Biochemistry, 2000 Aug 22, 39(33), 10207 - 18 Tri-partite assay for studying exon ligation by the ai5gamma group II intron; Bar-Shalom A et al.; Group II introns self-splice via a two-step mechanism: cleavage at the 5' splice site followed by exon ligation at the 3' splice site . The second step has been difficult to study in vitro because it is generally faster than the first . Herein we describe development and partial kinetic characterization of a novel assay for studying the second step in isolation . In this system, a truncated linear intron (nucleotides 1-881) mediates exon ligation between two oligonucleotide substrates: a 19 nt 5' exon and a 3' substrate consisting of the last 6 nucleotides of the intron plus a 6 nucleotide 3' exon . We found that neither the exact structure of domain 6 nor the identity of nucleotides flanking the 3' splice site is critical for accurate 3' splice site choice by the ai5gamma group II intron . The multiple turnover k(cat) (0.14 min(-)(1)) is slower than the single turnover k(obs) (0.6-0.7 min(-)(1)), consistent with rate-limiting product release under steady-state conditions . Decreased single turnover rates at lower pHs were more consistent with loss of catalytic activity than with rate-limiting chemistry . Binding of the 3' substrate (K(m) = 2.6 microM) could be improved by changing a long-range A:U base pair involving the last intronic nucleotide (the gamma-gamma' interaction) to G:C (K(m(3)(')(substrate)) = 1 microM). Biochemistry, 2000 Aug 22, 39(33), 10045 - 54 Cooperative ordering in homeodomain-DNA recognition: solution structure and dynamics of the MATa1 homeodomain; Anderson JS et al.; The mating type homeodomain proteins, MATa1 and MATalpha2, combine to form a heterodimer to bind DNA in diploid yeast cells . The a1-alpha2 heterodimer tightly and specifically binds haploid-specific gene operators to repress transcription . On its own, however, the a1 homeodomain does not bind DNA in a sequence-specific manner . To help understand this interaction, we describe the solution structure and backbone dynamics of the free a1 homeodomain . Free a1 in solution is an ensemble of structures having flexible hinges at the two turns in the small protein fold . Conformational changes in the a1 homeodomain upon ternary complex formation are located in the loop between helix 1 and helix 2, where the C-terminal tail of alpha2 binds to form the heterodimer, and at the C-terminus of helix 3, the DNA recognition helix . The observed differences, comparing the free and bound a1 structures, suggest a mechanism linking van der Waals stacking changes to the ordering of a final turn in the DNA-binding helix of a1 . The tail of alpha2 induces changes in loop 1 of a1 that push it toward a properly folded DNA binding conformation. J Biol Chem, 2000 Nov 10, 275(45), 35162 - 9 Very long-chain acyl-CoA synthetases . Human "bubblegum" represents a new family of proteins capable of activating very long-chain fatty acids; Steinberg SJ et al.; Activation by thioesterification to coenzyme A is a prerequisite for most reactions involving fatty acids . Enzymes catalyzing activation, acyl-CoA synthetases, have been classified by their chain length specificities . The most recently identified family is the very long-chain acyl-CoA synthetases (VLCS) . Although several members of this group are capable of activating very long-chain fatty acids (VLCFA), one is a bile acid-CoA synthetase, and others have been characterized as fatty acid transport proteins . It was reported that the Drosophila melanogaster mutant bubblegum (BGM) had elevated VLCFA and that the product of the defective gene had sequence homology to acyl-CoA synthetases . Therefore, we cloned full-length cDNA for a human homolog of BGM, and we investigated the properties of its protein product, hsBG, to determine whether it had VLCS activity . Northern blot analysis showed that hsBG is expressed primarily in brain . Compared with vector-transfected cells, COS-1 cells expressing hsBG had increased acyl-CoA synthetase activity with either long-chain fatty acid (2.4-fold) or VLCFA (2.6-fold) substrates . Despite this increased VLCFA activation, hsBG-expressing cells did not have increased rates of VLCFA degradation . Confocal microscopy showed that hsBG had a cytoplasmic localization in some COS-1 cells expressing the protein, whereas it appeared to associate with plasma membrane in others . Fractionation of these cells revealed that most of the hsBG-dependent acyl-CoA synthetase activity was soluble and not membrane-bound . Immunoaffinity-purified hsBG from transfected COS-1 cells was enzymatically active . hsBG and hsVLCS are only 15% identical, and comparison with sequences of two conserved motifs from all known families of acyl-CoA synthetases revealed that hsBG along with the D . melanogaster and murine homologs comprise a new family of acyl-CoA synthetases . Thus, two protein families are now known that contain enzymes capable of activating VLCFA . Because hsBG is expressed in brain but previously described VLCSs were not highly expressed in this organ, hsBG may play a central role in brain VLCFA metabolism and myelinogenesis. J Biol Chem, 2000 Dec 1, 275(48), 38059 - 66 Kinetic role for mammalian SF1/BBP in spliceosome assembly and function after polypyrimidine tract recognition by U2AF; Guth S et al.; Two sequences important for pre-mRNA splicing precede the 3' end of introns in higher eukaryotes, the branch point (BP) and the polypyrimidine (Py) tract . Initial recognition of these signals involves cooperative binding of the splicing factor SF1/mammalian branch point binding protein (mBBP) to the BP and of U2AF(65) to the Py tract . Both factors are required for recruitment of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) to the BP in reactions reconstituted from purified components . In contrast, extensive depletion of ST1/BBP in Saccharomyces cerevisiae does not compromise spliceosome assembly or splicing significantly . As BP sequences are less conserved in mammals, these discrepancies could reflect more stringent requirements for SF1/BBP in this system . We report here that extensive depletion of SF1/mBBP from nuclear extracts of HeLa cells results in only modest reduction of their activity in spliceosome assembly and splicing . Some of these effects reflect differences in the kinetics of U2 snRNP binding . Although U2AF(65) binding was reduced in the depleted extracts, the defects caused by SF1/mBBP depletion could not be fully restored by an increase in occupancy of the Py tract by exogenously added U2AF(65), arguing for a role of SF1/mBBP in U2 snRNP recruitment distinct from promoting U2AF(65) binding. Curr Genet, 2000 Jul, 38(1), 23 - 32 Recombination between divergent sequences leads to cell death in a mismatch-repair-independent manner; Inbar O et al.; Homologous recombination is an important DNA repair mechanism in vegetative cells . During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences, thus creating a gene-conversion event . Gene conversion of a functional member of a gene family, which uses an inactive member (such as a pseudogene) as a template, might have deleterious consequences . It is therefore important for the cell to prevent recombination between divergent sequences . We have studied the repair of a double-strand break by recombination in a haploid yeast strain carrying 99% identical alleles located on different chromosomes . The fate of the broken chromosome was followed in the whole cell population without imposing selective constraints . Our results show that all the cells were able to repair the broken chromosome by gene conversion . During the repair, the cells arrest in the cell cycle with a "dumbbell" configuration characteristic of G2/M-arrested cells . Surprisingly, although all the cells repaired the broken chromosome, 60% of them were unable to resume growth and to form colonies after the repair was completed . The low level of cell recovery was due to the 1% divergence between the alleles, but was not dependent on the function of the mismatch-repair system . Cell death, however, could be prevented by the presence of an alternative source of perfect homology located on a different chromosome. Mol Pharmacol, 2000 Sep, 58(3), 560 - 8 Probing the interaction of the cytotoxic bisdioxopiperazine ICRF-193 with the closed enzyme clamp of human topoisomerase IIalpha; Patel S et al.; Topoisomerase II is an ATP-operated protein clamp that captures a DNA helix and transports it through another DNA duplex, allowing chromosome segregation at mitosis . A number of cytotoxic bisdioxopiperazines such as ICRF-193 target topoisomerase II by binding and trapping the closed enzyme clamp . To investigate this unusual mode of action, we have used yeast to select plasmid-borne human topoisomerase IIalpha alleles resistant to ICRF-193 . Mutations in topoisomerase IIalpha of Leu-169 to Phe (L169F) (in the N-terminal ATPase domain) and Ala-648 to Pro (A648P) (in the core domain) were identified as conferring >50-fold and 5-fold resistance to ICRF-193 in vivo, respectively . The L169F mutation, located next to the Walker A box ATP-binding sequence, resulted in a mutant enzyme displaying ICRF-193-resistant topoisomerase and ATPase activities and whose closed clamp was refractory to ICRF-193-mediated trapping as an annulus on closed circular DNA . These data imply that the mutation interferes directly with ICRF-193 binding to the N-terminal ATPase gate . In contrast, the A648P enzyme displayed topoisomerase activities exhibiting wild-type sensitivity to ICRF-193 . We suggest that the inefficient trapping of the A648P closed clamp results either from the observed increased ATP requirement, or more likely, from lowered salt stability, perhaps involving destabilization of ICRF-193 interactions with the B'-B' interface in the core domain . These results provide evidence for at least two different phenotypic classes of ICRF-193 resistance mutations and suggest that bisdioxopiperazine action involves the interplay of both the ATPase and core domains of topoisomerase IIalpha. Mol Pharmacol, 2000 Sep, 58(3), 542 - 51 Human microsomal epoxide hydrolase is the target of germander-induced autoantibodies on the surface of human hepatocytes; De Berardinis V et al.; Germander, a plant used in folk medicine, caused an epidemic of cytolytic hepatitis in France . In about half of these patients, a rechallenge caused early recurrence, suggesting an immunoallergic type of hepatitis . Teucrin A (TA) was found responsible for the hepatotoxicity via metabolic activation by CYP3A . In this study, we describe the presence of anti-microsomal epoxide hydrolase (EH) autoantibodies in the sera of patients who drank germander teas for a long period of time . By Western blotting and immunocytochemistry, human microsomal EH was shown to be present in purified plasma membranes of both human hepatocytes and transformed spheroplasts and to be exposed on the cell surface where affinity-purified germander autoantibodies recognized it as their autoantigen . Immunoprecipitation of EH activity by germander-induced autoantibodies confirmed this finding . These autoantibodies were not immunoinhibitory . The plasma membrane-located EH was catalytically competent and may act as target for reactive metabolites from TA . To test this hypothesis CYP3A4 and EH were expressed with human cytochrome P450 reductase and cytochrome b(5) in a "humanized" yeast strain . In the absence of EH only one metabolite was formed . In the presence of EH, two additional metabolites were formed, and a time-dependent inactivation of EH was detected, suggesting that a reactive oxide derived from TA could alkylate the enzyme and trigger an immune response . Antibodies were found to recognize TA-alkylated EH . Recognition of EH present at the surface of human hepatocytes could suggest an (auto)antibody participation in an immune cell destruction. J Cell Biol, 2000 Aug 21, 150(4), 895 - 904 In vivo importance of actin nucleotide exchange catalyzed by profilin; Wolven AK et al.; The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly . Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP(2) and to polyproline stretches on several proteins . Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested . To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization . The actin-binding region of profilin was shown to be critical in vivo . act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding . In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover . However, Act1-157p circumvented the requirement for profilin . Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin-cofilin generated during filament disassembly. J Cell Biol, 2000 Aug 21, 150(4), 695 - 706 Binding of the Mex67p/Mtr2p heterodimer to FXFG, GLFG, and FG repeat nucleoporins is essential for nuclear mRNA export; Strasser K et al.; It is not known how Mex67p and Mtr2p, which form a heterodimer essential for mRNA export, transport mRNPs through the nuclear pore . Here, we show that the Mex67p/Mtr2p complex binds to all of the repeat types (GLFG, FXFG, and FG) found in nucleoporins . For this interaction, complex formation between Mex67p and Mtr2p has to occur . MEX67 and MTR2 also genetically interact with different types of repeat nucleoporins, such as Nup116p, Nup159p, Nsp1p, and Rip1p/Nup40p . These data suggest a model in which nuclear mRNA export requires the Mex67p/Mtr2p heterodimeric complex to directly contact several repeat nucleoporins, organized in different nuclear pore complex subcomplexes, as it carries the mRNP cargo through the nuclear pore. J Exp Biol, 2000 Sep, 203(Pt 18), 2747 - 56 Temporal and spatial expression of the cell-cycle regulator cul-1 in Drosophila and its stimulation by radiation-induced apoptosis; Filippov V et al.; Cul-1 protein is part of the ubiquitin ligase complex that is conserved from yeast to humans . This complex specifically marks cell-cycle regulators for their subsequent destruction . Two null mutations of the cul-1 gene are known, in budding yeast and in nematodes . Although in both these organisms the cul-1 gene executes essentially the same function, the manifestation of its lack-of-function mutations differs considerably . In yeast the mutation causes arrest at the G(1)/S-phase transition, whereas in nematodes excessive cell divisions occur because mutant cells are unable to exit the mitotic cycle . We isolated cul-1 orthologues from two model organisms, Drosophila melanogaster and mouse . We show that the Drosophila full-length cul-1 gene restores the yeast mutant's inability to pass through the G(1)/S-phase transition . We also characterize expression of this gene at the transcript and protein levels during Drosophila development and show that cul-1 gene is maternally supplied as a protein, but not as an RNA transcript . Zygotic transcription of the gene, however, resumes at early stages of embryogenesis . We also found an increase in cul-1 transcription in cultured cells treated with a lethal dose of gamma-irradiation. Nature, 2000 Aug 3, 406(6795), 541 - 4 A chromatin remodelling complex involved in transcription and DNA processing; Shen X et al.; The packaging of the eukaryotic genome in chromatin presents barriers that restrict the access of enzymes that process DNA . To overcome these barriers, cells possess a number of multi-protein, ATP-dependent chromatin remodelling complexes, each containing an ATPase subunit from the SNF2/SWI2 superfamily . Chromatin remodelling complexes function by increasing nucleosome mobility and are clearly implicated in transcription . Here we have analysed SNF2/SWI2- and ISWI-related proteins to identify remodelling complexes that potentially assist other DNA transactions . We purified a complex from Saccharomyces cerevisiae that contains the Ino80 ATPase . The INO80 complex contains about 12 polypeptides including two proteins related to the bacterial RuvB DNA helicase, which catalyses branch migration of Holliday junctions . The purified complex remodels chromatin, facilitates transcription in vitro and displays 3' to 5' DNA helicase activity . Mutants of ino80 show hypersensitivity to agents that cause DNA damage, in addition to defects in transcription . These results indicate that chromatin remodelling driven by the Ino80 ATPase may be connected to transcription as well as DNA damage repair. Immunopharmacol Immunotoxicol, 2000 May, 22(2), 373 - 86 Cocaine inhibits human neutrophil phagocytosis and phagolysosomal acidification in vitro; Mukunda BN et al.; Cocaine, used intravenously, increases the risk of infections, but its effects on neutrophil phagocytosis have not been examined in vitro . Human neutrophils were suspended in cocaine hydrochloride 0, 1, 10, 50, 100 or 200 microg/ml in Hank's balanced salt solution to which was added a phagocytic meal of killed Saccharomyces cerevisiae stained with the pH indicator dye bromcresol purple . Yeast per phagocytosing neutrophil and the percent neutrophils phagocytosing yeast were reduced in neutrophils treated with cocaine 100 and 200 microg/ml (P < 0.05) . When examined for percent of yeast phagocytosed after 10 minutes, neutrophils treated with cocaine 1-200 microg/ml demonstrated a decrease (P < 0.05) . However, at 60 minutes only neutrophils treated with cocaine 50 and 100 microg/ml still showed a decrease in percent of yeast phagocytosed . Phagolysosomal acidification was impaired in neutrophils treated with 50, 100 and 200 microg/ml cocaine . Thus, cocaine inhibits neutrophil phagocytosis and phagolysosomal acidification in vitro, offering a reason for cocaine users/abusers to have impaired host defense and to be potentially at higher risk for infections. Immunopharmacol Immunotoxicol, 2000 May, 22(2), 357 - 72 Omeprazole inhibits phagocytosis and acidification of phagolysosomes of normal human neutrophils in vitro; Agastya G et al.; We postulated omeprazole inhibition of the neutrophil proton pump, impairing phagocytosis and phagolysosomal acidification . Neutrophils from healthy human beings were treated with omeprazole prodrug 0.5 mM/l or acid activated omeprazole 0.5 mM/l, then incubated with killed Saccharomyces cerevisiae stained with bromcresol purple . Wet mounts were done at 10, 30 and 60 minutes . Percent neutrophils phagocytosing, percent yeast phagocytosed, and yeast per phagocytosing neutrophil were significantly decreased in acid activated omeprazole compared to controls and omeprazole prodrug . In contrast, percent acidification of intracellular yeast was significantly lower in both omeprazole prodrug and acid activated omeprazole compared to controls . Over time, control neutrophils showed an increase in percent yeast phagocytosed and yeast per phagocytosing neutrophil . When treated with acid activated omeprazole, the percent of neutrophils phagocytosing progressively decreased over time . We observed 1) omeprazole prodrug does not inhibit neutrophil phagocytosis but does inhibit phagolysosomal acidification, whereas 2) acid activated omeprazole inhibits both neutrophil phagocytosis and phagolysosome acidification . We conclude that omeprazole impairs these neutrophil functions in vitro. Oncogene, 2000 Aug 10, 19(34), 3948 - 54 The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway; Saavedra HI et al.; Activating mutations of RAS are thought to be early events in the evolution of thyroid follicular neoplasms . We used a doxycycline-inducible expression system to explore the acute effects of H-RAS12 on genomic stability in thyroid PCCL3 cells . At 2-3 days (first or second cell cycle) there was a significant increase in the frequency of micronucleation . Treatment of cells with YVAD-CHO inhibited RAS-induced apoptosis, but had no effect on micronucleation . The effects of H-RAS(V12) were mediated by activation of MAPK, as treatment with PD98059 at concentrations verified to selectively inhibit MEK1 reduced the frequency of prevalence of cells with micronuclei . In addition, doxycycline-inducible expression of a constitutively active MEK1, but not of a mutant RAC1, mimicked the effects of H-RAS(V12) . The effects of H-RAS(V12) on genome destabilization were apparent even though the sequence of p53 in PCCL3 cells was confirmed to be wild-type . Acute activation of H-RAS(V12) evoked a proportional increase in both CREST negative and CREST positive micronuclei, indicating that both clastogenic and aneugenic effects were involved . H-RAS(V12) and activated MEK1 also induced centrosome amplification, and chromosome misalignment . Evidence that acute expression of constitutively activated RAS destabilizes the genome of PCCL3 cells is consistent with a mode of tumor initiation in which this oncogene promotes phenotypic progression by predisposing to large scale genomic abnormalities. Oncogene, 2000 Aug 10, 19(34), 3914 - 24 Notch(ic)-ER chimeras display hormone-dependent transformation, nuclear accumulation, phosphorylation and CBF1 activation; Ronchini C et al.; Notch genes encode a family of evolutionarily conserved transmembrane receptors that are involved in many distinct cellular processes such as differentiation, proliferation and apoptosis . Notch function has been shown to be required both during development and in adult life . Moreover, several studies on spontaneous human tumors and in experimental models demonstrate that three of the four mammalian Notch genes can act as oncogenes . The mechanism by which Notch proteins induce neoplastic transformation is not known . In order to determine the early signaling events mediated by Notch during cellular transformation we constructed several inducible alleles of Notch(ic) by fusing portions of Nic to the hormone-binding domain of the estrogen receptor . Here we show that Notch(ic)-ER chimeras are conditionally activated by 4-Hydroxytamoxifen (OHT) in a dose-dependent manner . Clonal RKE cell lines expressing Notch(ic)-ER chimeras display hormone-dependent transformation in vitro . Transformation mediated by Notch(ic)-ER is reversible and chronic stimulation is necessary for the maintenance of the transformed phenotype . In response to hormone activation Notch(ic)-ER chimeras become hyperphosphorylated and accumulate in the nucleus of the cell; indicating that both phosphorylation and nuclear localization are required for Notch transforming activity. Biochem Cell Biol, 2000, 78(3), 289 - 98 Prohormone transport through the secretory pathway of neuroendocrine cells; Kuiper RP et al.; En route through the secretory pathway of neuroendocrine cells, prohormones pass a series of membrane-bounded compartments . During this transport, the prohormones are sorted to secretory granules and proteolytically cleaved to bioactive peptides . Recently, progress has been made in a number of aspects concerning secretory protein transport and sorting, particularly with respect to transport events in the early regions of the secretory pathway . In this review we will deal with some of these aspects, including: i) selective exit from the endoplasmic reticulum via COPII-coated vesicles and the potential role of p24 putative cargo receptors in this process, ii) cisternal maturation as an alternative model for protein transport through the Golgi complex, and iii) the mechanisms that may be involved in the sorting of regulated secretory proteins to secretory granules . Although much remains to be learned, interesting new insights into the functioning of the secretory pathway have been obtained. Mol Cell, 2000 Jul, 6(1), 197 - 201 Mediator-nucleosome interaction; Lorch Y et al.; Mediator, a multiprotein complex involved in the regulation of RNA polymerase II transcription, binds to nucleosomes and acetylates histones . Three lines of evidence identify the Nut1 subunit of Mediator as responsible for the histone acetyltransferase (HAT) activity . An "in-gel" HAT assay reveals a single band of the appropriate size . Sequence alignment shows significant similarity of Nut1 to the GCN5-related N-acetyltransferase superfamily . Finally, recombinant Nut1 exhibits HAT activity in an in-gel assay. Mol Cell, 2000 Jul, 6(1), 1 - 10 The Bub2p spindle checkpoint links nuclear migration with mitotic exit; Pereira G et al.; Bfa1p and Bub2p are spindle checkpoint proteins that likely have GTPase activation activity and are associated with the budding yeast spindle pole body (SPB) . Here, we show that Bfa1p and Bub2p bind the Ras-like GTPase Tem1p, a component of the mitotic exit network, to the cytoplasmic face of the SPB that enters the bud, whereas the GDP/GTP exchange factor Lte1p is associated with the cortex of the bud . Migration of the SPB into the bud probably allows activation of Tem1p through Lte1p, thereby linking nuclear migration with mitotic exit . Since components of the Bub2p checkpoint are conserved in other organisms, we propose that the position of the SPB or mammalian centrosome controls the timing of mitotic exit. Biochem J, 2000 Sep 1, 350 Pt 2, 337 - 52 Sac phosphatase domain proteins; Hughes WE et al.; Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids . One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins . Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p . The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p . These proteins therefore contain both Sac phosphatase and 5-phosphatase domains . This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae. J Clin Endocrinol Metab, 2000 Aug, 85(8), 2733 - 9 Prevalence and distribution of ret/ptc 1, 2, and 3 in papillary thyroid carcinoma in New Caledonia and Australia; Chua EL et al.; The world's highest incidence of thyroid cancer has been reported among females in New Caledonia, a French overseas territory in the Pacific located between Australia and Fiji . To date, no molecular genetic studies in this population are available . Over the past few years, the oncogenic rearrangement of the ret protooncogene (ret/ptc) has been studied in papillary carcinomas in different populations . In this study, we investigated the prevalence and distribution of ret/ptc1, 2, and 3 in papillary thyroid carcinoma from the New Caledonian population and compared the pattern with that of an Australian population . Fresh-frozen and paraffin-embedded papillary carcinomas from 27 New Caledonian and 20 Australian patients were examined for ret rearrangements by means of RT-PCR with primers flanking the chimeric region, followed by hybridization with radioactive probes . ret/ptc was present in 70% of the New Caledonian and in 85% of the Australian samples . Multiple rearrangements were detected and confirmed by sequencing in 19 cases, 4 of which had 3 types of rearrangements in the same tumor . This study demonstrates a high prevalence of ret/ptc in New Caledonian and Australian papillary carcinoma . The findings of multiple ret/ptc in the same tumor suggest that some thyroid neoplasms may indeed be polyclonal. J Biol Chem, 2000 Nov 10, 275(45), 34983 - 8 Identification and characterization of the human ORC6 homolog; Dhar SK et al.; A new protein was cloned and identified as the sixth member of the human origin recognition complex (ORC) . The newly identified 30-kDa protein hsORC6 is 28% identical and 49% similar to ORC6p from Drosophila melanogaster, which is consistent with the identities and similarities found among the other ORC members reported in the two species . The human ORC6 gene is located on chromosome 16q12 . ORC6 protein level did not change through the cell cycle . Like ORC1, ORC6 did not co-immunoprecipitate with other ORC subunits but was localized in the nucleus along with the other ORC subunits . Several cellular proteins co-immunoprecipitated with ORC6, including a 65-kDa protein that was hyperphosphorylated in G(1) and dephosphorylated in