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| Eur J Cell Biol, 1999 Jun, 78(6), 357 - 74 Identification and characterization of the human peroxin PEX3; Soukupova M et al.; The biogenesis of peroxisomes requires the interaction of several peroxins, encoded by PEX genes and is well conserved between yeast and humans . We have cloned the human cDNA of PEX3 based on its homology to different yeast PEX3 genes . The deduced peroxin HsPEX3 is a peroxisomal membrane protein with a calculated molecular mass of 42.1 kDa . We created N- and C-terminal tagged PEX3 to assay its topology at the peroxisomal membrane by immunofluorescence microscopy . Our results and the one predicted transmembrane spanning region are in line with the assumption that H sPEX3 is an integral peroxisomal membrane protein with the N-terminus inside the peroxisome and the C-terminus facing the cytoplasm . The farnesylated peroxisomal membrane protein PEX19 interacts with HsPEX3 in a mammalian two-hybrid assay in human fibroblasts . The physical interaction could be confirmed by coimmunoprecipitation of the two in vitro transcribed and translated proteins . To address the targeting of PEX3 to the peroxisomal membrane, the expression of different N- and C-terminal PEX3 truncations fused to green fluorescent protein (GFP) was investigated in human fibroblasts . The N-terminal 33 amino acids of PEX3 were necessary and sufficient to direct the reporter protein GFP to peroxisomes and seemed to be integrated into the peroxisomal membrane . The expression of a 1-16 PEX3-GFP fusion protein did not result in a peroxisomal localization, but interestingly, this and several other truncated PEX3 fusion proteins were also localized to tubular and/or vesicular structures representing mitochondria. Eur J Biochem, 1999 Jul, 263(1), 14 - 9 The metaphase to anaphase transition: a case of productive destruction; Farr KA et al.; The metaphase to anaphase transition is a point of no return; the duplicated sister chromatids segregate to the future daughter cells, and any mistake in this process may be deleterious to both progeny . At the heart of this process lies the anaphase inhibitor, which must be degraded in order for this transition to take place . The degradation of the anaphase inhibitor occurs via the ubiquitin-degradation pathway, and it involves the activity of the cyclosome/anaphase promoting complex (APC) . The fidelity of the metaphase to anaphase transition is ensured by several different regulatory mechanisms that modulate the activity of the cyclosome/APC . Great advancements have been made in this field in the past few years, but many questions still remain to be answered. EMBO J, 1999 Aug 2, 18(15), 4332 - 47 Dbp5, a DEAD-box protein required for mRNA export, is recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p; Schmitt C et al.; Dbp5 is a DEAD-box protein essential for mRNA export from the nucleus in yeast . Here we report the isolation of a cDNA encoding human Dbp5 (hDbp5) which is 46% identical to yDbp5p . Like its yeast homologue, hDbp5 is localized within the cytoplasm and at the nuclear rim . By immunoelectron microscopy, the nuclear envelope-bound fraction of Dbp5 has been localized to the cytoplasmic fibrils of the nuclear pore complex (NPC) . Consistent with this localization, we show that both the human and yeast proteins directly interact with an N-terminal region of the nucleoporins CAN/Nup159p . In a conditional yeast strain in which Nup159p is degraded when shifted to the nonpermissive temperature, yDbp5p dissociates from the NPC and localizes to the cytoplasm . Thus, Dbp5 is recruited to the NPC via a conserved interaction with CAN/Nup159p . To investigate its function, we generated defective hDbp5 mutants and analysed their effects in RNA export by microinjection in Xenopus oocytes . A mutant protein containing a Glu-->Gln change in the conserved DEAD-box inhibited the nuclear exit of mRNAs . Together, our data indicate that Dbp5 is a conserved RNA-dependent ATPase which is recruited to the cytoplasmic fibrils of the NPC where it participates in the export of mRNAs out of the nucleus. EMBO J, 1999 Aug 2, 18(15), 4210 - 21 Sin1: an evolutionarily conserved component of the eukaryotic SAPK pathway; Wilkinson MG et al.; The fission yeast Sty1/Spc1 mitogen-activated protein (MAP) kinase is a member of the eukaryotic stress-activated MAP kinase (SAPK) family . We have identified a protein, Sin1, that interacts with Sty1/Spc1 which is a member of a new evolutionarily conserved gene family . Cells lacking Sin1 display many, but not all, of the phenotypes of cells lacking the Sty1/Spc1 MAP kinase including sterility, multiple stress sensitivity and a cell-cycle delay . Sin1 is phosphorylated after stress but this is not Sty1/Spc1-dependent . Importantly, Sin1 is not required for activation of Sty1/Spc1 but is required for stress-dependent transcription via its substrate, Atf1 . We find that in the absence of Sin1, Sty1/Spc1 appears to translocate to the nucleus but Atf1 is not fully phosphorylated and becomes unstable in response to environmental stress . Sin1 is also required for effective transcription via the AP-1 factor Pap1 but does not prevent its nuclear translocation . Remarkably chimaeric fusions of sin1 with chicken sin1 sequences rescue loss of sin1 function . We conclude that Sin1 is a novel component of the eukaryotic SAPK pathway. J Biol Chem, 1999 Aug 6, 274(32), 22289 - 95 The roles of two amino acid residues in the active site of L-lactate monooxygenase . Mutation of arginine 187 to methionine and histidine 240 to glutamine; Sanders SA et al.; Lactate monooxygenase (LMO) catalyzes the conversion of L-lactate to acetate, CO(2), and water with the incorporation of molecular oxygen . Arginine 187 of LMO is highly conserved within the family of L-alpha-hydroxyacid oxidizing enzymes (Le, K . H . D., and Lederer, F . (1991) J . Biol . Chem . 266, 20877-20881) . By comparison with the equivalent residue in flavocytochrome b(2) from Saccharomyces cerevisiae (Pike, A . D., Chapman, S . K, Manson, F . D . C, . Reid, G . A . , Gondry, M., and Lederer, F . (1996) in Flavins and Flavoproteins (Stevenson, K . J., Massey, V., and Williams, C . H., Jr., eds) pp . 571-574, University of Calgary Press, Calgary, AB, Canada), arginine 187 might be expected to have an important role in catalytic efficiency and substrate binding in LMO . Histidine 240 is predicted to be close to the substrate binding site of LMO, although it is not conserved within the enzyme family . Arginine 187 has been replaced with methionine (R187M), and histidine 240 has been replaced with glutamine (H240Q) . L-Lactate oxidation by R187M is very slow . The binding of L-lactate to the mutant enzyme appears to be very weak, as is the binding of oxalate, a transition state analogue . The binding of pyruvate to the reduced enzyme is also very weak, resulting in complete uncoupling of enzyme turnover, with H(2)O(2) and pyruvate as the final products . In addition, anionic forms of the flavin are unstable . The K(d) for sulfite is increased nearly 400-fold by this mutation . The semiquinone form of R187M is also thermodynamically unstable, although the overall midpoint potential for the two-electron reduction of R187M is only 34 mV lower than for the wild-type enzyme . H240Q more closely resembles the wild-type enzyme . The steady-state activity of H240Q is completely coupled . The k(cat) is similar to that for the wild-type enzyme. J Biol Chem, 1999 Aug 6, 274(32), 22646 - 51 Nup192p is a conserved nucleoporin with a preferential location at the inner site of the nuclear membrane; Kosova B et al.; Human Nup93, the homologue of yeast Nic96p, is associated with a 205-kDa protein whose intracellular location and function is unknown . We show here that the yeast open reading frame YJL039c, which is homologous to this human p205, encodes the so far largest yeast nucleoporin . Accordingly, green fluorescent protein (GFP)-tagged YJL039c was localized to the nuclear pores and therefore named Nup192p . Affinity purification of ProtA-Nic96p from glutaraldehyde-fixed spheroplasts reveals association with Nup192p . NUP192 is essential for cell growth . A temperature-sensitive mutant nup192-15 is neither impaired in nuclear import of a SV40 nuclear localization sequence-containing reporter protein nor in mRNA export, but association of Nup49-GFP with nuclear pores is inhibited at the non-permissive temperature . By immunoelectron microscopy, Nup192p-ProtA is seen at the inner site of the nuclear pores, at a distance of 60 +/- 15 nm from the central plane of the pore . This suggests that Nup192p is an evolutionarily conserved structural component of the nuclear pore complex with a preferential location at the inner site of the nuclear membrane. J Biol Chem, 1999 Aug 6, 274(32), 22563 - 8 Retinoid-dependent recruitment of a histone H1 displacement activity by retinoic acid receptor; Nagpal S et al.; Targeted recruitment of histone acetyltransferase (HAT) activities by sequence-specific transcription factors, including the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), has been proposed to lead to destabilization of nucleosomal cores by acetylation of core histones . However, biochemical evidence indicates that destabilization and depletion of linker H1 histones must also occur at the promoter regions of actively transcribing genes . Mechanisms by which nuclear receptors and other transcription factors affect the removal of histone H1 from transcriptionally silent chromatin have not been previously described . In this report, we show that RARs interact in a ligand-dependent manner with HMG-I, which is known to displace histone H1 from chromatin . We further show that HMG-I and a novel related protein, HMG-R, also interact with other transcription factors . Using sense and antisense constructs of HMG-I/R in transient transfection assays with a retinoid responsive reporter, we also demonstrate that HMG-I/R is important for retinoid dependent transcriptional activity of RAR . These findings suggest a step wise mechanism by which RARs and other transcription factors can cause a targeted unfolding of compact chromatin as a first step in transcriptional activation, which would then be followed by recruitment of HAT activity and subsequent events. J Biol Chem, 1999 Aug 6, 274(32), 22225 - 30 Critical aspartic acid residues in pseudouridine synthases; Ramamurthy V et al.; The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules . Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E . V . (1996) Nucleic Acids Res . 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P . J., and Santi, D . V . (1996) Nucleic Acids Res . 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other . Upon further scrutiny (Huang, H . L., Pookanjanatavip, M., Gu, X . G., and Santi, D . V . (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase . To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate . We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases . Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function. J Biol Chem, 1999 Aug 6, 274(32), 22191 - 6 Mutations in actin subdomain 3 that impair thin filament regulation by troponin and tropomyosin; Korman VL et al.; Thin filament-mediated regulation of striated muscle contraction involves conformational switching among a few quaternary structures, with transitions induced by binding of Ca(2+) and myosin . We establish and exploit Saccharomyces cerevisiae actin as a model system to investigate this process . Ca(2+)-sensitive troponin-tropomyosin binding affinities for wild type yeast actin are seen to closely resemble those for muscle actin, and these hybrid thin filaments produce Ca(2+)-sensitive regulation of the myosin S-1 MgATPase rate . Yeast actin filament inner domain mutant K315A/E316A depresses Ca(2+) activation of the MgATPase rate, producing a 4-fold weakening of the apparent Ca(2+) affinity and a 50% decrease in the MgATPase rate at saturating Ca(2+) concentration . Observed destabilization of troponin-tropomyosin binding to actin in the presence of Ca(2+), a 1.4-fold effect, provides a partial explanation . Despite the decrease in apparent MgATPase Ca(2+) affinity, there was no detectable change in the true Ca(2+) affinity of the thin filament, measured using fluorophore-labeled troponin . Another inner domain mutant, E311A/R312A, decreased the MgATPase rate but did not change the apparent Ca(2+) affinity . These results suggest that charged residues on the surface of the actin inner domain are important in Ca(2+)- and myosin-induced thin filament activation. Cell, 1999 Jul 23, 98(2), 261 - 71 Spo76p is a conserved chromosome morphogenesis protein that links the mitotic and meiotic programs; van Heemst D et al.; Spo76p is conserved and related to the fungal proteins Pds5p and BIMD and the human AS3 prostate proliferative shutoff-associated protein . Spo76p localizes to mitotic and meiotic chromosomes, except at metaphase(s) and anaphase(s) . During meiotic prophase, Spo76p assembles into strong lines in correlation with axial element formation . As inferred from spo76-1 mutant phenotypes, Spo76p is required for sister chromatid cohesiveness, chromosome axis morphogenesis, and chromatin condensation during critical transitions at mitotic prometaphase and meiotic midprophase . Spo76p is also required for meiotic interhomolog recombination, likely at postinitiation stage(s) . We propose that a disruptive force coordinately promotes chromosomal axial compaction and destabilization of sister connections and that Spo76p restrains and channels the effects of this force into appropriate morphogenetic mitotic and meiotic outcomes. J Cell Biol, 1999 Jul 26, 146(2), 415 - 25 Slk19p is a centromere protein that functions to stabilize mitotic spindles; Zeng X et al.; We have identified a novel centromere-associated gene product from Saccharomyces cerevisiae that plays a role in spindle assembly and stability . Strains with a deletion of SLK19 (synthetic lethal Kar3p gene) exhibit abnormally short mitotic spindles, increased numbers of astral microtubules, and require the presence of the kinesin motor Kar3p for viability . When cells are deprived of both Slk19p and Kar3p, rapid spindle breakdown and mitotic arrest is observed . A functional fusion of Slk19p to green fluorescent protein (GFP) localizes to kinetochores and, during anaphase, to the spindle midzone, whereas Kar3p-GFP was found at the nuclear side of the spindle pole body . Thus, these proteins seem to play overlapping roles in stabilizing spindle structure while acting from opposite ends of the microtubules. J Cell Biol, 1999 Jul 26, 146(2), 333 - 44 Sec1p binds to SNARE complexes and concentrates at sites of secretion; Carr CM et al.; Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin . We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates . The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly . Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function . The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes . The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion. J Cell Biol, 1999 Jul 26, 146(2), 321 - 31 Biogenesis of Tom40, core component of the TOM complex of mitochondria; Rapaport D et al.; Tom40 is an essential component of the preprotein translocase of the mitochondrial outer membrane (TOM complex) in which it constitutes the core element of the protein conducting pore . We have investigated the biogenesis of Tom40 . Tom40 is inserted into the outer membrane by the TOM complex . Initially, Tom40 is bound as a monomer at the mitochondrial surface . The import receptor Tom20 is involved in this initial step; it stimulates both binding and efficient insertion of the Tom40 precursor . This step is followed by the formation of a further intermediate at which the Tom40 precursor is partially inserted into the outer membrane . Finally, Tom40 is integrated into preexisting TOM complexes . Efficient import appears to require the Tom40 precursor to be in a partially folded conformation . Neither the NH(2) nor the COOH termini are necessary to target Tom40 to the outer membrane . However, the NH(2)-terminal segment is required for Tom40 to become assembled into the TOM complex . A model for the biogenesis of Tom40 is presented. Nat Struct Biol, 1999 Aug, 6(8), 724 - 9 Crystal structure of the copper chaperone for superoxide dismutase; Lamb AL et al.; Cellular systems for handling transition metal ions have been identified, but little is known about the structure and function of the specific trafficking proteins . The 1.8 A resolution structure of the yeast copper chaperone for superoxide dismutase (yCCS) reveals a protein composed of two domains . The N-terminal domain is very similar to the metallochaperone protein Atx1 and is likely to play a role in copper delivery and/or uptake . The second domain resembles the physiological target of yCCS, superoxide dismutase I (SOD1), in overall fold, but lacks all of the structural elements involved in catalysis . In the crystal, two SOD1-like domains interact to form a dimer . The subunit interface is remarkably similar to that in SOD1, suggesting a structural basis for target recognition by this metallochaperone. Nat Struct Biol, 1999 Aug, 6(8), 709 - 11 Helping copper find a home; Poulos TL; The crystal structure of the superoxide dismutase copper chaperone provides some key insights into the molecular mechanism of copper trafficking. Histol Histopathol, 1999 Jul, 14(3), 887 - 94 Regulation of the MAPK family members: role of subcellular localization and architectural organization; Fanger GR; The members of the mitogen-activated protein kinase (MAPK) family are regulated by a diverse array of extracellular cues ranging from cytokines, growth factors and neuropeptides, which activate cell surface receptors, to stresses such as cold, heat, osmolarity changes and irradiation . The MAPK pathways control genetic expression by modifying transcription factor activity and cue important cell fate decisions including survival, proliferation, and programmed cell death (apoptosis) . One interesting feature of the MAPK pathways is that the components are evolutionarily conserved from yeast to human, and many of the pathways are similarly organized and regulated . Unlike previously imagined, architectural organization or the multimeric organization of signaling proteins into complexes which are localized to distinct subcellular regions is an important mechanism that influences the regulation of these pathways . In addition, extracellular stimuli can induce relocalization of specific signal transduction proteins . The formation of multimeric signaling complexes, as well as the dynamic movement of signaling proteins, contribute to determine signaling specificity and efficacy . This review describes what is currently known about the subcellular localization of MAPK pathway signaling proteins and the relocalization that occurs during events associated with activation of the MAPK family members. Protein Expr Purif, 1999 Aug, 16(3), 424 - 31 A "stripping" ligand tactic for use with the kinetic locking-on strategy: its use in the resolution and bioaffinity chromatographic purification of NAD(+)-dependent dehydrogenases; O'Flaherty M et al.; The kinetic locking-on strategy utilizes soluble analogues of the target enzymes' specific substrate to promote selective adsorption of individual NAD(+)-dependent dehydrogenases on their complementary immobilized cofactor derivative . Application of this strategy to the purification of NAD(+)-dependent dehydrogenases from crude extracts has proven that it can yield bioaffinity systems capable of producing one-chromatographic-step purifications with yields approaching 100% . However, in some cases the purified enzyme preparation was found to be contaminated with other proteins weakly bound to the immobilized cofactor derivative through binary complex formation and/or nonspecific interactions, which continuously "dribbled" off the matrix during the chromatographic procedure . The fact that this problem can be overcome by including a short pulse of 5'-AMP (stripping ligand) in the irrigant a couple of column volumes prior to the discontinuation of the specific substrate analogue (locking-on ligand) is clear from the results presented in this report . The general effectiveness of this auxiliary tactic has been assessed using model studies and through incorporation into an actual purification from a crude cellular extract . The results confirm the usefulness of the stripping-ligand tactic for the resolution and purification of NAD(+)-dependent dehydrogenases when using the locking-on strategy . These studies have been carried out using bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3), yeast alcohol dehydrogenase (YADH, EC 1.1.1.1), porcine heart mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37), and bovine heart L-lactate dehydrogenase (l-LDH, EC 1.1.1.27) . J Nat Prod, 1999 Jul, 62(7), 976 - 83 Synthesis and biological evaluation of analogues of cryptolepine, an alkaloid isolated from the Suriname rainforest; Yang SW et al.; Bioassay-guided fractionation of an extract of a mixture of Microphilis guyanensis and Genipa americanacollected in the rainforest of Suriname yielded the known alkaloid cryptolepine (2) as the major active compound in a yeast bioassay for potential DNA-damaging agents; the same compound was later reisolated from M . guyanensis . The structure of cryptolepine was identified unambiguously by spectral data and by its total synthesis . Several cryptolepine derivatives (3-29, 32-41) were synthesized based on modifications of the C-2, N-5, N-10, and C-11 positions . Two cryptolepine dimers (30, 31) were also prepared . The structure modifications did not result in compounds with a higher potency than the parent compound cryptolepine in the yeast assay system, although some derivatives did show significant activity . Selected compounds (6, 7, 17, 22, 23, 26, and 27) were also tested for cytotoxicity in mammalian cell culture, and two compounds showed significant cytotoxic activity. Phytochemistry, 1999 Aug, 51(7), 899 - 902 A chromene and prenylated benzoic acid from Piper aduncum; Baldoqui DC et al.; In addition to nerolidol, 2',6'-dihydroxy-4'-methoxydihydrochalcone, methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate, methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate and methyl 8-hydroxy-2,2-dimethyl-2H-1-chromene-6-carboxylate, two new natural products were isolated from the leaves of Piper aduncum, 2,2-dimethyl-2H-1-chromene-6-carboxylic acid and 3-(3',7'-dimethyl-2',6'-octadienyl)-4-methoxybenzoic acid . The structures of the isolates were established based on analysis of spectroscopic data, including ES-MS . The DNA-damaging activity of the isolated compounds was also investigated against mutant strains of Saccharomyces cerevisiae. Protein Sci, 1999 Jul, 8(7), 1381 - 92 Electrostatic interactions in the GCN4 leucine zipper: substantial contributions arise from intramolecular interactions enhanced on binding; Hendsch ZS et al.; The GCN4 leucine zipper is a peptide homodimer that has been the subject of a number of experimental and theoretical investigations into the determinants of affinity and specificity . Here, we utilize this model system to investigate electrostatic effects in protein binding using continuum calculations . A particularly novel feature of the computations made here is that they provide an interaction-by-interaction breakdown of the electrostatic contributions to the free energy of docking that includes changes in the interaction of each functional group with solvent and changes in interactions between all pairs of functional groups on binding . The results show that (1) electrostatic effects disfavor binding by roughly 15 kcal/mol due to desolvation effects that are incompletely compensated in the bound state, (2) while no groups strongly stabilize binding, the groups that are most destabilizing are charged and polar side chains at the interface that have been implicated in determining binding specificity, and (3) attractive intramolecular interactions (e.g., backbone hydrogen bonds) that are enhanced on binding due to reduced solvent screening in the bound state contribute significantly to affinity and are likely to be a general effect in other complexes . A comparison is made between the results obtained in an electrostatic analysis carried out calculationally and simulated results corresponding to idealized data from a scanning mutagenesis experiment . It is shown that scanning experiments provide incomplete information on interactions and, if overinterpreted, tend to overestimate the energetic effect of individual side chains that make attractive interactions . Finally, a comparison is made between the results available from a continuum electrostatic model and from a simpler surface-area dependent solvation model . In this case, although the simpler model neglects certain interactions, on average it performs rather well. Genes Cells, 1999 Jun, 4(6), 311 - 24 Hyperosmotic stress-induced reorganization of actin bundles in Dictyostelium cells over-expressing cofilin; Aizawa H et al.; BACKGROUND: Cofilin is a low-molecular weight actin-modulating protein, which binds to, severs, and depolymerizes actin filaments in vitro . Aip1, an actin-interacting protein, was recently identified as a product of a gene on a multicopy plasmid which suppresses the temperature-sensitive phenotype of a cofilin mutant in Saccharomyces cerevisiae . Actin cytoskeleton plays an essential role in resistance to hyperosmotic stress in Dictyostelium discoideum . The roles of cofilin and Aip1 in this resistance are not known . RESULTS: In response to hyperosmotic stress, D . discoideum cells round up . This stress-induced morphological change involves the redistribution of cofilin, together with actin filaments, into cortical contractile portions of the cells, followed by their contraction . Over-expression of cofilin increases and thickens cortical actin bundles in cells . The bundles become tight and are reorganized into a ring-shaped structure in response to hyperosmotic stress . The ring structure of actin bundles had two characteristic bands across them; bright and dark bands, heavily stained and not stained with phalloidin . In the bundles, straight filaments with a diameter of 5.3-nm were aligned parallel by cross-bridge structures . In cells lacking the myosin-II heavy chain, the bundles, which were induced by an over-expression of cofilin, shortened and became straight following hyperosmotic stress, forming a polygonal structure . D . discoideum Aip1/Wrp2 enhanced the severing of actin