkappa gene of the contig . This Vkappa gene, which was termed Z0, occurred on three YACs, albeit at distances smaller than 140 kb; this was probably due to deletions in the YACs caused by abundant repetitive sequences at the borders of the locus . According to its sequence and to the restriction map of its surroundings, Z0 is an orphon gene of the so-called Z family, of which several members are known to be dispersed throughout the genome . The possibility that Z0 has been the parent of the other Z orphons is discussed.

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