mitosis . Therefore, unlike the tight stoichiometric association of six yeast ORC subunits in one holo-complex, only a small fraction of human ORC1 and ORC6 is likely to be associated with a subcomplex of ORC2, 3, 4, and 5, suggesting differences in the architecture and regulation of human ORC. J Biol Chem, 2000 Nov 17, 275(46), 35684 - 91 Ku entry into DNA inhibits inward DNA transactions in vitro; Frit P et al.; Association of the DNA end-binding Ku70/Ku80 heterodimer with the 460-kDa serine/threonine kinase catalytic subunit forms the DNA-dependent protein kinase (DNA-PK) that is required for double-strand break repair by non-homologous recombination in mammalian cells . Recently, we have proposed a model in which the kinase activity is required for translocation of the DNA end-binding subunit Ku along the DNA helix when DNA-PK assembles on DNA ends . Here, we have questioned the consequences of Ku entry into DNA on local DNA processes by using human nuclear cell extracts incubated in the presence of linearized plasmid DNA . As two model processes, we have chosen nucleotide excision repair (NER) of UVC DNA lesions and transcription from viral promoters . We show that although NER efficiency is strongly reduced on linear DNA, it can be fully restored in the presence of DNA-PK inhibitors . Simultaneously, the amount of NER proteins bound to the UVC-damaged linear DNA is increased and the amount of Ku bound to the same DNA molecules is decreased . Similarly, the poor transcription efficiency exhibited by viral promoters on linear DNA is enhanced in the presence of DNA-PK inhibitor concentrations that prevent Ku entry into the DNA substrate molecule . The present results show that DNA-PK catalytic activity can regulate DNA transactions including transcription in the vicinity of double-strand breaks by controlling Ku entry into DNA. Biosci Biotechnol Biochem, 2000 Jul, 64(7), 1472 - 6 Transfructosylation of thiol group by beta-fructofuranosidases; Nakano H et al.; Beta-fructofuranosidase fructosylated not only the hydroxyl group but also the thiol group of 2-mercaptoethanol in a transfer reaction using sucrose as a donor substrate . The enzymes from Candida utilis and Saccharomyces cerevisiae (bakers' yeast) were effective catalysts for the thio-fructofuranosylation . The thio-fructosylation product was isolated by activated carbon chromatography and its structure was confirmed by Fab-mass spectrometry and NMR spectroscopy . The thio-fructofuranoside was synthesized effectively at around 3.0 M for the acceptor concentration . The product increased with the sucrose concentration at least up to 1.9 M . O-Fructofuranoside was simultaneously synthesized at an early stage of the reaction, although it was hydrolyzed on further incubation . On the contrary, the thio-fructofuranoside accumulated efficiently after synthesis, indicating it was very stable against the hydrolytic action of the beta-fructofranosidase. Biosci Biotechnol Biochem, 2000 Jul, 64(7), 1416 - 21 Pyrithiamine resistance gene (ptrA) of Aspergillus oryzae: cloning, characterization and application as a dominant selectable marker for transformation; Kubodera T et al.; A pyrithiamine (PT) resistance gene (ptrA) was cloned from a genomic DNA library prepared from a PT resistant mutant of Aspergillus oryzae . It conferred high resistance to PT on an A . oryzae industrial strain as well as A . nidulans . Nucleotide sequence analysis showed that the ptrA gene contained one intron (58-bp) and encodes 327 amino acid (aa) residues . Additionally, the deduced aa sequence has 72% and 63% identity to Fusarium solani sti35 encoding a stress-inducible protein and Saccharomyces cerevisiae THI4 encoding an enzyme involved in thiamine biosynthesis, respectively, indicating that ptrA is a mutated allele of a gene belonging to the THI4 family . The mutation point was identified in the conserved motif in 5'-flanking region of these three THI4 homologous genes (ptrA, sti35, and THI4) . The introduction of the ptrA gene allowed an A . oryzae industrial strain to grow on the minimum medium containing PT (0.1 mg/l) on which an untransformed strain did not grow . This result indicates that the ptrA is applicable as a dominant selectable marker for transformation of A . oryzae. DNA Cell Biol, 2000 Jul, 19(7), 447 - 57 Loss control of Mcm5 interaction with chromatin in cdc6-1 mutated in CDC-NTP motif; Feng L et al.; Saccharomyces cerevisiae Cdc6 plays an essential role in establishing and maintaining the prereplicative complex (pre-RC) by interacting with the origin recognition complex (ORC) and associating with chromatin origins . These interactions are required to load minichromosome maintenance proteins (MCMs) and other initiator proteins onto replication origins . Although the temperature-sensitive cdc6 mutant, cdc6-1, has been widely used for these studies, the molecular mechanism of the cdc6-1 mutation has been unclear . In this study, we have identified a base substitution at Gly260-->Asp, near the CDC-NTP motif . Using a chromatin immunoprecipitation assay (CHIP), we found that cdc6-1 fails to load Mcm5 onto the replication origins . Chromatin fractions were used to study Mcm5 binding in both the wildtype and mutant background . These studies indicated that Cdc6 is also involved in unloading Mcm5 from chromatin . Specifically, the cdc6-1 mutation protein, cdc6(G260D), which failed to load Mcm5 onto replication origins, also failed to unload the Mcm5 protein . Furthermore, the overexpression of wildtype CDC6 accelerated the unloading of Mcm5 from chromatin fractions . In the absence of functional Cdc6, the Mcm5 protein showed nonorigin binding to chromatin with the cell cycle arrested at the G1S phase transition . Our results suggested that the cdc6(G260D) mutant protein fails to assemble an operational replicative complex and that wildtype Cdc6 plays a role in preventing re-replication by controlling the unloading the MCMs from chromatin origins. Bioessays, 2000 Sep, 22(9), 786 - 9 Transcriptional regulation: a new dominion for inositol phosphate signaling? Shears SB. The diverse phosphorylation patterns of the six-carbon inositol ring generates a mesmerizing wealth of inositol phosphates but we have little insight into the precise cellular roles of most members of this family . Therefore, new information on these roles is very welcome . The discovery by two independent groups(1, 2) that the Arg82 transcriptional regulator from Saccharomyces cerevisiae has inositol phosphate kinase activity is intriguing in this respect . One group proposes that these events directly affect the function of a specific, multimeric transcriptional complex.(2) It will be argued here, however, that available data do not entirely support such a direct role for Arg82 in transcription . The potential relevance of these findings to higher organisms will also be discussed. J Biol Chem, 2000 Oct 27, 275(43), 33771 - 6 Copper activation of superoxide dismutase 1 (SOD1) in vivo . Role for protein-protein interactions with the copper chaperone for SOD1; Schmidt PJ et al.; Insertion of copper into superoxide dismutase 1 (SOD1) in vivo requires the copper chaperone for SOD1 (CCS) . CCS encompasses three protein domains: copper binding Domains I and III at the amino and carboxyl termini, and a central Domain II homologous to SOD1 . Using a yeast interaction mating system, yeast CCS was seen to physically interact with SOD1, and this interaction required sequences at the predicted dimer interface of CCS Domain II . Interactions with SOD1 also required sequences of Domain III, but not Domain I . Mutations were introduced at the dimer interface of yeast SOD1, and the corresponding mutant failed to interact with CCS . When loaded with copper independent of CCS, this mutant SOD1 exhibited superoxide scavenging activity, but was normally inactive in vivo because CCS failed to recognize the enzyme . Activation of SOD1 by CCS was also examined using an in vivo assay for copper incorporation into SOD1 . Yeast CCS was observed to insert copper into a pre-existing pool of apoSOD1 without the need for new SOD1 synthesis or for protein unfolding by the major SSA cytosolic heat shock proteins . Our data are consistent with a model in which prefolded dimers of apoSOD1 serve as substrate for the CCS copper chaperone. J Biol Chem, 2000 Nov 3, 275(44), 34359 - 64 CREB-binding protein/p300 activates MyoD by acetylation; Polesskaya A et al.; The myogenic protein MyoD requires two nuclear histone acetyltransferases, CREB-binding protein (CBP)/p300 and PCAF, to transactivate muscle promoters . MyoD is acetylated by PCAF in vitro, which seems to increase its affinity for DNA . We here show that MyoD is constitutively acetylated in muscle cells . In vitro, MyoD is acetylated both by CBP/p300 and by PCAF on two lysines located at the boundary of the DNA binding domain . MyoD acetylation by CBP/p300 (as well as by PCAF) increases its activity on a muscle-specific promoter, as assessed by microinjection experiments . MyoD mutants that cannot be acetylated in vitro are not activated in the functional assay . Our results provide direct evidence that MyoD acetylation functionally activates the protein and show that both PCAF and CBP/p300 are candidate enzymes for MyoD acetylation in vivo. Biochem Biophys Res Commun, 2000 Aug 18, 275(1), 228 - 32 Dbf4 motifs: conserved motifs in activation subunits for Cdc7 kinases essential for S-phase; Masai H et al.; Dbf4 and its related molecules were originally identified as cyclin-like partners for Cdc7 kinases, essential for S-phase . Recent reports and database search indicate the presence of multiple Dbf4-related molecules with distinct functions . We have identified three stretches of amino acids which are conserved in various Dbf4-related molecules and possibly play distinct functions in binding to and activation of the catalytic subunits as well as in interactions with other proteins . Discovery of conserved motifs for this possible new protein family would serve as a useful framework for future identification of new members of this family as well as for probing their functions . Biochem Biophys Res Commun, 2000 Aug 18, 275(1), 37 - 42 Cloning, genomic organization, and tissue distribution of human Ssf-1; Suarez-Huerta N et al.; During the screening of a human placenta cDNA library, realized in order to isolate the P2Y(11) coding sequence, an unrelated cDNA was cloned . We identified a 1422 bp open reading frame encoding a human protein displaying 40% amino acid identity with the Saccharomyces cerevisiae Ssf-1, a protein involved in the second step of mRNA splicing . Sequencing of the corresponding genomic DNA showed that the gene encoding human Ssf-1 is located upstream to the P2Y(11) gene on chromosome 19p31 . Comparison of the cDNA and genomic DNA sequences revealed that the human Ssf-1 gene is split into 12 exons . Northern blotting experiments showed that the 1.7 kb Ssf-1 mRNA presents an ubiquitous tissue expression . We also show that, in HL-60 human promyelocytic leukemia cells, Ssf-1 mRNA is rapidly upregulated following a treatment by granulocyte-colony stimulating factor and dibutyryl-cyclicAMP, two agents known to induce the granulocytic differentiation of these cells . Proc Natl Acad Sci U S A, 2000 Aug 15, 97(17), 9402 - 7 A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion; Seals DF et al.; Yeast vacuoles undergo priming, docking, and homotypic fusion, although little has been known of the connections between these reactions . Vacuole-associated Vam2p and Vam6p (Vam2/6p) are components of a 65S complex containing SNARE proteins . Upon priming by Sec18p/NSF and ATP, Vam2/6p is released as a 38S subcomplex that binds Ypt7p to initiate docking . We now report that the 38S complex consists of both Vam2/6p and the class C Vps proteins {Reider, S . E . and Emr, S . D . (1997) Mol . Biol . Cell 8, 2307-2327} . This complex includes Vps33p, a member of the Sec1 family of proteins that bind t-SNAREs . We term this 38S complex HOPS, for homotypic fusion and vacuole protein sorting . This unexpected finding explains how Vam2/6p associates with SNAREs and provides a mechanistic link of the class C Vps proteins to Ypt/Rab action . HOPS initially associates with vacuole SNAREs in "cis" and, after release by priming, hops to Ypt7p, activating this Ypt/Rab switch to initiate docking. EMBO J, 2000 Aug 15, 19(16), 4298 - 309 Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein; Tanimoto K et al.; In normoxic cells the hypoxia-inducible factor-1 alpha (HIF-1 alpha) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1 alpha to a functional form requires protein stabilization . Here we show that the product of the von Hippel-Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF-1 alpha under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1 alpha . The region of VHL mediating interaction with HIF-1 alpha overlapped with a putative macromolecular binding site observed within the crystal structure of VHL . This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF-1 alpha and subsequent degradation . Interestingly, the VHL binding site within HIF-1 alpha overlapped with one of the minimal transactivation domains . Protection of HIF-1 alpha against degradation by VHL was a multistep mechanism, including hypoxia-induced nuclear translocation of HIF-1 alpha and an intranuclear hypoxia-dependent signal . VHL was not released from HIF-1 alpha during this process . Finally, stabilization of HIF-1 alpha protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response. EMBO J, 2000 Aug 15, 19(16), 4281 - 91 An intact SH3 domain is required for myosin I-induced actin polymerization; Geli MI et al.; The yeast type I myosins (MYO3 and MYO5) are involved in endocytosis and in the polarization of the actin cytoskeleton . The tail of these proteins contains a Tail Homology 2 (TH2) domain