filaments by cofilin in vitro and colocalized with cofilin in cells, including those that were over-expressing cofilin before and after exposure to hyperosmotic stress . CONCLUSIONS: Cofilin plays a pivotal role in concert with Aip1/Wrp2 in the reorganization of actin architectures into bundles that contract in a myosin-II-independent manner, in response to hyperosmotic stress. Nature, 1999 Jul 15, 400(6741), 284 - 8 The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins; Orphanides G et al.; The regulation of gene expression depends critically upon chromatin structure . Transcription of protein-coding genes can be reconstituted on naked DNA with only the general transcription factors and RNA polymerase II . This minimal system cannot transcribe DNA packaged into chromatin, indicating that accessory factors may facilitate access to DNA . Two classes of accessory factor, ATP-dependent chromatin-remodelling enzymes and histone acetyltransferases, facilitate transcription initiation from chromatin templates . FACT (for facilitates chromatin transcription) is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro . Here we show that FACT comprises a new human homologue of the Saccharomyces cerevisiae Spt16/Cdc68 protein and the high-mobility group-1-like protein structure-specific recognition protein-1 . Yeast SPT16/CDC68 is an essential gene that has been implicated in transcription and cell-cycle regulation . Consistent with our biochemical analysis of FACT, we provide evidence that Spt16/Cdc68 is involved in transcript elongation in vivo . Moreover, FACT specifically interacts with nucleosomes and histone H2A/H2B dimers, indicating that it may work by promoting nucleosome disassembly upon transcription . In support of this model, we show that FACT activity is abrogated by covalently crosslinking nucleosomal histones. Curr Biol, 1999 Jul 15, 9(14), R531 - 4 Chromosome segregation: Samurai separation of Siamese sisters; Glotzer M; How do cells ensure that sister chromatids are precisely partitioned in mitosis? New studies on budding yeast have revealed that sister chromatid separation at anaphase requires endoproteolytic cleavage of a protein that maintains the association between sister chromatids. J Biol Chem, 1999 Jul 30, 274(31), 21986 - 94 Sequence selectivity of c-Myb in vivo . Resolution of a DNA target specificity paradox; Andersson KB et al.; We have investigated the basis for the striking difference between the broad DNA sequence selectivity of the c-Myb transcription factor minimal DNA-binding domain R(2)R(3) in vitro and the more restricted preference of a R(2)R(3)VP16 protein for Myb-specific recognition elements (MREs) in a Saccharomyces cerevisiae transactivation system . We show that sequence discrimination in yeast is highly dependent on the expression level of Myb effector protein . Full-length c-Myb and a C-terminally truncated protein (residues 1-360) were also included in the study . All of the tested Myb proteins displayed very similar DNA binding properties in electrophoretic mobility shift assays . Only minor differences between full-length c-Myb and truncated c-Myb(1-360) were observed . In transactivation studies in CV-1 cells, the MRE selectivity was highest at low expression levels of Myb effector proteins . However, the discrimination between MRE variants was rapidly lost with high input levels of effector plasmid . In c-Myb-expressing K-562 cells, the high degree of MRE selectivity was retained, thereby confirming the relevance of the results obtained in the yeast system . These data suggest that the MRE selectivity of c-Myb is an intrinsic property of only the R(2)R(3) domain itself and that the transactivation response of a specific MRE in vivo may be highly dependent on the expression level of the Myb protein in the cell. J Biol Chem, 1999 Jul 30, 274(31), 21981 - 5 Cloning and characterization of the EAP30 subunit of the ELL complex that confers derepression of transcription by RNA polymerase II; Schmidt AE et al.; The product of the human oncogene ELL encodes an RNA polymerase II transcription factor that undergoes frequent translocation in acute myeloid leukemia (AML) . In addition to its elongation activity, ELL contains a novel type of RNA polymerase II interaction domain that is capable of repressing polymerase activity in promoter-specific transcription . Remarkably, the ELL translocation that is found in patients with AML results in the deletion of exactly this functional domain . Here we report that the EAP30 subunit of the ELL complex has sequence homology to the Saccharomyces cerevisiae SNF8, whose genetic analysis suggests its involvement in the derepression of gene expression . Remarkably, EAP30 can interact with ELL and derepress ELL's inhibitory activity in vitro . This finding may reveal a key role for EAP30 in the pathogenesis of human leukemia. J Biol Chem, 1999 Jul 30, 274(31), 21659 - 64 Dissociation of mismatch recognition and ATPase activity by hMSH2-hMSH3; Wilson T et al.; MSH2-MSH3 directs the repair of insertion/deletion loops of up to 13 nucleotides in vivo and in vitro . To examine the biochemical basis of this repair specificity, we characterized the mispair binding and ATPase activity of hMSH2-hMSH3 . The ATPase was found to be regulated by a mismatch-stimulated ADP --> ATP exchange, which induces a conformational transition by the protein complex . We demonstrated strong binding of hMSH2-hMSH3 to an insertion/deletion loop containing 24 nucleotides that is incapable of provoking ADP --> ATP exchange, suggesting that mismatch recognition appears to be necessary but not sufficient to induce the intrinsic ATPase . These studies support the idea that hMSH2-hMSH3 functions as an adenosine nucleotide-regulated molecular switch that must be activated by mismatched nucleotides for classical mismatch repair to occur. J Biol Chem, 1999 Jul 30, 274(31), 21645 - 50 Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression; Nishiyama A et al.; Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena . Thioredoxin (TRX) is one of the major components of the thiol reducing system and plays multiple roles in cellular processes such as proliferation, apoptosis, and gene expression . To investigate the molecular mechanism of TRX action, we used a yeast two-hybrid system to identify TRX-binding proteins . One of the candidates, designated as thioredoxin-binding protein-2 (TBP-2), was identical to vitamin D(3) up-regulated protein 1 (VDUP1) . The association of TRX with TBP-2/VDUP1 was observed in vitro and in vivo . TBP-2/VDUP1 bound to reduced TRX but not to oxidized TRX nor to mutant TRX, in which two redox active cysteine residues are substituted by serine . Thus, the catalytic center of TRX seems to be important for the interaction . Insulin reducing activity of TRX was inhibited by the addition of recombinant TBP-2/VDUP1 protein in vitro . In COS-7 and HEK293 cells transiently transfected with TBP-2/VDUP1 expression vector, decrease of insulin reducing activity of TRX and diminishment of TRX expression was observed . These results suggested that TBP-2/VDUP1 serves as a negative regulator of the biological function and expression of TRX . Treatment of HL-60 cells with 1alpha, 25-dihydroxyvitamin D(3) caused an increase of TBP-2/VDUP1 expression and down-regulation of the expression and the reducing activity of TRX . Therefore, the TRX-TBP-2/VDUP1 interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid and macrophage lineages. Planta Med, 1999 Jun, 65(5), 437 - 9 alpha-Glucosidase inhibitors from Commelina communis; Kim HS et al.; A methanolic extract of Commelina communis showed potent inhibitory activity against alpha-glucosidase . One pyrrolidine alkaloid, 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP, 1) and four piperidine alkaloids, 1-deoxymannojirimycin (2), 1-deoxynojirimycin (3), alpha-homonojirimycin (4) and 7-O-beta-D-glucopyranosyl alpha-homonojirimycin (5) were isolated by bioassay-directed fractionation and separation . These compounds have been identified for the first time from Commelina communis, supporting the pharmacological basis of this plant that has been used as a traditional herbal medicine for the treatment of diabetes. Proc Assoc Am Physicians, 1999 Jul-Aug, 111(4), 347 - 56 Characterization of a multicopy family of genes encoding a surface-expressed serine endoprotease in rat Pneumocystis carinii; Russian DA et al.; A unique family of genes encoding serine endoproteases related to the Saccharomyces cerevisiae serine endoprotease kexin was identified in Pneumocystis carinii . Unlike previously described serine endoprotease genes that are single copies, multiple copies of the P . carinii serine endoprotease are distributed throughout the genome . The proteins predicted by these variant genes demonstrate sequence variability, but they retain the conserved active sites associated with endoprotease activity . The serine endoprotease was localized to the organism surface by immunohistochemical and immunofluorescence studies and to the electron lucent layer of the cyst wall by immunoelectron microscopy . The findings of multiple copies of the serine endoprotease gene in the P . carinii genome, and its localization to the cell surface, suggest that this protease plays an important role in the biology of P . carinii and that antigenic variation of the surface-expressed serine endoprotease may be a strategy for immune evasion . P . carinii serine endoprotease provides a novel target for chemotherapeutic and immune-based approaches to the treatment of P . carinii pneumonia. Plant J, 1999 Jul, 19(1), 43 - 53 Vacuolar processing enzyme is up-regulated in the lytic vacuoles of vegetative tissues during senescence and under various stressed conditions; Kinoshita T et al.; Vacuolar processing enzyme (VPE) has been shown to be responsible for maturation of various seed proteins in protein-storage vacuoles . Arabidopsis has three VPE homologues; betaVPE is specific to seeds and alphaVPE and gammaVPE are specific to vegetative organs . To investigate the activity of the vegetative VPE, we expressed the gammaVPE in a pep4 strain of the yeast Saccharomyces cerevisiae and found that gammaVPE has the ability to cleave the peptide bond at the carbonyl side of asparagine residues . An immunocytochemical analysis revealed the specific localization of the gammaVPE in the lytic vacuoles of Arabidopsis leaves that had been treated with wounding . These findings indicate that gammaVPE functions in the lytic vacuoles as the betaVPE does in the protein-storage vacuoles . The betaVPE promoter was found to direct the expression of the beta-glucuronidase reporter gene in seeds and the root tip of transgenic Arabidopsis plants . On the other hand, both the alphaVPE and gammaVPE promoters directed the expression in senescent tissues, but not in young intact tissues . The mRNA levels of both alphaVPE and gammaVPE were increased in the primary leaves during senescence in parallel with the increase of the mRNA level of a senescence-associated gene (SAG2) . Treatment with wounding, ethylene and salicylic acid up-regulated the expression of alphaVPE and gammaVPE, while jasmonate slightly up-regulated the expression of gammaVPE . These gene expression patterns of the VPEs were associated with the accumulation of vacuolar proteins that are known to respond to these treatments . Taken together, the results suggest that vegetative VPE might regulate the activation of some functional proteins in the lytic vacuoles. Plant J, 1999 Jun, 18(6), 663 - 8 Transcriptional activation by Arabidopsis GT-1 may be through interaction with TFIIA-TBP-TATA complex; Le Gourrierec J et al.; GT-1 belongs to the class of trihelix DNA-binding proteins and binds to a promoter sequence found in many different genes . Data presented in this report show that GT-1 contains a trans-activation function in yeast and in plant cells . However, in tobacco BY-2 protoplasts, this activity functions only when an internal region containing the DNA-binding domain is deleted . Gel-shift and co-immunoprecipitation assays have revealed that GT-1 can interact with and stabilize the TFIIA-TBP-TATA complex . These results suggest that GT-1 may activate transcription through direct inter- action with the transcriptional pre-initiation complex. Biochem J, 1999 Aug 1, 341 ( Pt 3), 665 - 8 APS, an adapter protein with a PH and SH2 domain, is a substrate for the insulin receptor kinase; Ahmed Z et al.; APS (adapter protein with a PH and SH2 domain) is the newest member of a family of tyrosine kinase adapter proteins including SH2-B and Lnk . We previously identified SH2-B as an insulin-receptor-binding protein and substrate {Kotani, Wilden and Pillay (1998) Biochem J . 335, 103-109} . Here we show that APS interacts with the insulin receptor kinase activation loop through its SH2 domain and insulin stimulates the tyrosine-phosphorylation of APS . Furthermore, the phosphorylation of activation-loop tyrosine residues 1158 and 1162 are required for this interaction. Cancer Res, 1999 Jul 15, 59(14), 3442 - 50 Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform; Wessel I et al.; Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II, with at least two effects on the enzyme: namely, locking it in a closed-clamp form and inhibiting its ATPase activity . This is in contrast to topoisomerase II poisons as etoposide and amsacrine (m-AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA . Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change in expression of the beta isoform . Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug-selected mutation described at this site . Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines . Alkaline elution assay demonstrated a lack of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells . Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant demonstrated that R162Q conferred resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or m-AMSA . Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q enzyme than with the wt . The R162Q enzyme has a 20-25% decreased catalytic capacity compared to the wt and was almost inactive at <0.25 mM ATP compared to the wt . Kinetoplast DNA decatenation by the R162Q enzyme at 1 mM ATP was not resistant to ICRF-187 compared to wt, whereas it was clearly less sensitive than wt to ICRF-187 at low ATP concentrations . This suggests that it is a shift in the equilibrium to an open-clamp state in the enzyme's catalytic cycle caused by a decreased ATP binding by the mutated enzyme that is responsible for bisdioxopiperazine resistance. Ann N Y Acad Sci, 1999 May 18, 870, 108 - 18 Fidelity of retrotransposon replication; Gabriel A et al.; Ty1, the genetically tractable retrotransposable element found in the yeast Saccharomyces cerevisiae, closely resembles vertebrate retroviruses both in structure and in mechanism of replication . By direct sequence analysis, we examined the rate and spectrum of new mutations appearing during a single cycle of Ty1 replication . The rate of new mutations was comparable to those seen for replicating retroviruses . All observed changes were base substitutions, and their location suggested that template ends may be hot spots for generating these mutations . To test this, we developed methods to examine, at the nucleotide level, the end structure of the expected Ty1 replication intermediates . Our results demonstrate that Ty1 reverse transcriptase can add terminal non-templated bases in vivo during each step in replication . Furthermore, Ty1 RNAse H creates multiple template ends by imprecisely cleaving RNA . This expands the range of sites of subsequent non-templated base addition . Finally, on reaching template ends, Ty1 reverse transcriptase can strand transfer to inappropriate templates . Taken together, these mutagenic mechanisms may influence the evolution of particular regions of the Ty1 genome and serve as a mechanism to regulate the overall level of Ty1 transposition in its host cell. Gene, 1999 Jul 22, 235(1-2), 59 - 67 Molecular cloning and characterization of splice variants of human RAD50 gene; Kim KK et al.; In this report, splice variants of human RAD50 (hRAD50) were cloned and characterized . A Northern blot survey identified two transcripts that hybridized to a hRAD50 cDNA clone, an upper faint band (5.9kb) and lower dense band (4.6kb) . cDNA clones (hRAD50-2, 4.6kb) encompassing the entire hRAD50 transcript but having a shorter 3'-untranslated region (3'UTR) than the previously reported hRAD50-1 cDNA (5.9kb; Dolganov, G.M., Maser, R.S., Novikov, A., Tosto, L., Chong, S., Bressan, D.A., Petrini, J.H.J., 1996 . Human Rad50 is physically associated with human Mre11: Identification of a conserved multiprotein complex implicated in recombinational DNA repair . Mol . Cell . Biol . 16, 4832-4841.) were isolated . The presence of AU-rich sequences in the 3'UTR of hRAD50-1, which define mRNA instability and Northern results, suggest that hRAD50-2 is the major transcript of hRAD50 . A third alternative splice variant that lacks the ATP-binding domain was also identified (hRAD50-3, approximately 4.5kb) . Expression of hRAD50-3 transcript was detected in all tissues examined by RT-PCR (reverse transcriptase-polymerase chain reaction) and nested DNA-PCR analyses . Expression of hRAD50 partially rescued the MMS (methyl methanesulfonate)-sensitive phenotype in rad50 mutant yeast, whereas hRAD50-3 did not show complementation . These data suggest that the hRAD50-3 does not repair DNA double-strand breaks most likely due to its inability to bind ATP, and to bind damaged DNA . The existence of these alternative splice forms is potentially important in regulation of the biological activity of the DNA recombinational repair gene, hRAD50. Anal Biochem, 1999 Aug 1, 272(2), 263 - 9 Characterization of a temperature-sensitive mutant of a ubiquitin-conjugating enzyme and its use as a heat-inducible degradation signal; Tongaonkar P et al.; The ubiquitin/proteasome pathway is a highly conserved mechanism of proteolysis in all eukaryotes . Ubiquitin (Ub) is conjugated to proteolytic substrates through the sequential action of ubiquitin-activating (E1/Uba) and ubiquitin-conjugating (E2/Ubc) enzymes . The mechanism of substrate recognition and ubiquitination is an area of active investigation, and we have begun a site-directed mutagenesis approach to define the biochemical and biophysical properties of ubiquitin-conjugating enzymes . We have characterized a specific mutation in Ubc4 (Ubc4(P62S)) which was previously shown to cause a temperature-sensitive growth defect in several other Ubc's . Ubc4(P62S) was rapidly degraded in vivo, contributing to the loss of function . However, reconstitution experiments revealed that the catalytic activity of Ubc4(P62S) was reversibly inactivated at 37 degrees C, demonstrating that the primary defect of Ubc4(P62S) is its inability to form a ubiquitin thioester bond at high temperature . The in vivo defect is compounded by increased susceptibility of Ubc4(P62S) to degradation by the ubiquitin/proteasome pathway . We have exploited the temperature-dependent degradation of the P62S mutant to destabilize an otherwise stable test protein (glutathione S-transferase) . The use of this mutant may provide a useful cis-acting temperature-inducible degradation signal . Exp Cell Res, 1999 Aug 1, 250(2), 401 - 13 The nuclear localization signal of the human Ku70 is a variant bipartite type recognized by the two components of nuclear pore-targeting complex; Koike M et al.; Ku protein is a complex of two subunits, Ku70 and Ku80 . Ku is suspected to participate in both DNA double-strand break repair and transcription . Since both of these processes take place in the cell nucleus, we have been investigating the subcellular localization and nuclear transport of Ku proteins . In the present study, we analyzed the subcellular localization and nuclear localization signal (NLS) of Ku70 . Fusion proteins of Ku70 and green fluorescent protein (GFP) transiently expressed in cells were clearly localized in the nuclei of interphase cells . Ku70 staining was distributed throughout both the nucleus and the cytoplasm in late telophase to early G1 phase cells . The NLS of Ku70 was located at the region composed of 18 amino acid residues (positions 539 to 556) . This region overlapped with the Ku80-independent DNA-binding domain reported previously . The Ku70 NLS consisted of two basic subregions and a nonbasic intervening region . All the subregions were necessary for complete NLS activity . The amino acids in the nonbasic intervening region of Ku70 might be important for full NLS activity not only to provide sufficient length between the two separated clusters of basic amino acids but also to have an adequate amino acid sequence . All of the basic amino acid residues in the basic subregions were conserved among mammalian and avian homologues, confirming their importance in the nuclear translocation of Ku70 . The structure of the Ku70 NLS resembled the consensus of a bipartite-type NLS . The Ku70 NLS was mediated to target to the nuclear rim by two components of the nuclear pore-targeting complex, PTAC58 and PTAC97 . J Cell Sci, 1999 Aug, 112 ( Pt 16), 2647 - 56 The casein kinase Ialpha isoform is both physically positioned and functionally competent to regulate multiple events of mRNA metabolism; Gross SD et al.; Casein kinase I is a highly conserved family of serine/threonine protein kinases present in every organism tested from yeast to humans . To date, little is known about the function of the higher eukaryotic isoforms in this family . The CKI isoforms in Saccharomyces cerevisiae, however, have been genetically linked to the regulation of DNA repair, cell cycle progression and cytokinesis . It has also been established that the nuclear localization of two of these isoforms is essential for their function . The work presented here demonstrates that the higher eukaryotic CKIalpha isoform is also present within nuclei of certain established cell lines and associated with discrete nuclear structures . The nature of its nuclear localization was characterized . In this regard, CKIalpha was shown to colocalize with factors involved in pre-mRNA splicing at nuclear speckles and that its association with these structures exhibited several biochemical properties in common with known splicing factors . The kinase was also shown to be associated with a complex that contained certain splicing factors . Finally, in vitro, CKIalpha was shown to be capable of phosphorylating particular splicing factors within a region rich in serine/arginine dipeptide repeat motifs suggesting that it has both the opportunity and the capacity to regulate one or more steps of mRNA metabolism. Biochemistry, 1999 Jul 20, 38(29), 9198 - 208 Glutamic acid 472 and lysine 480 of the sodium pump alpha 1 subunit are essential for activity . Their conservation in pyrophosphatases suggests their involvement in recognition of ATP phosphates; Scheiner-Bobis G et al.; P-type ATPases such as the Na+,K+-ATPase (sodium pump) hydrolyze ATP to pump ions through biological membranes against their electrochemical gradients . The mechanisms that couple ATP hydrolysis to the vectorial ion transport are not yet understood, but unveiling structures that participate in ATP binding and in the formation of the ionophore might help to gain insight into this process . Looking at the alpha- and beta-phosphates of ATP as a pyrophosphate molecule, we found that peptides highly conserved among all soluble inorganic pyrophosphatases are also present in ion-transporting ATPases . Included therein are Glu48 and Lys56 of the Saccharomyces cerevisiae pyrophosphatase (SCE1-PPase) that are essential for the activity of this enzyme and have been shown in crystallographic analysis to interact with phosphate molecules . To test the hypothesis that equivalent amino acids are also essential for the activity of ion-transporting ATPases, Glu472 and Lys480 of the sodium pump alpha 1 subunit corresponding to Glu48 and Lys56 of SCE1-PPase were mutated to various amino acids . Mutants of the sodium pump alpha1 subunit were expressed in yeast and analyzed for their ATPase activity and their ability to bind ouabain in the presence of either ATP, Mg2+, and Na+ or phosphate and Mg2+ . All four mutants investigated, Glu472Ala, Glu472Asp, Lys480Ala, and Lys480Arg, display only a fraction of the ATPase activity obtained with the wild-type enzyme . The same applies with respect to their ability to bind ouabain, where maximum ouabain binding to the mutants accounts for only about 10% of the binding obtained with the wild-type enzyme . On the basis of our results, we conclude that Glu472 and Lys480 are essential for the activity of the sodium pump . Their function is probably to arrest the alpha- and beta-phosphate groups of ATP in a proper position prior to hydrolysis of the gamma-phosphate group . The identification of these amino acids as essential components of the ATP-recognizing mechanism of the pump has resulted in a testable hypothesis for the initial interactions of the sodium pump, and possibly of other P-type ATPases, with ATP. FEBS Lett, 1999 Jul 2, 454(1-2), 131 - 6 Human release factor eRF1: structural organisation of the unique functional gene on chromosome 5 and of the three processed pseudogenes; Guenet L et al.; In lower and higher eukaryotes, a family of tightly related proteins designated eRF1 (for eukaryotic release factor 1) catalyses termination of protein synthesis at all three stop codons . The human genome contains four eRF1 homologous sequences localised on chromosomes 5, 6, 7 and X . We report here the cloning and the structural analysis of the human eRF1 gene family . It appears that the gene located on chromosome 5 alone is potentially functional, whereas the other three sequences resemble processed pseudogenes . This is the first description of the structural organisation of the human eRF1 gene, which has been remarkably conserved during evolution and which is essential in the translation termination process. Cell, 1999 Jul 9, 98(1), 81 - 90 Vesicular tubular clusters between the ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles; Martinez-Menarguez JA et al.; We have determined the concentrations of the secretory proteins amylase and chymotrypsinogen and the membrane proteins KDELr and rBet1 in COPII- and COPI-coated pre-Golgi compartments of pancreatic cells by quantitative immunoelectron microscopy . COPII was confined to ER membrane buds and adjacent vesicles . COPI occurred on vesicular tubular clusters (VTCs), Golgi cisternae, the trans-Golgi network, and immature secretory granules . Both secretory proteins exhibited a first, significant concentration step in noncoated segments of VTC tubules and were excluded from COPI-coated tips . By contrast, KDELr and rBet1 showed a first, significant concentration in COPII-coated ER buds and vesicles and were prominently present in COPI-coated tips of VTC tubules . These data suggest an important role of VTCs in soluble cargo concentration by exclusion from COPI-coated domains. Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8567 - 72 "Mutagenesis" by peptide aptamers identifies genetic network members and pathway connections; Geyer CR et al.; We selected peptide aptamers from combinatorial libraries that disrupted cell-cycle arrest caused by mating pheromone in yeast . We used these aptamers as baits in two-hybrid hunts to identify genes involved in cell-cycle arrest . These experiments identified genes known to function in the pathway, as well as a protein kinase, the CBK1 product, whose function was not known . We used a modified two-hybrid system to identify specific interactions disrupted by these aptamers . These experiments demonstrate a means to perform "genetics" on the protein complement of a cell without altering its genetic material . Peptide aptamers can be identified that disrupt a process . These aptamers can then be used as affinity reagents to identify individual proteins and protein interactions needed for the process . Forward genetic analysis with peptide aptamer "mutagens" should be particularly useful in elucidating genetic networks in organisms and processes for which classical genetics is not feasible. Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8493 - 8 Retention of the BUB3 checkpoint protein on lagging chromosomes; Martinez-Exposito MJ et al.; Accurate chromosome segregation at mitosis is ensured both by the intrinsic fidelity of the mitotic machinery and by the operation of checkpoints that monitor chromosome-microtubule attachment . When unattached kinetochores are present, anaphase is delayed and the time available for chromosome-microtubule capture increases . Genes required for this delay first were identified in budding yeast (the MAD and BUB genes), but it is not yet known how the checkpoint senses unattached chromosomes or how it signals cell-cycle arrest . We report the isolation and analysis of a murine homologue of BUB3, a gene whose deletion abolishes mitotic checkpoint function in Saccharomyces cerevisiae . mBub3 belongs to a small gene family that has been highly conserved through evolution . By expressing recombinant proteins in insect cells, we show that mBub3, like yeast Bub3p, binds to Bub1 to form a complex with protein kinase activity . During prophase and prometaphase, preceding kinetochore-microtubule attachment, Bub3 localizes to kinetochores . High levels of mBub3 remain associated with lagging chromosomes but not with correctly aligned chromosomes during metaphase, consistent with a role for Bub3 in sensing microtubule attachment . Intriguingly, the number of lagging chromosomes with high Bub3 staining increases dramatically in cells treated with low (and pharmacologically relevant) concentrations of the chemotherapeutic taxol and the microtubule poison nocodazole. Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8483 - 8 Development of gene-switch transgenic mice that inducibly express transforming growth factor beta1 in the epidermis; Wang XJ et al.; Previous attempts to establish transgenic mouse models to study the functions of transforming growth factor beta1 (TGFbeta1) in the skin revealed controversial roles for TGFbeta1 in epidermal growth (inhibition vs . stimulation) and resulted in neonatal lethality in one instance . To establish a viable transgenic model for studying functions of TGFbeta1 in the skin, we have now developed transgenic mice, which allow focal induction of the TGFbeta1 transgene in the epidermis at different expression levels and at different developmental stages . This system, termed "gene-switch," consists of two transgenic lines . The mouse loricrin vector targets the GLVPc transactivator (a fusion molecule of the truncated progesterone receptor and the GAL4 DNA binding domain), and a thymidine kinase promoter drives the TGFbeta1 target gene with GAL4 binding sites upstream of the promoter . These two transgenic lines were mated to generate bigenic mice, and TGFbeta1 transgene expression was controlled by topical application of an antiprogestin . On epidermal-specific induction of the TGFbeta1 transgene, the BrdUrd labeling index in the transgenic epidermis decreased 6-fold compared with controls . Induction of the TGFbeta1 transgene expression also caused epidermal resistance to phorbol 12-myristate 13-acetate-induced hyperplasia, with a reduction in both epidermal thickness and BrdUrd labeling compared with those in controls . In addition, TGFbeta1 transgene expression induced an increase in angiogenesis in the dermis . Given that the TGFbeta1 transgene can affect both the epidermis and dermis, this transgenic model will provide a useful tool for studying roles of TGFbeta1 in wound-healing and skin carcinogenesis in the future. Mol Cell Biol, 1999 Aug, 19(8), 5685 - 95 Activity of the c-myc replicator at an ectopic chromosomal location; Malott M et al.; DNA replication starts at multiple discrete sites across the human chromosomal c-myc region, including two or more sites within 2.4 kb upstream of the c-myc gene . The corresponding 2.4-kb c-myc origin fragment confers autonomously replicating sequence (ARS) activity on plasmids, which specifically initiate replication in the origin fragment in vitro and in vivo . To test whether the region that displays plasmid replicator activity also acts as a chromosomal replicator, HeLa cell sublines that each contain a single copy of the Saccharomyces cerevisiae FLP recombinase target (FRT) sequence flanked by selectable markers were constructed . A clonal line containing a single unrearranged copy of the transduced c-myc origin was produced by cotransfecting a donor plasmid containing the 2.4-kb c-myc origin fragment and FRT, along with a plasmid expressing the yeast FLP recombinase, into cells containing a chromosomal FRT acceptor site . The amount of short nascent DNA strands at the chromosomal acceptor site was quantitated before and after targeted integration of the origin fragment . Competitive PCR quantitation showed that the c-myc origin construct substantially increased the amount of nascent DNA relative to that at the unoccupied acceptor site and to that after the insertion of non-myc DNA . The abundance of nascent strands was greatest close to the c-myc insert of the integrated donor plasmid, and significant increases in nascent strand abundance were observed at sites flanking the insertion . These results provide biochemical and genetic evidence for the existence of chromosomal replicators in metazoan cells and are consistent with the presence of chromosomal replicator activity in the 2.4-kb region of c-myc origin DNA. J Biol Chem, 1999 Jul 23, 274(30), 21223 - 7 Ku binds telomeric DNA in vitro; Bianchi A et al.; Ku is a heterodimeric protein with high binding affinity for ends, nicks, and gaps in double-stranded DNA . Both in mammalian cells and in budding yeast, Ku plays a role in nonhomologous end joining in the double strand break repair pathway . However, Ku has a more significant role in DNA repair in mammalian cells compared with yeast, in which a homology-dependent pathway is the predominant one . Recently Ku has been shown to be a likely component of the telomeric complex in yeast, suggesting the possibility of a similar role for Ku at mammalian telomeres . However, long single-stranded G-rich overhangs are continuously present at mammalian but not at yeast telomeres . These overhangs have the potential to fold in vitro into G-G base-paired conformations, such as G-quartets, that might prevent Ku from recognizing telomeric ends and thus offer a mechanism to sequester the telomere from the prevalent double strand break repair pathway in mammals . We show here that Ku binds to mammalian telomeric DNA ends in vitro and that G-quartet conformations are unable to prevent Ku from binding with high affinity to the DNA . Our results indicate that the DNA binding characteristics of Ku are consistent with its direct interaction with telomeric DNA in mammalian cells and its proposed role as a telomere end factor. Science, 1999 Jul 16, 285(5426), 418 - 22 Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis; Zou H et al.; A vertebrate securin (vSecurin) was identified on the basis of its biochemical analogy to the Pds1p protein of budding yeast and the Cut2p protein of fission yeast . The vSecurin protein bound to a vertebrate homolog of yeast separins Esp1p and Cut1p and was degraded by proteolysis mediated by an anaphase-promoting complex in a manner dependent on a destruction motif . Furthermore, expression of a stable Xenopus securin mutant protein blocked sister-chromatid separation but did not block the embryonic cell cycle . The vSecurin proteins share extensive sequence similarity with each other but show no sequence similarity to either of their yeast counterparts . Human securin is identical to the product of the gene called pituitary tumor-transforming gene (PTTG), which is overexpressed in some tumors and exhibits transforming activity in NIH 3T3 cells . The oncogenic nature of increased expression of vSecurin may result from chromosome gain or loss, produced by errors in chromatid separation. RNA, 1999 Jul, 5(7), 876 - 82 Trans-complementation of the second step of pre-mRNA splicing by exogenous 5' exons; Chanfreau G et al.; During splicing of nuclear pre-mRNAs, the first step liberates the 5' exon (exon 1) and yields a lariat intron-3'exon (intron-exon 2) intermediate . The second step results in exon ligation . Previous results indicated that severe truncations of the 5' exon of the actin pre-mRNA result in a block to the second splicing step in vitro in yeast extracts, leading to an accumulation of intron-exon 2 lariat intermediates . We show that exogenous exon 1 RNA oligonucleotides can chase these stalled intermediates into lariat intron and spliced exons . This reaction requires some of the cis elements and trans-acting factors that are required for a normal second step . There is no strong sequence requirement for the exon 1 added in trans, but oligonucleotides with complementarity to the U5 snRNA conserved loop perform the chase more efficiently . Using a dominant negative mutant of the DEAH-box ATPase Prp16p and ATP depletion, we show that the stalled intermediate is blocked after the Prp16p-dependent step . These results show that exogenous RNAs with various sequences but containing no splicing signals can be incorporated into spliceosomes and undergo RNA recombination and exon shuffling during the second step of pre-mRNA splicing. Mol Cell Biol, 1999 Aug, 19(8), 5732 - 42 Dual requirement for the EcR/USP nuclear receptor and the dGATAb factor in an ecdysone response in Drosophila melanogaster; Brodu V et al.; The EcR/USP nuclear receptor controls Drosophila metamorphosis by activating complex cascades of gene transcription in response to pulses of the steroid hormone ecdysone at the end of larval development . Ecdysone release provides a ubiquitous signal for the activation of the receptor, but a number of its target genes are induced in a tissue- and stage-specific manner . Little is known about the molecular mechanisms involved in this developmental modulation of the EcR/USP-mediated pathway . Fbp1 is a good model of primary ecdysone response gene expressed in the fat body for addressing this question . We show here that the dGATAb factor binds to three target sites flanking an EcR/USP binding site in a 70-bp enhancer that controls the tissue and stage specificity of Fbp1 transcription . We demonstrate that one of these sites and proper expression of dGATAb are required for specific activation of the enhancer in the fat body . In addition, we provide further evidence that EcR/USP plays an essential role as a hormonal timer . Our study provides a striking example of the integration of molecular pathways at the level of a tissue-specific hormone response unit. Mol Cell Biol, 1999 Aug, 19(8), 5652 - 8 Transcription factor TFIIH is required for promoter melting in vivo; Guzman E et al.; The Rad25 protein in yeast is a DNA helicase and a subunit of the general transcription factor TFIIH . While in vitro studies have led to the hypothesis that TFIIH helicase activity plays a role in promoter melting, in vivo tests are lacking . Using potassium permanganate, which preferentially modifies single-stranded DNA, we show that a temperature-sensitive rad25(ts) mutant severely reduces the normally extensive promoter melting observed in vivo on the highly expressed genes TDH2 and PDC1 and on the induced heat shock gene HSP82 . Loss of promoter melting can be observed in as little as 30 s after a shift to the nonpermissive temperature and is accompanied by a dramatic reduction in transcription . These effects on the promoter are specific, since the mutation does not affect TATA box occupancy or, in the case of HSP82, recruitment of TATA-binding protein to the TATA element or that of heat shock factor to heat shock elements . Additionally, using the technique of formaldehyde cross-linking coupled with restriction