that constitutes a putative actin-binding site . Because of the important mechanistic implications of a second ATP-independent actin-binding site, we analyzed its functional relevance in vivo . Even though the myosin tail interacts with actin, and this interaction seems functionally important, deletion of a major portion of the TH2 domain did not abolish interaction . In contrast, we found that the SH3 domain of Myo5p significantly contributes to this interaction, implicating other proteins . We found that Vrp1p, the yeast homolog of WIP {Wiskott-Aldrich syndrome protein (WASP)-interacting protein}, seems necessary to sustain the Myo5p tail-F-actin interaction . Consistent with recent results implicating the yeast type I myosins in regulating actin polymerization in vivo, we demonstrate that the C-terminal domain of Myo5p is able to induce cytosol-dependent actin polymerization in vitro, and that this activity requires both an intact Myo5p SH3 domain and Vrp1p. RNA, 2000 Aug, 6(8), 1142 - 56 Substrate recognition by a eukaryotic RNase III: the double-stranded RNA-binding domain of Rnt1p selectively binds RNA containing a 5'-AGNN-3' tetraloop; Nagel R et al.; Rnt1p is an RNase III homolog from budding yeast, required for processing snRNAs, snoRNAs, and rRNA . Numerous Rnt1p RNA substrates share potential to form a duplex structure with a terminal four-base loop with the sequence AGNN . Using a synthetic RNA modeled after the 25S rRNA 3' ETS cleavage site we find that the AGNN loop is an important determinant of substrate selectivity . When this loop sequence is altered, the rate of Rnt1p cleavage is reduced . The reduction in cleavage rate can be attributed to reduced binding of the mutant substrate as measured by a gel-shift assay . Deletion of the nonconserved N-terminal domain of Rnt1p does not affect cleavage site choice or the ability of the enzyme to distinguish substrates that contain the AGNN loop, indicating that this region is not required for selective cleavage . Strikingly, a recombinant fragment of Rnt1p containing little more than the dsRBD is able to discriminate between wild-type and mutant loop sequences in a binding assay . We propose that a major determinant of AGNN loop recognition by Rnt1p is present in its dsRBD. RNA, 2000 Aug, 6(8), 1120 - 30 Identification of a U2/U6 helix la mutant that influences 3' splice site selection during nuclear pre-mRNA splicing; Chang JS et al.; Base substitutions in U2/U6 helix I, a conserved base-pairing interaction between the U6 and U2 snRNAs, have previously been found to specifically block the second catalytic step of nuclear pre-mRNA splicing . To further assess the role of U2/U6 helix I in the second catalytic step, we have screened mutations in U2/U6 helix I to identify those that influence 3' splice site selection using a derivative of the yeast actin pre-mRNA . In these derivatives, the spacing between the branch site adenosine and 3' splice site has been reduced from 43 to 12 nt and this results in enhanced splicing of mutants in the conserved 3' terminal intron residue . In this context, mutation of the conserved 3' intron terminal G to a C also results in the partial activation of a nearby cryptic 3' splice site with U as the 3' terminal intron nucleotide . Using this highly sensitive mutant substrate, we have identified a mutation in the U6 snRNA (U57A) that significantly increases the selection of the cryptic 3' splice site over the normal 3' splice site and augments its utilization relative to that observed with the wild-type U2 or U6 snRNAs . In a previous study, we found that the same U6 mutation suppressed the effects of an A-to-G branch site mutation in an allele-specific fashion . The ability of U6-U57 mutants to influence the fidelity of both branch site and 3' splice site recognition suggests that this nucleotide may participate in the formation of the active site(s) of the spliceosome. Cell, 2000 Jul 21, 102(2), 245 - 55 Zip3 provides a link between recombination enzymes and synaptonemal complex proteins; Agarwal S et al.; In budding yeast, absence of the meiosis-specific Zip3 protein (also known as Cst9) causes synaptonemal complex formation to be delayed and incomplete . The Zip3 protein colocalizes with Zip2 at discrete foci on meiotic chromosomes, corresponding to the sites where synapsis initiates . Observations suggest that Zip3 promotes synapsis by recruiting the Zip2 protein to chromosomes and/or stabilizing the association of Zip2 with chromosomes . Zip3 interacts with a number of gene products involved in meiotic recombination, including proteins that act at both early (Mre11, Rad51, and Rad57) and late (Msh4 and Msh5) steps in the exchange process . We speculate that Zip3 is a component of recombination nodules and serves to link the initiation of synapsis to meiotic recombination. J Biol Chem, 2000 Nov 17, 275(46), 36341 - 9 Lysyl oxidase activates the transcription activity of human collagene III promoter . Possible involvement of Ku antigen; Giampuzzi M et al.; Lysyl oxidase is an extracellular enzyme that controls the maturation of collagen and elastin . Lysyl oxidase and collagen III often show similar expression patterns in fibrotic tissues . Therefore, we investigated the influence of lysyl oxidase overexpression on the promoter activity of human COL3A1 gene . Our results showed that when COS-7 cells overexpressed the mature form of lysyl oxidase, the activity of the human COL3A1 promoter was increased up to an average of 12 times when tested by luciferase reporter assay . The effect was specific, because other promoters were not affected . Moreover, lysyl oxidase effect was abolished by beta-aminopropionitrile, a specific inhibitor of its catalytic activity . Electrophoretic mobility shift assay showed a binding activity in the region from -101 to -77 that was significantly increased by lysyl oxidase overexpression . The binding was specifically competed by the cold probe, and the mutagenesis of this region abolished both the binding activity in gel retardation and lysyl oxidase stimulation of COL3A1 promoter in transfection experiments . We identified the binding activity as Ku antigen in its two components: Ku80 and Ku70 . This study suggests a new coordinated mechanism by which lysyl oxidase might control the development of fibrosis. Hum Mol Genet, 2000 Aug 12, 9(13), 1995 - 9 Identification of PEX3 as the gene mutated in a Zellweger syndrome patient lacking peroxisomal remnant structures; Shimozawa N et al.; Peroxisome biogenesis disorders, of which 13 complementation groups have been identified, are subdivided with regard to two major dysfunctions: peroxisomal matrix protein import and peroxisomal membrane synthesis . Detectable remnant membrane structures are evident only in the former . Molecular defects have been defined in 10 PEX genes, including eight related to protein import and two to membrane synthesis . We now have evidence that the human complete cDNA encoding Pex3p, a peroxisomal membrane protein (PMP) factor for the proper localization of PMPs, rescues the import of both PMP and the matrix protein in fibroblasts from a Zellweger syndrome patient of complementation group G . This patient was homozygous for a 1 base insertion in the codon for V182, which resulted in a change of codon (182-183) and introduced a termination codon (184), which inactivated PMP and matrix protein import by Pex3p . A PEX3-defective CHO mutant clone, ZPG208, was of the same complementation group as group G. Biotechnol Bioeng, 2000 Oct 5, 70(1), 41 - 53 Characterization of an encapsulation device for the production of monodisperse alginate beads for cell immobilization; Serp D et al.; An encapsulation device, designed on the basis of the laminar jet break-up technique, is characterized for cell immobilization with different types of alginate . The principle of operation of the completely sterilizable encapsulator, together with techniques for the continuous production of beads from 250 microm to 1 mm in diameter, with a size distribution below 5%, at a flow rate of 1-15 mL/min, is described . A modification of the device, to incorporate an electrostatic potential between the alginate droplets and an internal electrode, results in enhanced monodispersity with no adverse effects on cell viability . The maximum cell loading capacity of the beads strongly depends on the nozzle diameter as well as the cells used . For the yeast Phaffia rhodozyma, it is possible to generate 700 microm alginate beads with an initial cell concentration of 1 x 10(8) cells/mL of alginate whereas only 1 x 10(6) cells/ml could be entrapped within 400 microm beads . The alginate beads have been characterized with respect to mechanical resistance and size distribution immediately after production and as a function of storage conditions . The beads remain stable in the presence of acetic acid, hydrochloric acid, water, basic water, and sodium ions . The latter stability applies when the ratio of sodium: calcium ions is less than 1/5 . Complexing agents such as sodium citrate result in the rapid solubilization of the beads due to calcium removal . The presence of cells does not affect the mechanical resistance of the beads . Finally, the mechanical resistance of alginate beads can be doubled by treatment with 5-10 kDa chitosan, resulting in reduced leaching of cells . Gene, 2000 Aug 8, 253(2), 189 - 96 Identification and molecular analysis of BANP; Birot A et al.; BTG3 belongs to a family of structurally related genes whose biochemical functions remain elusive . In order to investigate the mechanism underlying BTG3-mediated functions, we tried to identify BTG3 potential partners . The use of the yeast 'two-hybrid system', with BTG3 as bait, enabled us to isolate BANP (BTG3 Associated Nuclear Protein) . Other commonly used protein-binding assays did not confirm this yeast interaction . However, BANP had never been described before, and this prompted us to further characterise this gene . In this paper, we present data on its molecular organization in mouse, then we speculate on the nature of this nuclear protein, and finally we localise BANP on the human chromosome 16q24 subregion; we discuss the fact that frequent loss of heterozygosity within this region has been observed in different tumours. FEBS Lett, 2000 Aug 11, 479(1-2), 15 - 8 High affinity insulin binding by soluble insulin receptor extracellular domain fused to a leucine zipper; Hoyne PA et al.; Insulin receptors (IRs) that are truncated at the end of the ectodomain form dimers that bind insulin with different characteristics to wild type receptors . These soluble IRs have lowered affinity for insulin compared with full-length IR, and exhibit linear Scatchard plots in contrast to the curvilinear plots obtained with full-length IR, IR truncated at the C-terminus of the transmembrane region and IR ectodomains fused to the self-associating constant domains from Fc or lambda immunoglobulins . In this report, we have fused the IR ectodomain to the 33 residue leucine zipper from the transcriptional activator GCN4 of Saccharomyces cerevisiae . This fusion protein binds insulin with high affinity in a manner comparable to native receptor . The respective dissociation constants were Kd1 8.2 X 10(-11) M and Kd2 1.6 x 10(-8) M for hIRedZip and Kd1 5.7 x 10(-11) M and Kd2 6.3 x 10(-9) M for membrane-anchored, native receptor. J Biol Chem, 2000 Nov 24, 275(47), 36541 - 9 Structural basis for the species-specific activity of TFIIS; Shimasaki NB et al.; Many proteins involved in eukaryotic transcription are similar in function and in sequence between organisms . Despite the sequence similarities, there are many factors that do not function across species . For example, transcript elongation factor TFIIS is highly conserved among eukaryotes, and yet the TFIIS protein from Saccharomyces cerevisiae cannot function with mammalian RNA polymerase II and vice versa . To determine the reason for this species specificity, chimeras were constructed linking three structurally independent regions of the TFIIS proteins from yeast and human cells . Two independently folding domains, II and III, have been examined previously using NMR () . Yeast domain II alone is able to bind yeast RNA polymerase II with the same affinity as the full-length TFIIS protein, and this domain was expected to confer the species selectivity . Domain III has previously been shown to be readily exchanged between mammalian and yeast factors . However, the results presented here indicate that domain II is insufficient to confer species selectivity, and a primary determinant lies in a 30-amino acid highly conserved linker region connecting domain II with domain III . These 30 amino acids may physically orient domains II and III to support functional interactions between TFIIS and RNA polymerase II. J Biol Chem, 2000 Nov 17, 275(46), 35727 - 33 Tripartite regulation of Gln3p by TOR, Ure2p, and phosphatases; Bertram PG et al.; Gln3p is a GATA-type transcription factor responsive to different nitrogen nutrients and starvation in yeast Saccharomyces cerevisiae . Recent evidence has linked TOR signaling to Gln3p . Rapamycin causes dephosphorylation and nuclear translocation of Gln3p, thereby activating nitrogen catabolite repressible-sensitive genes . However, a detailed mechanistic understanding of this process is lacking . In this study, we show that Tor1p physically interacts with Gln3p . An intact TOR kinase domain is essential for the phosphorylation of Gln3p, inhibition of Gln3p nuclear entry and repression of Gln3p-dependent transcription . In contrast, at least two distinct protein phosphatases, Pph3p and the Tap42p-dependent phosphatases, are involved in the activation of Gln3p . The yeast pro-prion protein Ure2p binds to both hyper- and hypo-phosphorylated Gln3p . In contrast to the free Gln3p, the Ure2p-bound Gln3p is signifcantly resistant