endonuclease cleavage and ligation-mediated PCR, we were able to map the polymerase density on the promoter of HSP82 . This high-resolution mapping allowed us to determine that the polymerase II (Pol II) density on the promoter is also dramatically reduced after inactivation of TFIIH . These data provide strong support for the hypothesis that TFIIH functions with Pol II in the transcriptionally required step of promoter melting and show, surprisingly, that the extent of TFIIH-dependent promoter melting observed in vivo is several times larger than that seen in vitro. Mol Cell Biol, 1999 Aug, 19(8), 5441 - 52 Imp3p and Imp4p, two specific components of the U3 small nucleolar ribonucleoprotein that are essential for pre-18S rRNA processing; Lee SJ et al.; The function of the U3 small nucleolar ribonucleoprotein (snoRNP) is central to the events surrounding pre-rRNA processing, as evidenced by the severe defects in cleavage of pre-18S rRNA precursors observed upon depletion of the U3 RNA and its unique protein components . Although the precise function of each component remains unclear, since U3 snoRNA levels remain unchanged upon genetic depletion of these proteins, it is likely that the proteins themselves have significant roles in the cleavage reactions . Here we report the identification of two previously undescribed protein components of the U3 snoRNP, representing the first snoRNP components identified by using the two-hybrid methodology . By screening for proteins that physically associate with the U3 snoRNP-specific protein, Mpp10p, we have identified Imp3p (22 kDa) and Imp4p (34 kDa) (named for interacting with Mpp10p) . The genes encoding both proteins are essential in yeast . Genetic depletion reveals that both proteins are critical for U3 snoRNP function in pre-18S rRNA processing at the A0, A1, and A2 sites in the pre-rRNA . Both Imp proteins associate with Mpp10p in vivo, and both are complexed only with the U3 snoRNA . Conservation of RNA binding domains between Imp3p and the S4 family of ribosomal proteins suggests that it might associate with RNA directly . However, as with other U3 snoRNP-specific proteins, neither Imp3p nor Imp4p is required for maintenance of U3 snoRNA integrity . Imp3p and Imp4p are therefore novel protein components specific to the U3 snoRNP with critical roles in pre-rRNA cleavage events. Mol Cell Biol, 1999 Aug, 19(8), 5417 - 28 Hec1p, an evolutionarily conserved coiled-coil protein, modulates chromosome segregation through interaction with SMC proteins; Zheng L et al.; hsHec1p, a Homo sapiens coiled-coil-enriched protein, plays an important role in M-phase progression in mammalian cells . A Saccharomyces cerevisiae protein, identical to Tid3p/Ndc80p and here designated scHec1p, has similarities in structure and biological function to hsHec1p . Budding yeast cells deleted in the scHEC1/NDC80 allele are not viable, but this lethal phenotype can be rescued by hsHEC1 under control of the endogenous scHEC1 promoter . At the nonpermissive temperature, significant mitotic delay, chromosomal missegregation, and decreased viability were observed in yeast cells with temperature-sensitive (ts) alleles of hsHEC1 . In the hshec1-113 ts mutant, we found a single-point mutation changing Trp395 to a stop codon, which resulted in the expression of a C-terminally truncated 45-kDa protein . The binding of this mutated protein, hshec1-113p, to five identified hsHec1p-associated proteins was unchanged, while its binding to human SMC1 protein and yeast Smc1p was ts . Hec1p also interacts with Smc2p, and the binding of the mutated hshec1-113p to Smc2p was not ts . Overexpression of either hsHEC1 or scHEC1 suppressed the lethal phenotype of smc1-2 and smc2-6 at nonpermissive temperatures, suggesting that the interactions between Hec1p and Smc1p and -2p are biologically significant . These results suggest that Hec1 proteins play a critical role in modulating chromosomal segregation, in part, through their interactions with SMC proteins. Mol Cell Biol, 1999 Aug, 19(8), 5393 - 404 Transcriptional elements involved in the repression of ribosomal protein synthesis; Li B et al.; The ribosomal proteins (RPs) of Saccharomyces cerevisiae are encoded by 137 genes that are among the most transcriptionally active in the genome . These genes are coordinately regulated: a shift up in temperature leads to a rapid, but temporary, decline in RP mRNA levels . A defect in any part of the secretory pathway leads to greatly reduced ribosome synthesis, including the rapid loss of RP mRNA . Here we demonstrate that the loss of RP mRNA is due to the rapid transcriptional silencing of the RP genes, coupled to the naturally short lifetime of their transcripts . The data suggest further that a global inhibition of polymerase II transcription leads to overestimates of the stability of individual mRNAs . The transcription of most RP genes is activated by two Rap1p binding sites, 250 to 400 bp upstream from the initiation of transcription . Rap1p is both an activator and a silencer of transcription . The swapping of promoters between RPL30 and ACT1 or GAL1 demonstrated that the Rap1p binding sites of RPL30 are sufficient to silence the transcription of ACT1 in response to a defect in the secretory pathway . Sir3p and Sir4p, implicated in the Rap1p-mediated repression of silent mating type genes and of telomere-proximal genes, do not influence such silencing of RP genes . Sir2p, implicated in the silencing both of the silent mating type genes and of genes within the ribosomal DNA locus, does not influence the repression of either RP or rRNA genes . Surprisingly, the 180-bp sequence of RPL30 that lies between the Rap1p sites and the transcription initiation site is also sufficient to silence the Gal4p-driven transcription in response to a defect in the secretory pathway, by a mechanism that requires the silencing region of Rap1p . We conclude that for Rap1p to activate the transcription of an RP gene it must bind to upstream sequences; yet for Rap1p to repress the transcription of an RP gene it need not bind to the gene directly . Thus, the cell has evolved a two-pronged approach to effect the rapid extinction of RP synthesis in response to the stress imposed by a heat shock or by a failure of the secretory pathway . Calculations based on recent transcriptome data and on the half-life of the RP mRNAs suggest that in a rapidly growing cell the transcription of RP mRNAs accounts for nearly 50% of the total transcriptional events initiated by RNA polymerase II . Thus, the sudden silencing of the RP genes must have a dramatic effect on the overall transcriptional economy of the cell. Mol Cell Biol, 1999 Aug, 19(8), 5373 - 82 A novel role in DNA metabolism for the binding of Fen1/Rad27 to PCNA and implications for genetic risk; Gary R et al.; Fen1/Rad27 nuclease activity, which is important in DNA metabolism, is stimulated by proliferating cell nuclear antigen (PCNA) in vitro . The in vivo role of the PCNA interaction was investigated in the yeast Rad27 . A nuclease-defective rad27 mutation had a dominant-negative effect that was suppressed by a mutation in the PCNA binding site, thereby demonstrating the importance of the Rad27-PCNA interaction . The PCNA-binding defect alone had little effect on mutation, recombination, and the methyl methanesulfonate (MMS) response in repair-competent cells, but it greatly amplified the MMS sensitivity of a rad51 mutant . Furthermore, the PCNA binding mutation resulted in lethality when combined with a homozygous or even a heterozygous pol3-01 mutation in the 3'-->5' exonuclease domain of DNA polymerase delta . These results suggest that phenotypically mild polymorphisms in DNA metabolic proteins can have dramatic consequences when combined. Mol Cell Biol, 1999 Aug, 19(8), 5257 - 66 Mutations in elongation factor 1beta, a guanine nucleotide exchange factor, enhance translational fidelity; Carr-Schmid A et al.; Translation elongation factor 1beta (EF-1beta) is a member of the family of guanine nucleotide exchange factors, proteins whose activities are important for the regulation of G proteins critical to many cellular processes . EF-1beta is a highly conserved protein that catalyzes the exchange of bound GDP for GTP on EF-1alpha, a required step to ensure continued protein synthesis . In this work, we demonstrate that the highly conserved C-terminal region of Saccharomyces cerevisiae EF-1beta is sufficient for normal cell growth . This region of yeast and metazoan EF-1beta and the metazoan EF-1beta-like protein EF-1delta is highly conserved . Human EF-1beta, but not human EF-1delta, is functional in place of yeast EF-1beta, even though both EF-1beta and EF-1delta have previously been shown to have guanine nucleotide exchange activity in vitro . Based on the sequence and functional homology, mutagenesis of two C-terminal residues identical in all EF-1beta protein sequences was performed, resulting in mutants with growth defects and sensitivity to translation inhibitors . These mutants also enhance translational fidelity at nonsense codons, which correlates with a reduction in total protein synthesis . These results indicate the critical function of EF-1beta in regulating EF-1alpha activity, cell growth, translation rates, and translational fidelity. J Biol Chem, 1999 Jul 23, 274(30), 21149 - 54 Interaction of the alpha(1B)-adrenergic receptor with gC1q-R, a multifunctional protein; Xu Z et al.; gC1q-R, a multifunctional protein, was found to bind with the carboxyl-terminal cytoplasmic domain of the alpha(1B)-adrenergic receptor (173 amino acids, amino acids 344-516) in a yeast two-hybrid screen of a cDNA library prepared from the rat liver . In a series of studies with deletion mutants in this region, the ten arginine-rich amino acids (amino acids 369-378) were identified as the site of interaction . The interaction was confirmed by specific co-immunoprecipitation of gC1q-R with full-length alpha(1B)-adrenergic receptors expressed on transfected COS-7 cells, as well as by fluorescence confocal laser scanning microscopy, which showed co-localization of these proteins in intact cells . Interestingly, the alpha(1B)-adrenergic receptors were exclusively localized to the region of the plasma membrane in COS-7 cells that expressed the alpha(1B)-adrenergic receptor alone, whereas gC1q-R was localized in the cytoplasm in COS-7 cells that expressed gC1q-R alone; however, in cells that co-expressed alpha(1B)-adrenergic receptors and gC1q-R, most of the alpha(1B)-adrenergic receptors were co-localized with gC1q-R in the intracellular region, and a remarkable down-regulation of receptor expression was observed . These observations suggest a new role for the previously identified complement regulatory molecule, gC1q-R, in regulating the cellular localization and expression of the alpha(1B)-adrenergic receptors. J Biol Chem, 1999 Jul 23, 274(30), 21029 - 36 Distinct regions of the Swi5 and Ace2 transcription factors are required for specific gene activation; McBride HJ et al.; Swi5 and Ace2 are cell cycle-regulated transcription factors that activate expression of early G(1)-specific genes in Saccharomyces cerevisiae . Swi5 and Ace2 have zinc finger DNA-binding domains that are highly conserved, and the two proteins bind to the same DNA sequences in vitro . Despite this similarity in DNA binding, Swi5 and Ace2 activate different genes in vivo, with Swi5 activating the HO gene and Ace2 activating CTS1 expression . In this report we have used chimeric fusions between Swi5 and Ace2 to determine what regions of these proteins are necessary for promoter-specific activation of HO and CTS1 . We have identified specific regions of Swi5 and Ace2 that are required for activation of HO and CTS1, respectively . The Swi5 protein binds HO promoter DNA cooperatively with the Pho2 homeodomain protein, and the HO specificity region of Swi5 identified in the chimeric analysis coincides with the region of Swi5 previously identified that interacts with Pho2 in vitro . Swi5 and Ace2 also activate expression of a number of other genes expressed in G(1) phase of the cell cycle, including ASH1, CDC6, EGT2, PCL2, PCL9, RME1, and SIC1 . Analysis of the Swi5/Ace2 chimeras shows that distinct regions of Swi5 and Ace2 contribute to the transcriptional activation of some of these other G(1)-regulated genes. J Biol Chem, 1999 Jul 23, 274(30), 20937 - 42 The DNA helicase, Hmi1p, is transported into mitochondria by a C-terminal cleavable targeting signal; Lee CM et al.; We have identified a novel mitochondrial targeting signal in the precursor of the DNA helicase Hmi1p of Saccharomyces cerevisiae that is located at the C terminus of the protein . Similar to classical N-terminal presequences, this C-terminal targeting signal consists of a stretch of positively charged amino acids that has the potential to form an amphipathic alpha-helix . Deletion of the C-terminal 36 amino acids of helicase resulted in loss of import into mitochondria, while deletion of the N-terminal 40 amino acids had no effect . When C-terminal regions of the helicase were placed at the C terminus of a passenger protein, dihydrofolate reductase, the resulting fusion proteins were directed into the mitochondrial matrix, and the C-terminal region of helicase became proteolytically processed . Import of helicase occurs in a C- to N-terminal direction; it requires a membrane potential and the TIM17-23 translocase together with mitochondrial Hsp70 . Helicase is the only mitochondrial matrix protein identified thus far with a cleavable targeting signal at its C terminus. J Biol Chem, 1999 Jul 23, 274(30), 20855 - 60 Regulation of apoptotic protease activating factor-1 oligomerization and apoptosis by the WD-40 repeat region; Adrain C et al.; Apoptotic protease activating factor-1 (Apaf-1) has been identified as a proximal activator of caspase-9 in cell death pathways that trigger mitochondrial damage and cytochrome c release . The mechanism of Apaf-1 action is unclear but has been proposed to involve the clustering of caspase-9 molecules, thereby facilitating autoprocessing of adjacent zymogens . Here we show that Apaf-1 can dimerize via the CED-4 homologous and linker domains of the molecule providing a means by which Apaf-1 can promote the clustering of caspase-9 and facilitate its activation . Apaf-1 dimerization was repressed by the C-terminal half of the molecule, which contains multiple WD-40 repeats, but this repression was overcome in the presence of cytochrome c and dATP . Removal of the WD-40 repeat region resulted in a constitutively active Apaf-1 that exhibited greater cytotoxicity in transient transfection assays when compared with full-length Apaf-1 . These data suggest a mechanism for Apaf-1 function and reveal an important regulatory role for the WD-40 repeat region. Biotechniques, 1999 Jul, 27(1), 106 - 10 Eukaryotic conditional expression system; Sourisseau T et al.; Existing conditional expression systems can be classified in two major categories that are based either on the induction or on the de-repression of transcription . The system described here combines both mechanisms, since a unique transcription factor can be shifted from a repression to a stimulation activity by simply changing its ligand . The resulting advantage of this system is the complete absence of basal expression before active induction . The principle of this method is based on the unexpected ability of the chimeric protein containing the DNA-binding domain of the yeast Gal4 transcription factor fused to the COOH half of the estradiol receptor (GalER), to act as a repressor when bound to the drug 4OH-tamoxifen, in the context of a previously described optimized Gal4-responsive promoter . The efficacy of this system has been assessed in transient expression assays using the chloramphenicol acetyl transferase (CAT), and in situ, through the activity of a Gal4 responsive beta-galactosidase gene. EMBO J, 1999 Jul 15, 18(14), 4068 - 75 Repressor binding to a dorsal regulatory site traps human eIF4E in a high cap-affinity state; Ptushkina M et al.; Eukaryotic translation initiation involves recognition of the 5' end of cellular mRNA by the cap-binding complex known as eukaryotic initiation factor 4F (eIF4F) . Initiation is a key point of regulation in gene expression in response to mechanisms mediated by signal transduction pathways . We have investigated the molecular interactions underlying inhibition of human eIF4E function by regulatable repressors called 4E-binding proteins (4E-BPs) . Two essential components of eIF4F are the cap-binding protein eIF4E, and eIF4G, a multi-functional protein that binds both eIF4E and other essential eIFs . We show that the 4E-BPs 1 and 2 block the interaction between eIF4G and eIF4E by competing for binding to a dorsal site on eIF4E . Remarkably, binding of the 4E-BPs at this dorsal site enhances cap-binding via the ventral cap-binding slot, thus trapping eIF4E in inactive complexes with high affinity for capped mRNA . The binding contacts and affinities for the interactions between 4E-BP1/2 and eIF4E are distinct (estimated K(d) values of 10(-8) and 3x10(-9) for 4E-BP1 and 2, respectively), and the differences in these properties are determined by three amino acids within an otherwise conserved motif . These data provide a quantitative framework for a new molecular model of translational regulation. EMBO J, 1999 Jul 15, 18(14), 3981 - 9 The mitochondrial proteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe/S proteins; Kispal G et al.; Iron-sulfur (Fe/S) cluster-containing proteins catalyse a number of electron transfer and metabolic reactions . Little is known about the biogenesis of Fe/S clusters in the eukaryotic cell . Here, we demonstrate that mitochondria perform an essential role in the synthesis of both intra- and extra-mitochondrial Fe/S proteins . Nfs1p represents the yeast orthologue of the bacterial cysteine desulfurase NifS that initiates biogenesis by producing elemental sulfur . The matrix-localized protein is required for synthesis of both mitochondrial and cytosolic Fe/S proteins . The ATP-binding cassette (ABC) transporter Atm1p of the mitochondrial inner membrane performs an essential function only in the generation of cytosolic Fe/S proteins by mediating export of Fe/S cluster precursors synthesized by Nfs1p and other mitochondrial proteins . Assembly of cellular Fe/S clusters constitutes an indispensable biosynthetic task of mitochondria with potential relevance for an iron-storage disease and the control of cellular iron uptake. EMBO J, 1999 Jul 15, 18(14), 3934 - 46 Got1p and Sft2p: membrane proteins involved in traffic to the Golgi complex; Conchon S et al.; Traffic through the yeast Golgi complex depends on a member of the syntaxin family of SNARE proteins, Sed5p, present in early Golgi cisternae . Sft2p is a non-essential tetra-spanning membrane protein, found mostly in the late Golgi, that can suppress some sed5 alleles . We screened for mutations that show synthetic lethality with sft2 and found one that affects a previously uncharacterized membrane protein, Got1p, as well as new alleles of sed5 and vps3 . Got1p is an evolutionarily conserved non-essential protein with a membrane topology similar to that of Sft2p . Immunofluorescence and subcellular fractionation indicate that it is present in early Golgi cisternae . got1 mutants, but not sft2 mutants, show a defect in an in vitro assay for ER-Golgi transport at a step after vesicle tethering to Golgi membranes . In vivo, inactivation of both Got1p and Sft2p results in phenotypes ascribable to a defect in endosome-Golgi traffic, while their complete removal results in an ER-Golgi transport defect . Thus the presence of either Got1p or Sft2p is required for vesicle fusion with the Golgi complex in vivo . We suggest that Got1p normally facilitates Sed5p-dependent fusion events, while Sft2p performs a related function in the late Golgi. EMBO J, 1999 Jul 15, 18(14), 3877 - 87 Structural basis for the specificity of ubiquitin C-terminal hydrolases; Johnston SC et al.; The release of ubiquitin from attachment to other proteins and adducts is critical for ubiquitin biosynthesis, proteasomal degradation and other cellular processes . De-ubiquitination is accomplished in part by members of the UCH (ubiquitin C-terminal hydrolase) family of enzymes . We have determined the 2.25 A resolution crystal structure of the yeast UCH, Yuh1, in a complex with the inhibitor ubiquitin aldehyde (Ubal) . The structure mimics the tetrahedral intermediate in the reaction pathway and explains the very high enzyme specificity . Comparison with a related, unliganded UCH structure indicates that ubiquitin binding is coupled to rearrangements which block the active-site cleft in the absence of authentic substrate . Remarkably, a 21-residue loop that becomes ordered upon binding Ubal lies directly over the active site . Efficiently processed substrates apparently pass through this loop, and constraints on the loop conformation probably function to control UCH specificity. Biochem Biophys Res Commun, 1999 Jul 22, 261(1), 156 - 62 Cloning and functional characterization of CYP94A2, a medium chain fatty acid hydroxylase from Vicia sativa; Le Bouquin R et al.; A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A2) was isolated from a Vicia sativa library . CYP94A2 displays 58% sequence identity with CYP94A1, a fatty acid omega-hydroxylase isolated from the same material . Heterologous expression of CYP94A2 in Saccharomyces cerevisiae yeast strain WAT11 shows that it catalyses the hydroxylation of myristic (C14) acid with a K(m(app)) of 4.0 microM and a turnover rate number of 80 min(-1) . In addition, lauric (C12) and palmitic (C16) acids were hydroxylated at a ten-fold lower rate, while C18 fatty acids were not oxidized . Remarkably, the regiospecificity of hydroxylation is different for the C12, C14, and C16 fatty acids and appears to be correlated with the length of the carbon chain . Northern blot analysis showed a low level of constitutive expression of CYP94A2 in V . sativa seedlings . In contrast to CYP94A1, transcript accumulation of CYP94A2 was only weakly enhanced in seedlings treated with clofibrate or methyl jasmonate, indicating that both substrate range and gene regulation of the two fatty acid hydroxylases are different . Nat Struct Biol, 1999 Jul, 6(7), 652 - 6 Rational design of a GCN4-derived mimetic of interleukin-4; Domingues H et al.; In this work we describe the rational design of two helix coiled coil peptide mimetics of interleukin-4 (IL-4) which are able to recognize and bind its high affinity receptor (IL-4R alpha) . We have used the leucine-zipper domain of the yeast transcription factor GCN4 as a scaffold into which the putative binding epitope of IL-4 for IL-4R alpha was transferred in a stepwise manner, using computer-aided molecular modeling . The resulting molecules bind IL-4R alpha with affinities ranging from 2 mM to 5 microM, depending on the fraction of the IL-4 binding site incorporated and on their stability . To our knowledge this is the first time a molecule capable of binding a cytokine receptor has been successfully designed in a rational manner. Nat Biotechnol, 1999 Jul, 17(7), 676 - 82 Direct analysis of protein complexes using mass spectrometry; Link AJ et al.; We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides . The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions . The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit . By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively. FEBS Lett, 1999 Jun 25, 453(3), 341 - 5 Plant cell growth and differentiation may involve GAP regulation of Rac activity; Borg S et al.; Two Rac GTPase cDNAs, LjRac1 and LjRac2, were identified in the legume Lotus japonicus . Two-hybrid screening with dominant-constitutive mutations in the two Rac GTPases target three plant cDNAs, LjRacGAP1, LjRacGAP2 and LjRacGAP3, that encode putative GTPase activating proteins of Rho-GTPase subfamily members . Employing Rac antiserum, purified recombinant LjRac GTPases and recombinant LjRacGAP1, for ligand overlay assays, in vitro GAP affinity assays and GTPase activation, we confirmed that eukaryote Rac/RacGAP interplay is conserved in plants . In this investigation we have developed some tools that can be used to characterize the role of enhanced LjRac2 expression in developing root nodules. FEBS Lett, 1999 Jun 25, 453(3), 299 - 304 Analysis of the in vivo interaction between a basic repressor and an acidic activator; Wellhausen A et al.; The artificial basic repressor SSB24 represses transcription of a reporter construct activated by GCN4 . We show that the positively charged SSB24 and the negatively charged acidic activator GCN4 interact in vitro and in vivo . However, deleting the interaction domain from the GCN4 activator does not result in loss of repression by SSB24 . Similarly, transcription activated by the holoenzyme component SRB2 is repressed, although SSB24 and SRB2 do not interact . Repression by SSB24 therefore does not depend on the observed protein-protein interaction between SSB24 and GCN4. FEBS Lett, 1999 Jun 25, 453(3), 273 - 7 The RNA polymerase II core subunit 11 interacts with keratin 19, a component of the intermediate filament proteins; Bruno T et al.; We have previously cloned the human RNA polymerase II subunit 11, as a doxorubicin sensitive gene product . We suggested multiple tasks for this subunit, including structural and regulatory roles . With the aim to clarify the human RNA polymerase II subunit 11 function, we have identified its interacting protein partners using the yeast two-hybrid system . Here, we show that human RNA polymerase II subunit 11 specifically binds keratin 19, a component of the intermediate filament protein family, which is expressed in a tissue and differentiation-specific manner . In particular, keratin 19 is a part of the nuclear matrix intermediate filaments . We provide evidence that human RNA polymerase II subunit 11 interacts with keratin 19 via its N-terminal alpha motif, the same motif necessary for its interaction with the human RNA polymerase II core subunit 3 . We found that keratin 19 contains two putative leucine zipper domains sharing peculiar homology with the alpha motif of human RNA polymerase II subunit 3 . Finally, we demonstrate that keratin 19 can compete for binding human RNA polymerase II subunit 11/human RNA polymerase II subunit 3 in vitro, suggesting a possible regulatory role for this molecule in RNA polymerase II assembly/activity. Comput Chem, 1999 Jun 15, 23(3-4), 275 - 82 Zones of low entropy in genomic sequences; Crochemore M et al.; We consider the problem of detecting regions having low entropy in