to dephosphorylation . Taken together, these results reveal a tripartite regulatory mechanism by which the phosphorylation of Gln3p is regulated. Annu Rev Biophys Biomol Struct, 2000, 29, 81 - 103 GCN5-related N-acetyltransferases: a structural overview; Dyda F et al.; Hundreds of acetyltransferases exist . All use a common acetyl donor--acetyl coenzyme A--and each exhibits remarkable specificity for acetyl acceptors, which include small molecules and proteins . Analysis of the primary sequences of these enzymes indicates that they can be sorted into several superfamilies . This review covers the three-dimensional structures of members of one of these superfamilies, now referred to in the literature as the GCN5-related N-acetyltransferases (GNAT), reflecting the importance of one functional category, the histone acetyltransferases . Despite the diversity of substrate specificities, members of the GNAT superfamily demonstrate remarkable similarity in protein topology and mode of acetyl coenzyme A binding, likely reflecting a conserved catalytic mechanism. Anal Chem, 2000 Jul 15, 72(14), 3349 - 54 Utility of accurate mass tags for proteome-wide protein identification; Conrads TP et al.; An enabling capability for proteomics would be the ability to study protein expression on a global scale . While several different separation and analysis options are being investigated to advance the practice of proteomics, mass spectrometry (MS) is rapidly becoming the core instrumental technology used to characterize the large number of proteins that constitute a proteome . To be most effective, proteomic measurements must be high-throughput, ideally allowing thousands of proteins to be identified on a time scale of hours . Most strategies of identification by MS rely on the analysis of enzymatically produced peptides originating from an isolated protein followed by either peptide mapping or tandem MS (MS/MS) to obtain sequence information for a single peptide . In the case of peptide mapping, several peptide masses are needed to unambiguously identify a protein with the typically achieved mass measurement accuracies (MMA) . The ability to identify proteins based on the mass of a single peptide (i.e., an accurate mass tag; AMT) is proposed and is largely dependent on the MMA that can be achieved . To determine the MMA necessary to enable the use of AMTs for proteome-wide protein identification, we analyzed the predicted proteins and their tryptic fragments from Saccharomyces cerevisiae and Caenorhabditis elegans . The results show that low ppm (i.e., approximately 1 ppm) level measurements have practical utility for analysis of small proteomes . Additionally, up to 85% of the peptides predicted from these organisms can function as AMTs at sub-ppm MMA levels attainable using Fourier transform ion cyclotron resonance MS . Additional information, such as sequence constraints, should enable even more complex proteomes to be studied at more modest mass measurement accuracies . Once AMTs are established, subsequent high-throughput measurements of proteomes (e.g., after perturbations) will be greatly facilitated. Plant Physiol, 2000 Aug, 123(4), 1449 - 58 Ethylene perception by the ERS1 protein in Arabidopsis; Hall AE et al.; Ethylene perception in Arabidopsis is controlled by a family of five genes, including ETR1, ERS1 (ethylene response sensor 1), ERS2, ETR2, and EIN4 . ERS1, the most highly conserved gene with ETR1, encodes a protein with 67% identity to ETR1 . To clarify the role of ERS1 in ethylene sensing, we biochemically characterized the ERS1 protein by heterologous expression in yeast . ERS1, like ETR1, forms a membrane-associated, disulfide-linked dimer . In addition, yeast expressing the ERS1 protein contains ethylene-binding sites, indicating ERS1 is also an ethylene-binding protein . This finding supports previous genetic evidence that isoforms of ETR1 also function in plants as ethylene receptors . Further, we used the ethylene antagonist 1-methylcyclopropene (1-MCP) to characterize the ethylene-binding sites of ERS1 and ETR1 . We found 1-MCP to be both a potent inhibitor of the ethylene-induced seedling triple response, as well as ethylene binding by yeast expressing ETR1 and ERS1 . Yeast expressing ETR1 and ERS1 showed nearly identical sensitivity to 1-MCP, suggesting that the ethylene-binding sites of ETR1 and ERS1 have similar affinities for ethylene. J Biol Chem, 2000 Nov 24, 275(47), 36550 - 5 Mismatch recognition and DNA-dependent stimulation of the ATPase activity of hMutSalpha is abolished by a single mutation in the hMSH6 subunit; Dufner P et al.; The most abundant mismatch binding factor in human cells, hMutSalpha, is a heterodimer of hMSH2 and hMSH6, two homologues of the bacterial MutS protein . The C-terminal portions of all MutS homologues contain an ATP binding motif and are highly conserved throughout evolution . Although the N termini are generally divergent, they too contain short conserved sequence elements . A phenylalanine --> alanine substitution within one such motif, GXFY(X)(5)DA, has been shown to abolish the mismatch binding activity of the MutS protein of Thermus aquaticus (Malkov, V . A., Biswas, I., Camerini-Otero, R . D., and Hsieh, P . (1997) J . Biol . Chem . 272, 23811-23817) . We introduced an identical mutation into one or both subunits of hMutSalpha . The Phe --> Ala substitution in hMSH2 had no effect on the biological activity of the heterodimer . In contrast, the in vitro mismatch binding and mismatch repair functions of hMutSalpha were severely attenuated when the hMSH6 subunit was mutated . Moreover, this variant heterodimer also displayed a general DNA binding defect . Correspondingly, its ATPase activity could not be stimulated by either heteroduplex or homoduplex DNA . Thus the N-terminal portion of hMSH6 appears to impart on hMutSalpha not only the specificity for recognition and binding of mismatched substrates but also the ability to bind to homoduplex DNA. Mol Cell Biol, 2000 Sep, 20(17), 6476 - 82 The Rad51 paralog Rad51B promotes homologous recombinational repair; Takata M et al.; The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination . Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51 . In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out . RAD51B(-/-) cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle . Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus . RAD51B(-/-) cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to gamma-rays . The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B(-/-) cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR . These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity. Mol Cell Biol, 2000 Sep, 20(17), 6399 - 409 Sgs1 helicase activity is required for mitotic but apparently not for meiotic functions; Miyajima A et al.; The SGS1 gene of Saccharomyces cerevisiae is a homologue for the Bloom's syndrome and Werner's syndrome genes . The disruption of the SGS1 gene resulted in very poor sporulation, and the majority of the cells were arrested at the mononucleated stage . The recombination frequency measured by a return-to-growth assay was reduced considerably in sgs1 disruptants . However, double-strand break formation, which is a key event in the initiation of meiotic DNA recombination, occurred; crossover and noncrossover products were observed in the disruptants, although the amounts of these products were slightly decreased compared with those in wild-type cells . The spores produced by sgs1 disruptants showed relatively high viability . The sgs1 spo13 double disruptants sporulated poorly, like the sgs1 disruptants, but spore viability was reduced much more than with either sgs1 or spo13 single disruptants . Disruption of the RED1 or RAD17 gene partially alleviated the poor-sporulation phenotype of sgs1 disruptants, indicating that portions of the population of sgs1 disruptants are blocked by the meiotic checkpoint . The poor sporulation of sgs1 disruptants was complemented with a mutated SGS1 gene encoding a protein lacking DNA helicase activity; however, the mutated gene could suppress neither the sensitivity of sgs1 disruptants to methyl methanesulfonate and hydroxyurea nor the mitotic hyperrecombination phenotype of sgs1 disruptants. Mol Cell Biol, 2000 Sep, 20(17), 6354 - 63 Coupling of PAK-interacting exchange factor PIX to GIT1 promotes focal complex disassembly; Zhao ZS et al.; The p21-activated kinase PAK is targeted to focal complexes (FCs) through interactions with the SH3 domains of the PAK-interacting exchange factor PIX and Nck . PIX is a Rac GTP exchange factor that also binds the G-protein-coupled receptor kinase-interacting protein known as GIT1 . Overexpression of GIT1 in fibroblasts or epithelial cells causes a loss of paxillin from FCs and stimulates cell motility . This is due to the direct interaction of a C-terminal 125-residue domain of GIT1 with paxillin, under the regulation of PIX . In its activated state, GIT1 can promote FC disassembly independent of actin-myosin contractile events . Additionally, GIT directly couples to a key component of FCs, focal adhesion kinase (FAK), via a conserved Spa2 homology domain . We propose that GIT1 and FAK cooperate to promote motility both by directly regulating focal complex dynamics and by the activation of Rac. Mol Cell Biol, 2000 Sep, 20(17), 6244 - 58 Gic2p may link activated Cdc42p to components involved in actin polarization, including Bni1p and Bud6p (Aip3p); Jaquenoud M et al.; Gic2p is a Cdc42p effector which functions during cytoskeletal organization at bud emergence and in response to pheromones, but it is not understood how Gic2p interacts with the actin cytoskeleton . Here we show that Gic2p displayed multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting that Gic2p may regulate their function in vivo . In support of this idea, Gic2p cofractionated with Bud6p and Spa2p and interacted with Bud6p by coimmunoprecipitation and two-hybrid analysis . Importantly, localization of Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the Gic proteins but did not require an intact actin cytoskeleton . We identified a conserved domain in Gic2p which was necessary for its polarization function but dispensable for binding to Cdc42p-GTP and its localization to the site of polarization . Expression of a mutant Gic2p harboring a single-amino-acid substitution in this domain (Gic2p(W23A)) interfered with polarized growth in a dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the incipient bud site . We propose that at bud emergence, Gic2p functions as an adaptor which may link activated Cdc42p to components involved in actin organization and polarized growth, including Bni1p, Spa2p, and Bud6p. Mol Cell Biol, 2000 Sep, 20(17), 6212 - 23 Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders; Hemmings-Mieszczak M et al.; We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M . Hemmings-Mieszczak and T . Hohn, RNA 5:1149-1157, 1999) . We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro . We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream . Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting . Furthermore, the stop codon context must not impede downstream reinitiation . Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI) . Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated . Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi. J Biol Chem, 2000 Nov 10, 275(45), 35506 - 11 RNA polymerase II subunit Rpb9 regulates transcription elongation in vivo; Hemming SA et al.; RNA polymerase II lacking the Rpb9 subunit uses alternate transcription initiation sites in vitro and in vivo and is unable to respond to the transcription elongation factor TFIIS in vitro . Here, we show that RPB9 has a synthetic phenotype with the TFIIS gene . Disruption of RPB9 in yeast also resulted in sensitivity to 6-azauracil, which is a phenotype linked to defects in transcription elongation . Expression of the TFIIS gene on a high-copy plasmid partially suppressed the 6-azauracil sensitivity of Deltarpb9 cells . We set out to determine the relevant cellular role of yeast Rpb9 by assessing the ability of 20 different site-directed and deletion mutants of RPB9 to complement the initiation and elongation defects of Deltarpb9 cells in vivo . Rpb9 is composed of two zinc ribbons . The N-terminal zinc ribbon restored the wild-type pattern of initiation start sites, but was unable to complement the growth defects associated with defects in elongation . Most of the site-directed mutants complemented the elongation-specific growth phenotypes and reconstituted the normal pattern of transcription initiation sites . The anti-correlation between the growth defects of cells disrupted for RPB9 and the selection of transcription start sites suggests that this is not the primary cellular role for Rpb9 . Genome-wide transcription profiling of Deltarpb9 cells revealed only a few changes, predominantly in genes related to metabolism. Biol Chem, 2000 May-Jun, 381(5-6), 487 - 95 Retrieval of the mrp2 gene encoded conjugate export pump from the canalicular membrane contributes to cholestasis induced by tert-butyl hydroperoxide and chloro-dinitrobenzene; Schmitt M et al.; Oxidative stress is known to induce cholestasis, but the underlying mechanisms are poorly understood . In this study we have characterized the short-term effects of tert-butyl hydroperoxide (t-BOOH)- and 1-chloro-2,4-dinitrobenzene (CDNB) on the mrp2 gene encoded canalicular export pump (Mrp2) . The effects of t-BOOH and CDNB on bile formation, tissue GSH levels and subcellular Mrp2 localization were studied in perfused rat liver . Both, t-BOOH (0.5 mM) and CDNB (0.1 mM) induced within 60 min a decrease of hepatic GSH levels by more than 90% and an almost complete cessation of bile flow . As revealed by confocal laser scanning microscopy, this cholestasis was accompanied by a loss of immunoreactive MRP2 from the canalicular membrane and its appearance inside the hepatocytes in putative intracellular vesicles . On the other hand, the intracellular distribution of dipeptidyl peptidase IV (DPPIV), another canalicular protein, and of zonula occludens associated polypeptide (ZO-1) remained unaffected, indicating selectivity of the Mrp2 retrieval pattern . Both, t-BOOH and CDNB induced a rapid net K+ efflux from the liver and a significant decrease of liver cell hydration . We conclude that severe glutathione depletion induces cholestasis by a retrieval of Mrp2, but not of DPPIV from the canalicular membrane . The underlying mechanism is unclear; however, a decrease in liver cell hydration, which occurs under these conditions, may contribute to this effect. Biol Chem, 2000 May-Jun, 381(5-6), 453 - 6 Msb4p, a protein involved in Cdc42p-dependent organization of the actin cytoskeleton, is a Ypt/Rab-specific GAP; Albert S et al.; Ypt/Rab proteins of the Ras superfamily are regulators of protein transport in exo- and endocytosis . Like Ras and Rho proteins, they have a slow intrinsic GTPase activity that can be accelerated by several orders of magnitude by GTPase-activating proteins (GAP) . Here we describe a new member of a family of Ypt/Rab-specific GAPs, Msb4p/Gyp4p, that shares with other Gyp family members significant homology in the catalytic domain, recently identified in Gyp1p and Gyp7p . Purified Msb4p/Gyp4p acts primarily on Sec4p, Ypt6p and Ypt7p and might have a role in polarized secretion. Biol Chem, 2000 May-Jun, 381(5-6), 427 - 31 A conserved Gbeta binding (GBB) sequence motif in Ste20p/PAK family protein kinases; Leberer E et al.; Serine/threonine protein kinases of the Ste20p/PAK family are highly conserved from yeast to man . These protein kinases have been implicated in the signaling from heterotrimeric G proteins to mitogen-activated protein (MAP) kinase cascades and to cytoskeletal components such as myosin-I . In the yeast Saccharomyces cerevisiae, Ste20p is involved in transmitting the mating-pheromone signal from the betagamma-subunits of a heterotrimeric G protein to a downstream MAP kinase cascade . We have previously shown that binding of the G-protein beta-subunit (Gbeta) to a short binding site in the non-catalytic carboxy-terminal region of Ste20p is essential fortransmitting the pheromone signal . In this study, we searched protein sequence databases for sequences that are similar to the Gbeta binding site in Ste20p . We identified a sequence motif with the consensus sequence S S L phi P L I/V x phi phi beta (x: any residue; phi: A, I, L, S, or T; beta: basic residues) that is solely present in members of Ste20p/PAK family protein kinases . We propose that this sequence motif, which we have designated GBB (Gbeta binding) motif, is specifically responsible for binding of Gbeta to Ste20p/PAK protein kinases in response to activation of heterotrimeric G protein coupled receptors . Thus, the GBB motif is a novel type of signaling domain that serves to link protein kinases of the Ste20p/PAK family to G protein coupled receptors. Ital J Neurol Sci, 1999 Dec, 20(6), 401 - 8 Nuclear gene defects in mitochondrial disorders; Zeviani M et al.; An increasing number of nuclear genes have been associated with abnormalities of oxidative phosphorylation and mitochondrial disorders . The protein products of these genes can be grouped into three categories: structural components of the respiratory chain, factors influencing the structural integrity or the copy number of mitochondrial DNA, and proteins which control the formation, assembly and turnover of the respiratory complexes . Loss-of-function mutations in SURF-1, a gene belonging to the third category, have been associated with Leigh syndrome with cytochrome c oxidase deficiency . Mature Surf-1 protein (Surf-1p) is a 30 kDa hydrophobic polypeptide whose function is still unknown . Using antibodies against human Surf-1p, we demonstrated that this protein is imported into mitochondria as a larger precursor . The same analysis revealed that no protein is present in cell lines harboring loss-of-function mutations of SURF-1, regardless of their type and position . We also generated several constructs with truncated or partially deleted SURF-1 cDNAs . None of these constructs, expressed into SURF-1 null mutant cells, were able to rescue the COX phenotype, suggesting that different regions of the protein are all essential for function . Finally, experiments based on 2D gel electrophoresis indicated that assembly of COX in SURF-1 null mutants is blocked at an early step, most likely before the incorporation of subunit II in the nascent intermediates composed of subunit I alone or subunit I plus subunit IV. Nat Cell Biol, 2000 Aug, 2(8), E153 - 7 Evolution and function of ubiquitin-like protein-conjugation systems; Hochstrasser M; Ubiquitin functions by covalently modifying other proteins . In the past few years, a surprising number of other proteins have been identified that, despite often being only slightly similar to ubiquitin, can also be attached to proteins . Newly discovered parallels between the activation of ubiquitin and the biosynthesis of certain enzyme cofactors now hint at the possible evolutionary origins of the ubiquitin system. Nat Cell Biol, 2000 Aug, 2(8), E139 - 41 Ubiquitin biology: an old dog learns an old trick; Pickart CM; Regulated protein degradation in eukaryotes occurs principally through covalent tagging of substrates with ubiquitin, thereby targeting them for destruction by 26S proteasomes . Classical allostery has now been added to the repertoire of mechanisms that can modulate ubiquitin tagging, allowing feed-forward regulation to be achieved through targeted protein destruction. Nat Cell Biol, 2000 Aug, 2(8), 492 - 9 Cohesin ensures bipolar attachment of microtubules to sister centromeres and resists their precocious separation; Tanaka T et al.; The multisubunit protein complex cohesin is required to establish cohesion between sister chromatids during S phase and to maintain it during G2 and M phases . Cohesin is essential for mitosis, and even partial defects cause very high rates of chromosome loss . In budding yeast, cohesin associates with specific sites which are distributed along the entire length of a chromosome but are more dense in the vicinity of the centromere . Real-time imaging of individual centromeres tagged with green fluorescent protein suggests that cohesin bound to centromeres is important for bipolar attachment to microtubules . This cohesin is, however, incapable of resisting the consequent force, which leads to sister centromere splitting and chromosome stretching . Meanwhile, cohesin bound to sequences flanking the centromeres prevents sister chromatids from completely unzipping and is required to pull back together sister centromeres that have already split . Cohesin therefore has a central role in generating a dynamic tension between microtubules and sister chromatid cohesion at centromeres, which lasts until chromosome segregation is finally promoted by separin-dependent cleavage of the cohesin subunit Scc1p. J Biol Chem, 2000 Nov 3, 275(44), 34744 - 9 Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2C alpha and beta 2 isoforms; Cheng A et al.; We previously reported that the activating phosphorylation on cyclin-dependent kinases in yeast (Cdc28p) and in humans (Cdk2) is removed by type 2C protein phosphatases . In this study, we characterize this PP2C-like activity in HeLa cell extract and determine that it is due to PP2C beta 2, a novel PP2C beta isoform, and to PP2C alpha . PP2C alpha and PP2C beta 2 co-purified with Mg(2+)-dependent Cdk2/Cdk6 phosphatase activity in DEAE-Sepharose, Superdex-200, and Mono Q chromatographies . Moreover, purified recombinant PP2C alpha and PP2C beta 2 proteins efficiently dephosphorylated monomeric Cdk2/Cdk6 in vitro . The dephosphorylation of Cdk2 and Cdk6 by PP2C isoforms was inhibited by the binding of cyclins . We found that the PP2C-like activity in HeLa cell extract, partially purified HeLa PP2C alpha and PP2C beta 2 isoforms, and the recombinant PP2Cs exhibited a comparable substrate preference for a phosphothreonine containing substrate, consistent with the conservation of threonine residues at the site of activating phosphorylation in CDKs. J Biol Chem, 2000 Oct 6, 275(40), 30801 - 5 Functional interaction between the p160 coactivator proteins and the transcriptional enhancer factor family of transcription factors; Belandia B et al.; SRC1, initially identified as a nuclear receptor coactivator, was found to interact with a member of the transcriptional enhancer factor (TEF) family of transcription factors, TEF-4 . The interaction, which occurs in both intact cells and in a cell-free system, is mediated by the highly conserved basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domain present in the N-terminal region of SRC1 . Moreover, all three members of the p160 family of nuclear receptor coactivators, SRC1, TIF2, and RAC3, are able to potentiate transcription from a TEF response element in transient transfection experiments, and this activation requires the presence of the bHLH-PAS domain . These results suggest that the p160 proteins could be bona fide coactivators of the TEF family of transcription factors. Arch Biochem Biophys, 2000 Aug 15, 380(2), 303 - 8 The decreased in vivo clearance of CYP2D6 substrates by CYP2D6*10 might be caused not only by the low-expression but also by low affinity of CYP2D6; Fukuda T et al.; CYP2D6 exhibits genetic polymorphism with interindividual differences in metabolic activity . We have found a significant influence on the pharmacokinetics of venlafaxine by the CYP2D6*10 allele in a Japanese population . CYP2D6.10, which is translated from CYP2D6*10, has two amino acid substitutions: Pro34 --> Ser and Ser486 --> Thr . In this study, CYP2D6.10 was expressed in Saccharomyces cerevisiae and its catalytic activity for CYP2D6 substrates was investigated . The CYP2D6*10B- and *10C-associated cDNA were isolated from human lymphocyte genotyped as CYP2D6*10 . In addition, three forms of CYP2D6, Pro34/Thr486 (PT), Ser34/Ser486 (SS), and Pro34/Ser486 (wild type, CYP2D6.1), were constructed by PCR-site mutagenesis to clarify the effects of the two amino-acid substitutions . The expression of CYP2D6 protein was confirmed by immunoblotting using CYP2D antibody . The absorbance at 450 nm was measured by CO-reduced difference spectra from five all microsome preparations . The CYP2D6 forms with Pro34 --> Ser amino acid substitution were at a lower expression than CYP2D6.1 from the findings of immunoblotting and spectral analysis . The apparent K(m) values of CYP2D6.1, CYP2D6.10A, and CYP2D6.10C were 1.7, 8.5, and 49.7 microM, respectively, for bufuralol 1'-hydroxylation, and 9.0, 51.9, and 117.4 microM, respectively, for venlafaxine O-demethylation, respectively . The V(max) values were not significantly different among the three variants . These findings suggest that the decreased in vivo clearance by CYP2D6*10 was caused not only by low expression of but also the increased K(m) value of CYP2D6 . Anal Biochem, 2000 Aug 15, 284(1), 42 - 8 In vivo determination of substrate specificity of hepatitis C virus NS3 protease: genetic assay for site-specific proteolysis; Kim SY et al.; Hepatitis C virus (HCV) NS3 protease is responsible for the processing of the viral polyprotein and is considered as a primary target for the development of anti-HCV therapy . We have developed a genetic method in yeast to screen for good substrate sequences of the NS3 protease . A library of fusion proteins was constructed with a transcription factor, GAL4, linked to the intracellular domain of an integral membrane protein, STE2, by a randomized protease substrate sequence . In yeast cells expressing NS3 protease, the substrate sequences in the fusion proteins were specifically recognized and cleaved . This cleavage resulted in the release of GAL4 from the cytoplasmic membrane and the subsequent activation of reporter genes by GAL4, which was detected by the growth of yeast cells on selective media . Based on the analysis of 69 isolated substrate sequences, a consensus sequence was deduced: (Glu/Asp)-X-Val-Val-(Leu/Pro)-Cys / (Ser/Ala), with the scissile bond being located between Cys and Ser or Ala and X not being determined . This is largely consistent with the previous results obtained by biochemical methods . An oligopeptide containing the deduced sequence was highly efficiently cleaved in vitro by the purified NS3 protease . These data demonstrated that the present genetic method could be used as an efficient tool for the in vivo determination of substrate specificity of proteases . Biochemistry, 2000 Aug 15, 39(32), 9909 - 16 Effects of 5' leader and 3' trailer structures on pre-tRNA processing by nuclear RNase P; Ziehler WA et al.; Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader preceding the aminoacyl acceptor stem and a 3' trailer extending beyond this stem . An early step in pre-tRNA maturation is removal of the 5' leader by the endoribonuclease, RNase P . Extensive pairing between leader and trailer sequences has previously been demonstrated to block RNase P cleavage, suggesting that the 5' leader and 3' trailer sequences might need to be separated for the substrate to be recognized by the eukaryotic holoenzyme . To address whether the nuclear RNase P holoenzyme recognizes the 5' leader and 3' trailer sequences independently, interactions of RNase P with pre-tRNA(Tyr) containing either the 5' leader, the 3' trailer, or both were examined . Kinetic analysis revealed little effect of the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage using the various pre-tRNA derivatives . However, the presence of a 3' trailer that pairs with the 5' leader increases the K(m) of pre-tRNA slightly, in agreement with previous results . Similarly, competition studies demonstrate that removal of a complementary 3' trailer lowers the apparent K(I), consistent with the structure between these two sequences interfering with their interaction with the enzyme . Deletion of both the 5' and 3' extensions to give mature termini resulted in the least effective competitor . Further studies showed that the nuclear holoenzyme, but not the B . subtilis holoenzyme, had a high affinity for single-stranded RNA in the absence of attached tRNA structure . The data suggest that yeast nuclear RNase P contains a minimum of two binding sites involved in substrate recognition, one that interacts with tRNA and one that interacts with the 3' trailer . Furthermore, base pairing between the 5' leader and 3' trailer hinders recognition. Immunity, 2000 Jul, 13(1), 85 - 94 Cabin1 represses MEF2-dependent Nur77 expression and T cell apoptosis by controlling association of histone deacetylases and acetylases with MEF2; Youn HD et al.; TCR signaling leading to thymocyte apoptosis is mediated through the expression of the Nur77 family of orphan nuclear receptors . MEF2 has been shown to be the major transcription factor responsible for calcium-dependent Nur77 transcription . Cabin1 was recently identified as a transcriptional repressor of MEF2, which can be released from MEF2 in a calcium-dependent fashion . The molecular basis of repression of MEF2 by Cabin1, however, has remained unknown . We report that Cabin1 represses MEF2 by two distinct mechanisms . Cabin1 recruits mSin3 and its associated histone deacetylases 1 and 2; Cabin1 also competes with p300 for binding to MEF2 . Thus, activation of MEF2 and the consequent transcription of Nur77 are controlled by the association of MEF2 with the histone deacetylases via the calcium-dependent repressor Cabin1. Nucleic Acids Res, 2000 Aug 15, 28(16), 3160 - 7 Combinatorial regulation of phospholipid biosynthetic gene expression by the UME6, SIN3 and RPD3 genes; Elkhaimi M et al.; The Ume6p-Sin3p-Rpd3p complex negatively regulates expression of genes containing a Ume6p binding site . However, these regulatory proteins also function independently to regulate gene expression both negatively and positively . The model system for this combinatorial regulation is the yeast phospholipid biosynthetic pathway . Sin3p negatively regulates the INO1, CHO1, CHO2 and OPI3 genes while Ume6p negatively regulates the INO1 gene and positively regulates the other genes . We have suggested that the positive regulation results from indirect effects on expression of the INO2 transcriptional activator gene . Here, we demonstrate that the effect of Ume6p on INO2 gene expression is also indirect . We also show that Rpd3p is a negative regulator of phospholipid biosynthetic gene expression . The ability of Ume6p, Sin3p and Rpd3p to differentially regulate expression of the phospholipid biosynthetic genes affects phospholipid composition . A sin3 mutant strain lacks detectable levels of phosphatidylethanolamine and elevated levels of phosphatidylcholine (PC) and a rpd3 mutant strain has reduced levels of PC . These alterations in membrane composition suggest that there may exist additional differences in regulation of phospholipid biosynthetic gene expression and that membrane compositions may be coordinated with other biological processes regulated by Ume6p, Sin3p and Rpd3p. Microbiology, 2000 Aug, 146 ( Pt 8), 1841 - 53 Fruiting body development in Coprinus cinereus: regulated expression of two galectins secreted by a non-classical pathway; Boulianne RP et al.; Fruiting body formation in the basidiomycete Coprinus cinereus is a developmental process that occurs as a response of the mycelium to external stimuli . First, localized, highly branched hyphal structures (knots) are formed as a reaction to nutritional depletion . Hyphal-knot formation is repressed by light; however, light signals are essential for the development of the hyphal knot into an embryonic fruiting body (primordium) as well as karyogamy, meiosis and fruiting body maturation . The role of the different environmental signals in the initial phases of fruiting body development was analysed . It was observed that two fungal galectins, Cgl1 and Cgl2, are differentially regulated during fruiting body formation . cgl2 expression initiated in early stages of fruiting body development (hyphal knot formation) and was maintained until maturation of the fruiting body, whereas cgl1 was specifically expressed in primordia and mature fruiting bodies . Immunofluorescence and immuno-electron microscopy studies detected galectins within specific fruiting body tissues . They localized in the extracellular matrix and the cell wall but also in membrane-bound bodies in the cytoplasm . Heterologous expression of Cgl2 in Saccharomyces cerevisiae indicated that secretion of this protein occurred independently of the classical secretory pathway. J Biol Chem, 2000 Oct 13, 275(41), 31914 - 20 Biochemical characterization of the TATA-binding protein-Gal4 activation domain complex; Xie Y et al.; It has been suggested that complexes between gene-specific activators and the TATA-binding protein (TBP) play an important role in the expression of many genes . However, few detailed studies of well defined activator-TBP complexes have been reported . An analysis of the biochemical properties of the complex formed by the acidic activation domain (AAD) of the yeast activator Gal4 and TBP is presented here . This is shown to be composed of two AAD and one TBP molecule . DNA binding experiments reveal that TATA-containing DNAs and the Gal4 AAD bind TBP competitively, suggesting that the AAD and TATA boxes recognize overlapping surfaces of TBP . The kinetics of the formation and dissociation of the AAD(2)-TBP complex is also probed . The impact of these findings on models for Gal4-mediated transcriptional activation is considered. Mol Microbiol, 2000 Aug, 37(3), 671 - 9 Individual functions of the HAK and TRK potassium transporters of Schwanniomyces occidentalis; Banuelos MA et al.; We have cloned the gene encoding the TRK transporter of the soil yeast Schwanniomyces occidentalis and obtained the HAK1 trk1 delta and the hak1 delta TRK1 mutant strains . Analyses of the transport capacities of these mutants have shown that (i) the HAK1 and the TRK1 potassium transporters are the only transporters operating at low and medium K+ concentrations (< 1 mM); (ii) the HAK1 transporter is functional at low pH but fails at high pH; and (iii) the TRK1 transporter functions at neutral and high pH and fails at low pH . At neutral pH, both transporters are functional, but HAK1 is not expressed, except at very low K+ concentrations (< 50 microM) where HAK1 is very effective . TRK1 is also involved in the control of the membrane potential. Mol Microbiol, 2000 Jun, 36(6), 1293 - 305 Upstream nucleosomes and Rgr1p are required for nucleosomal repression of transcription; Moss DR et al.; The mechanisms of transcription repression and derepression in vivo are not fully understood . We have obtained evidence that begins to clarify the minimum requirements for counteracting nucleosomal repression in vivo . Location of the TATA element near the nucleosome dyad does not block RNA polymerase II transcription in vivo if there is a nucleosome-free region located immediately upstream . However, location of the TATA element similarly within the nucleosome does block transcription if the region upstream of it is nucleosome bound . Histone H4 depletion derepresses transcription in the latter case, supporting the idea that the nucleosomes are responsible for the repression . These results raise the intriguing possibility that the minimum requirement for derepression of transcription in vivo is a nucleosome-free region upstream of the core promoter . Importantly, we find that a C-terminal deletion in RGR1, a component of the mediator/holoenzyme complex and a global repressor, can also derepress transcription. Mol Biol Cell, 2000 Aug, 11(8), 2617 - 29 The interaction between Ran and NTF2 is required for cell cycle progression; Quimby BB et al.; The small GTPase Ran is required for the trafficking of macromolecules into and out of the nucleus . Ran also has been implicated in cell cycle control, specifically in mitotic spindle assembly . In interphase cells, Ran is predominately nuclear and thought to be GTP bound, but it is also present in the cytoplasm, probably in the GDP-bound state . Nuclear transport factor 2 (NTF2) has been shown to import RanGDP into the nucleus . Here, we examine the in vivo role of NTF2 in Ran import and the effect that disruption of Ran imported into the nucleus has on the cell cycle . A temperature-sensitive (ts) mutant of Saccharomyces cerevisiae NTF2 that does not bind to Ran is unable to import Ran into the nucleus at the nonpermissive temperature . Moreover, when Ran is inefficiently imported into the nucleus, cells arrest in G(2) in a MAD2 checkpoint-dependent manner . These findings demonstrate that NTF2 is required to transport Ran into the nucleus in vivo . Furthermore, we present data that suggest that depletion of nuclear Ran triggers a spindle-assembly checkpoint-dependent cell cycle arrest. J Biol Chem, 2000 Nov 10, 275(45), 35200 - 8 Androgen receptor interacts with a novel MYST protein, HBO1; Sharma M et al.; The androgen receptor (AR), a member of the nuclear receptor superfamily, plays a central role in male sexual differentiation and prostate cell proliferation . Results of treating prostate cancer by androgen ablation indicate that signals mediated through AR are critical for the growth of these tumors . Like other nuclear receptors, AR exerts its transcriptional function by binding to cis-elements upstream of promoters and interacting with other transcriptional factors (e.g . activators, repressors and modulators) . To determine the mechanism of AR-regulated transcription, we used the yeast two-hybrid system to identify AR-associated proteins . One of the proteins we identified is identical to the human origin recognition complex-interacting protein termed HBO1 . A ligand-enhanced interaction between AR and HBO1 was further confirmed in vivo and in vitro . Immunofluorescence experiments showed that HBO1 is a nuclear protein, and Northern blot analysis revealed that it is ubiquitously expressed, with the highest levels present in human testis . HBO1 belongs to the MYST family, which is characterized by a highly conserved C2HC zinc finger and a putative histone acetyltransferase domain . Surprisingly, two yeast members of the MYST family, SAS2 and SAS3, have been shown to function as transcription silencers, despite the presence of the histone acetyltransferase domain . Using a GAL4 DNA-binding domain assay, we mapped a transcriptional repression domain within the N-terminal region of HBO1 . Transient transfection experiments revealed that HBO1 specifically repressed AR-mediated transcription in both CV-1 and PC-3 cells . These results indicate that HBO1 is a new AR-interacting protein capable of modulating AR activity . It could play a significant role in regulating AR-dependent genes in normal and prostate cancer cells. Cell, 2000 Jul 7, 102(1), 99 - 108 Structural basis for the interaction between FxFG nucleoporin repeats and importin-beta in nuclear trafficking; Bayliss R et al.; We describe the crystal structure of a complex between importin-beta residues 1-442 (Ib442) and five FxFG nucleoporin repeats from Nsp1p . Nucleoporin FxFG cores bind on the convex face of Ib442 to a primary site between the A helices of HEAT repeats 5 and 6, and to a secondary site between HEAT repeats 6 and 7 . Mutations at importin-beta Ile178 in the primary FxFG binding site reduce both binding and nuclear protein import, providing direct evidence for the functional significance of the importin-beta-FxFG interaction . The FxFG binding sites on importin-beta do not overlap with the RanGTP binding site . Instead, RanGTP may release importin-beta from FxFG nucleoporins by generating a conformational change that alters the structure of the FxFG binding site. Cell, 2000 Jul 7, 102(1), 21 - 31 A mechanism for coupling exit from mitosis to partitioning of the nucleus; Bardin AJ et al.; Exit from mitosis must not occur prior to partitioning of chromosomes between daughter cells . We find that the GTP binding protein Tem1, a regulator of mitotic exit, is present on the spindle pole body that migrates into the bud during S phase and mitosis . Tem1's exchange factor, Lte1, localizes to the bud . Thus, Tem1 and Lte1 are present in the same cellular compartment (the bud) only after the nucleus enters the bud during nuclear division . We also find that the presence of Tem1 and Lte1 in the bud is required for mitotic exit . Our results suggest that the spatial segregation of Tem1 and Lte1 ensures that exit from mitosis only occurs after the genetic material is partitioned between mother and daughter cell. Chromosoma, 2000 Jun, 109(3), 206 - 13 Cloning and characterisation of polymorphic heterochromatic segments of Brachycome dichromosomatica; Houben A et al.; After selective enrichment and differential hybridisation of Cot-1 DNA fractions of plants with and without polymorphic heterochromatic segments, a repetitive sequence (called Bds1) specific to the polymorphic chromosome segments of Brachycome dichromosomatica (Brachyscome dichromosomatica) was isolated . A single repeat unit of Bds1 is 92 bp long and is organised in tandem arrays at three different polymorphic segment sites on the chromosomes of cytodeme A2 . Although all three sites showed extensive polymorphism between plants, the karyotypes of all analysed mitotic root cells were stable within a single plant . Electron microscopy revealed heavily condensed chromatin structures at the most obvious polymorphic site . The mechanisms that generate and maintain the observed chromosome structure polymorphisms are discussed. Nucleic Acids Res, 2000 Aug 15, 28(16), 3056 - 64 CHL1 is a nuclear protein with an essential ATP binding site that exhibits a size-dependent effect on chromosome segregation; L Holloway S; Saccharomyces cerevisiae chl1 mutants have a significant increase in the rate of chromosome missegregation . CHL1 encodes a 99 kDa predicted protein with an ATP binding site consensus, a putative helix-turn-helix DNA binding motif, and homology to helicases . Using site-directed mutagenesis, I show that mutations that are predicted to abolish ATP binding in CHL1 inactivate its function in chromosome segregation . Furthermore, overexpression of these mutations interferes with chromosome transmission of a 125 kb chromosome fragment in a wild-type strain . Polyclonal antibodies against CHL1 show that CHL1 is predominantly in the nuclear fraction of S . CEREVISIAE: CHL1 function is more critical for the segregation of small chromosomes . In chl1Delta1/chl1Delta1 mutants, artificial circular or linear chromosomes <150 kb in size exhibit near random segregation (0.12 per cell division), whereas all chromosomes tested >225 kb were lost at rates (5 x 10(-)(3) per cell division) comparable to that observed for endogenous chromosome III . These results reveal an important role for ATPases/DNA helicases in chromosome segregation . Such enzymes may alter DNA topology to allow loading of proteins involved in maintaining sister chromatid cohesion. Plant J, 2000 Aug, 23(3), 375 - 83 Fungal cell wall phosphomannans facilitate the toxic activity of a plant PR-5 protein; Ibeas JI et al.; Osmotin is a plant PR-5 protein . It has a broad spectrum of antifungal activity, yet also exhibits specificity for certain fungal targets . The structural bases for this specificity remain unknown . We show here that full sensitivity of Saccharomyces cerevisiae cells to the PR-5 protein osmotin is dependent on the function of MNN2, MNN4 and MNN6 . MNN2 is an alpha-1, 2-mannosyltransferase catalyzing the addition of the first mannose to the branches on the poly l,6-mannose backbone of the outer chain of cell wall N-linked mannans . MNN4 and MNN6 are required for the transfer of mannosylphosphate to cell wall mannans . Null mnn2, mnn4 or mnn6 mutants lack phosphomannans and are defective in binding osmotin to the fungal cell wall . Both antimannoprotein antibody and the cationic dye alcian blue protect cells against osmotin cytotoxicity . MNN1 is an alpha-1,3-mannosyltransferase that adds the terminal mannose to the outer chain branches of N-linked mannan, masking mannosylphosphate . Null mnn1 cells exhibit enhanced osmotin binding and sensitivity . Several cell wall mannoproteins can bind to immobilized osmotin, suggesting that their polysaccharide constituent determines osmotin binding . Our results demonstrating a causal relationship between cell surface phosphomannan and the susceptibility of a yeast strain to osmotin suggest that cell surface polysaccharides of invading pathogens control target specificity of plant PR-5 proteins. Plant J, 2000 Jul, 23(2), 171 - 82 The roles of three functional sulphate transporters involved in uptake and translocation of sulphate in Arabidopsis thaliana; Takahashi H et al.; To investigate the uptake and long-distance translocation of sulphate in plants, we have characterized three cell-type-specific sulphate transporters, Sultr1;1, Sultr2;1 and Sultr2;2 in Arabidopsis thaliana . Heterologous expression in the yeast sulphate transporter mutant indicated that Sultr1;1 encodes a high-affinity sulphate transporter (Km for sulphate 3.6 +/- 0.6 microM), whereas Sultr2;1 and Sultr2;2 encode low-affinity sulphate transporters (Km for sulphate 0.41 +/- 0.07 mM and >/= 1.2 mM, respectively) . In Arabidopsis plants expressing the fusion gene construct of the Sultr1;1 promoter and green fluorescent protein (GFP), GFP was localized in the lateral root cap, root hairs, epidermis and cortex of roots . beta-glucuronidase (GUS) expressed with the Sultr2;1 promoter was specifically accumulated in the xylem parenchyma cells of roots and leaves, and in the root pericycles and leaf phloem . Expression of the Sultr2;2 promoter-GFP fusion gene showed specific localization of GFP in the root phloem and leaf vascular bundle sheath cells . Plants continuously grown with low sulphate concentrations accumulated high levels of Sultr1;1 and Sultr2;1 mRNA in roots and Sultr2;2 mRNA in leaves . The abundance of Sultr1;1 and Sultr2;1 mRNA was increased remarkably in roots by short-term stress caused by withdrawal of sulphate . Addition of selenate in the sulphate-sufficient medium increased the sulphate uptake capacity, tissue sulphate content and the abundance of Sultr1;1 and Sultr2;1 mRNA in roots . Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes. Plant J, 2000 Jul, 23(1), 143 - 57 Technical advance: the DNA-binding activity of gal4 is inhibited by methylation of the gal4 binding site in plant chromatin; Galweiler L et al.; Derivatives of the Saccharomyces cerevisiae transcription factor Gal4 which act as effective transcription activators in yeast, Drosophila, mammalian cells and plant protoplasts are shown to direct expression from a GUS reporter construct when expressed in transgenic tobacco . However, in comparison to 35S-GUS controls, Gal4-mediated expression of the reporter gene was relatively weak and extremely variable . GUS expression was lost as plants matured and it was almost undetectable in most of their progeny . Gal4-mediated gene expression could be restored by treating tissues with 5-aza-cytidine, implicating cytosine methylation in the loss of Gal4-mediated expression . Restoration of reporter expression was not accompanied by an increase in steady-state levels of the activator transcript . We propose that the DNA-binding activity of Gal4 is sensitive to methylation of its binding site in plant chromatin . The Gal4-DNA co-crystal predicts that 5-methylcytosine at either of the outer two positions of the binding site will effectively prevent Gal4 binding . We show that these positions become extensively methylated in transgenic plants and that methylation of Gal4-binding sites interferes with Gal4 binding in vitro . These observations suggest that the Gal4 DNA-binding domain is intrinsically sensitive to cytosine methylation and that, despite the success of Gal4-based expression systems in yeast and Drosophila, Gal4 is not ideal for use in plant gene expression technology. Anticancer Res, 2000 May-Jun, 20(3B), 1947 - 52 Immunohistochemical characterization of p57KIP2 expression in human esophageal squamous cell carcinoma; Matsumoto M et al.; Functional defects in the CIP/KIP family of cyclin-dependent kinase inhibitors (CDKIs) have been shown to be associated with human malignancies . We immunohistochemically examined p57KIP2 (p57) expression in 92 patients with human esophageal squamous cell carcinoma (SCC) to determine the relationship between this expression and those of cyclin D1 and E . The p57 labeling index (LI) (defined as the percentage of p57-positive cells) in esophageal SCC was 43.3 +/- 3.2% (mean +/- standard error of the mean) . In non-neoplastic esophageal epithelium, p57 staining was more frequently observed in the basal and parabasal cells than in surface layer cells . Immunostaining for cyclin D1 and E was observed in 28.2% (28/92) and 32.6% (30/92) of tumors, respectively . The median p57 LI in cyclin D1-positive cases was 66.2, and significantly higher than that in negative cases (31.9%) (p = 0.0009) . There was no significant relationship between p57 LI and cyclin E expression (p = 0.147) . As determined using Kaplan-Meier's method, loss of p57 immunoreactivity was not a prognostic factor for esophageal SCC (p = 0.548) . Our in vivo findings suggested that p57 protein expression was positively correlated with cyclin D1 expression and that loss of p57 protein expression alone does not affect progression of esophageal SCC. Science, 2000 Aug 4, 289(5480), 782 - 5 Role of adenine nucleotide translocator 1 in mtDNA maintenance; Kaukonen J et al.; Autosomal dominant progressive external ophthalmoplegia is a rare human disease that shows a Mendelian inheritance pattern, but is characterized by large-scale mitochondrial DNA (mtDNA) deletions . We have identified two heterozygous missense mutations in the nuclear gene encoding the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT1) in five families and one sporadic patient . The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein . The analogous mutation in yeast caused a respiratory defect . These results indicate that ANT has a role in mtDNA maintenance and that a mitochondrial disease can be caused by a dominant mechanism. Science, 2000 Aug 4, 289(5480), 774 - 9 Pol kappa: A DNA polymerase required for sister chromatid cohesion; Wang Z et al.; Establishment of cohesion between sister chromatids is coupled to replication fork passage through an unknown mechanism . Here we report that TRF4, an evolutionarily conserved gene necessary for chromosome segregation, encodes a DNA polymerase with beta-polymerase-like properties . A double mutant in the redundant homologs, TRF4 and TRF5, is unable to complete S phase, whereas a trf4 single mutant completes a presumably defective S phase that results in a failure of cohesion between the replicated sister chromatids . This suggests that TRFs are a key link in the coordination between DNA replication and sister chromatid cohesion . Trf4 and Trf5 represent the fourth class of essential nuclear DNA polymerases (designated DNA polymerase kappa) in Saccharomyces cerevisiae and probably in all eukaryotes. J Mol Biol, 2000 Aug 4, 301(1), 147 - 56 Crystal structure of the amino-terminal coiled-coil domain of the APC tumor suppressor; Day CL et al.; Coiled coils serve as dimerization domains for a wide variety of proteins, including the medically important oligomeric tumor suppressor protein, APC . Mutations in the APC gene are associated with an inherited susceptibility to colon cancer and with approximately 75 % of sporadic colorectal tumors . To define the basis for APC pairing and to explore the anatomy of dimeric coiled coils, we determined the 2.4 A resolution X-ray crystal structure of the N-terminal dimerization domain of APC . The peptide APC-55, encompassing the heptad repeats in APC residues 2-55, primarily forms an alpha-helical, coiled-coil dimer with newly observed core packing features . Correlated asymmetric packing of four core residues in distinct, standard rotamers is associated with a small shift in the helix register . At the C terminus, the helices splay apart and interact with a symmetry-related dimer in the crystal to form a short, anti-parallel, four-helix bundle . N-terminal fraying and C-terminal splaying of the helices, as well as the asymmetry and helix register shift describe unprecedented dynamic excursions of coiled coils . The low stability of APC-55 and divergence from the expected coiled-coil fold support the suggestion that the APC dimerization domain may extend beyond the first 55 residues . Biochim Biophys Acta, 2000 Jul 20, 1459(1), 218 - 29 A new microsystem for automated electrorotation measurements using laser tweezers; Reichle C et al.; We have developed a new microsystem for fast, automated studies of reactions and kinetics of single cells with biochemical or pharmacological agents . A cell spins in an external rotating electric field and the frequency dependence characterises the passive dielectric properties of membrane and cytoplasm . We use a planar microelectrode chip with microchannel (easily covered with a removable slip) for the application of frequencies exceeding 250 MHz to determine cytoplasmic properties in low and high conductivity electrolyte solutions . The laser tweezers serve as a bearing system, rotation is induced by microelectrodes and rotation speed is recorded automatically . This opens up new possibilities in biotechnology, e.g . for drug screening as demonstrated by measuring the influence of ionomycin on the passive dielectric properties of T-lymphoma cells . Additionally, a possible infrared-induced long-term cell damage could be observed by electrorotation and is discussed. J Biol Chem, 2000 Oct 20, 275(42), 33134 - 41 Human Ku antigen tightly binds and stabilizes a tetrahelical form of the Fragile X syndrome d(CGG)n expanded sequence; Uliel L et al.; Hairpin and tetrahelical structures of a d(CGG)(n) sequence in the FMR1 gene have been implicated in its expansion in fragile X syndrome . The identification of tetraplex d(CGG)(n) destabilizing proteins (Fry, M., and Loeb, L . A.(1999) J . Biol . Chem . 274, 12797-12803; Weisman-Shomer, P., Naot, Y., and Fry, M . (2000) J . Biol . Chem . 275, 2231-2238) suggested that proteins might modulate d(CGG)(n) folding and aggregation . We assayed human TK-6 lymphoblastoid cell extracts for d(CGG)(8) oligomer binding proteins . The principal binding protein was identified as Ku antigen by its partial amino acid sequence and antigenicity . The purified 88/75-kDa heterodimeric Ku bound with similar affinities (K(d) approximately 1 . 8-10.2 x 10(-9) mol/liter) to double-stranded d(CGG)(8).d(CCG)(8), hairpin d(CGG)(8), single-stranded d(CII)(8), or tetraplex structures of telomeric or IgG switch region sequences . However, Ku associated more tightly with bimolecular G'2 tetraplex d(CGG)(8) (K(d) approximately 0.35 x 10(-9) mol/liter) . Binding to Ku protected G'2 d(CGG)(8) against nuclease digestion and impeded its unwinding by the tetraplex destabilizing protein qTBP42 . Stabilization of d(CGG)(n) tetraplex domains in FMR1 by Ku or other proteins might promote d(CGG) expansion and FMR1 silencing. J Biol Chem, 2000 Oct 6, 275(40), 30833 - 8 The relationship between homology length and crossing over during the repair of a broken