DNA sequences, which is a particular case of searching for dos-DNA zones . The entropy is measured in linear time as the number of distinct segments occurring in the regions . As a consequence, we are able to determine regions containing a small number of repetitions of long segments or a large number of repetitions of short segments . The method provides an index on sequences that is applied to compare them without any alignment . Comparisons extract regions having similar combinatorial features that would not have been found by standard alignment methods . The present methodology is applied to Saccharomyces cerevisiae yeast chromosomes to show what the approach is able to produce. Structure Fold Des, 1999 Jun 15, 7(6), 605 - 17 Crystal structure of the Atx1 metallochaperone protein at 1.02 A resolution; Rosenzweig AC et al.; BACKGROUND: Metallochaperone proteins function in the trafficking and delivery of essential, yet potentially toxic, metal ions to distinct locations and particular proteins in eukaryotic cells . The Atx1 protein shuttles copper to the transport ATPase Ccc2 in yeast cells . Molecular mechanisms for copper delivery by Atx1 and similar human chaperones have been proposed, but detailed structural characterization is necessary to elucidate how Atx1 binds metal ions and how it might interact with Ccc2 to facilitate metal ion transfer . RESULTS: The 1.02 A resolution X-ray structure of the Hg(II) form of Atx1 (HgAtx1) reveals the overall secondary structure, the location of the metal-binding site, the detailed coordination geometry for Hg(II), and specific amino acid residues that may be important in interactions with Ccc2 . Metal ion transfer experiments establish that HgAtx1 is a functional model for the Cu(I) form of Atx1 (CuAtx1) . The metal-binding loop is flexible, changing conformation to form a disulfide bond in the oxidized apo form, the structure of which has been solved to 1.20 A resolution . CONCLUSIONS: The Atx1 structure represents the first structure of a metallochaperone protein, and is one of the largest unknown structures solved by direct methods . The structural features of the metal-binding site support the proposed Atx1 mechanism in which facile metal ion transfer occurs between metal-binding sites of the diffusible copper-donor and membrane-tethered copper-acceptor proteins . The Atx1 structural motif represents a prototypical metal ion trafficking unit that is likely to be employed in a variety of organisms for different metal ions. Biochem Biophys Res Commun, 1999 Jul 14, 260(3), 658 - 64 Casein kinase 2 binds to and phosphorylates BRCA1; O'Brien KA et al.; The BRCA1 gene encodes a complex protein that appears to be involved in some aspects of DNA repair, transcription, or cell cycle regulation . The phosphorylation of BRCA1 is enhanced following episodes of DNA damage or during cell cycle progression, indicating that phosphorylation may be an important regulatory mechanism . Through a yeast two hybrid assay, we found that the beta-subunit of casein kinase 2 (CK2) associated with a carboxy-terminal region of BRCA1 . This association was much weaker with the same fragment bearing a missense mutation (M1775R) that has been identified in breast tumors . The interaction was also evident in Sf9 cells . Subsequent studies showed that BRCA1 was phosphorylated in vitro by CK2 . An analysis by site directed mutagenesis of BRCA1 showed that in vitro phosphorylation by CK2 required a serine at aa1572 . These data implicate CK2 as a potential mediator of BRCA1 activity . Nature, 1999 Jul 1, 400(6739), 86 - 9 Solution structure of the catalytic domain of GCN5 histone acetyltransferase bound to coenzyme A; Lin Y et al.; Gene transcription requires the release of inactive DNA from its packaging of histone proteins . Following the discovery of the first transcription-associated histone acetyltransferase, tetrahymena GCN5, it was shown that yeast GCN5 is recruited to the promoter and causes hyper-acetylation of histones and transcriptional activation of target genes, establishing a direct connection between histone acetylation and transcriptional activation . Many other important transcription regulators have been found to have histone acetyltransferase activity, including TAFII230/250, p300/CBP and its associated factor PCAF . Here we present the solution structure of the catalytic domain of tGCN5 (residues 47-210) in complex with coenzyme A . The structure contains two domains; the amino-terminal domain is similar to those of other GCN5-related N-acetyltransferases but the carboxy-terminal domain is not . Coenzyme A binds in a deep hydrophobic pocket between the two domains . Chemical shift changes upon titration with histone H3 peptides indicate a binding site at the domain boundary opposite to the coenzyme A site . The structural data indicate a single-step acetyl-transfer reaction mechanism catalysed by a hydrogen bond to the backbone amide group of leucine 126 and the side-chain carboxyl group of a conserved acidic residue. Nature, 1999 Jul 1, 400(6739), 37 - 42 Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1; Uhlmann F et al.; Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin . Attachment of sister kinetochores to the mitotic spindle during mitosis generates forces that would immediately split sister chromatids were it not opposed by cohesion . Cohesion is essential for the alignment of chromosomes in metaphase but must be abolished for sister separation to start during anaphase . In the budding yeast Saccharomyces cerevisiae, loss of sister-chromatid cohesion depends on a separating protein (separin) called Esp1 and is accompanied by dissociation from the chromosomes of the cohesion subunit Scc1 . Here we show that Esp1 causes the dissociation of Scc1 from chromosomes by stimulating its cleavage by proteolysis . A mutant Scc1 is described that is resistant to Esp1-dependent cleavage and which blocks both sister-chromatid separation and the dissociation of Scc1 from chromosomes . The evolutionary conservation of separins indicates that the proteolytic cleavage of cohesion proteins might be a general mechanism for triggering anaphase. FEBS Lett, 1999 Jun 18, 453(1-2), 77 - 80 Ca2(+)-dependent interaction of N-copine, a member of the two C2 domain protein family, with OS-9, the product of a gene frequently amplified in osteosarcoma; Nakayama T et al.; N-copine is a novel two C2 domain protein that shows Ca2(+)-dependent phospholipid binding and membrane association . By using yeast two-hybrid assays, we identified OS-9 as a protein capable of interacting with N-copine . We further revealed that the second C2 domain of N-copine bound with the carboxy-terminal region of OS-9 . Their interaction in vivo was also confirmed by co-immunoprecipitation from 293E cells co-expressing transfected N-copine and OS-9 . In vitro binding assays showed that this interaction was Ca2(+)-dependent . By Northern blot analysis, N-copine and OS-9 were co-expressed in the same regions of human brain . These results reveal that OS-9 is a potential target of N-copine. Cell Biochem Biophys, 1999, 30(3), 369 - 87 Protein tyrosine kinase-mediated pathways in G protein-coupled receptor signaling; Dikic I et al.; Abundant evidence has indicated that protein tyrosine kinases (PTKs) convey signals from G protein-coupled receptors (GPCRs) to regulate cell proliferation, migration, adhesion, and potentially cellular transformation . Molecular mechanisms by which PTKs regulate such diverse effects in GPCR signaling are not well understood . Recently, an unifying theme has emerged where both growth factors and GPCRs utilize protein tyrosine kinase activity and the highly conserved Ras/MAP kinase pathway to control mitogenic signals . Additionally, PTKs are also involved in the regulation of signal transmission from GPCRs to activation of the JNK/SAPK kinase pathway . Furthermore novel insights in chemokine receptor-activated PTKs and their role in mediating cell functions are discussed in this review. Biologicals, 1998 Dec, 26(4), 331 - 46 Recombinant human albumin as a stabilizer for biological materials and for the preparation of international reference reagents; Tarelli E et al.; Recombinant human albumin expressed in Saccharomyces cerevisiae was compared with native human serum albumin in its physicochemical properties and in its use as a stabilizer in lyophilized preparations of thyroid-stimulating hormone (TSH), interleukin 15 (IL-15) and granulocyte colony-stimulating factor (G-CSF) . Advantages of recombinant albumin include its lack of potential human contaminants and infectious agents . When used at concentrations of 0.1-0.2% (w/v), recombinant albumin was equivalent to native serum albumin in its capacity to protect immunological, biological and biochemical properties of TSH, IL-15 and G-CSF . Physicochemical characteristics of the two forms of albumin including their binding to fatty acids were also similar . The recombinant form of albumin used in this study should be considered as a suitable stabilizer in the preparation of lyophilized products and reference reagents. J Cell Biol, 1999 Jul 12, 146(1), 165 - 80 Drosophila roadblock and Chlamydomonas LC7: a conserved family of dynein-associated proteins involved in axonal transport, flagellar motility, and mitosis; Bowman AB et al.; Eukaryotic organisms utilize microtubule-dependent motors of the kinesin and dynein superfamilies to generate intracellular movement . To identify new genes involved in the regulation of axonal transport in Drosophila melanogaster, we undertook a screen based upon the sluggish larval phenotype of known motor mutants . One of the mutants identified in this screen, roadblock (robl), exhibits diverse defects in intracellular transport including axonal transport and mitosis . These defects include intra-axonal accumulations of cargoes, severe axonal degeneration, and aberrant chromosome segregation . The gene identified by robl encodes a 97-amino acid polypeptide that is 57% identical (70% similar) to the 105-amino acid Chlamydomonas outer arm dynein-associated protein LC7, also reported here . Both robl and LC7 have homology to several other genes from fruit fly, nematode, and mammals, but not Saccharomyces cerevisiae . Furthermore, we demonstrate that members of this family of proteins are associated with both flagellar outer arm dynein and Drosophila and rat brain cytoplasmic dynein . We propose that roadblock/LC7 family members may modulate specific dynein functions. Cell Motil Cytoskeleton, 1999, 43(3), 186 - 98 MEKK1 interacts with alpha-actinin and localizes to stress fibers and focal adhesions; Christerson LB et al.; Mitogen-activated protein (MAP) kinases orchestrate the effects of many extracellular stimuli on cells . The serine/threonine protein kinase MEKK1 is an upstream activator of the MAP kinases c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), extracellular signal-regulated kinase (ERK), and p38 as well as NF-kappa B . In a yeast two-hybrid interaction screen to identify proteins that bind to an N-terminal fragment of MEKK1 (amino acids 1-719), the actin-crosslinking protein alpha-actinin was identified as a MEKK1-binding protein . Over-expressed MEKK1 co-immunoprecipitated with alpha-actinin in cell lysates . Both endogenous and over-expressed MEKK1 colocalized with alpha-actinin along actin stress fibers and at focal adhesions . Residues 221-559 of MEKK1 bound to purified alpha-actinin in vitro, indicating that the interaction is direct, and this fragment localized to actin filaments in cells . MEKK1 kinase activity was not required for association with actin filaments, because a catalytically inactive mutant of MEKK1 (MEKK1 D1369A) localized to stress fibers . These results provide strong evidence for the interaction between MEKK1 and alpha-actinin . Thus, restriction of the kinase to the actin cytoskeleton may serve to regulate its specificity towards downstream targets. Plant Cell, 1999 Jul, 11(7), 1293 - 306 Environmental signals controlling sexual development of the corn Smut fungus Ustilago maydis through the transcriptional regulator Prf1; Hartmann HA et al.; Environmental signals induce and coordinate discrete morphological transitions during sexual development of Ustilago maydis . In this fungus, mating of two compatible haploid sporidia is a prerequisite for plant infection . Cell fusion is governed by the action of pheromones and receptors, whereas the subsequent pathogenicity program is controlled by the combinatorial interaction of homeodomain proteins . The U . maydis pheromone response factor (Prf1) is a central regulator of both processes . We have analyzed the regulation of the prf1 gene and demonstrate that pheromone and cAMP signaling regulate prf1 post-transcriptionally . Transcriptional activation of prf1 was observed in the presence of carbon sources, such as glucose and fructose, allowing us to define the cis-acting element in the prf1 promoter that mediates these effects . The same element provides for negative control of prf1 gene transcription at high cAMP levels . A protein that specifically binds to this element was purified and analyzed for its role in prf1 gene regulation . On the basis of these results, we present a model in which prf1 integrates different environmental signals to control development in U . maydis. Hum Mol Genet, 1999 Aug, 8(8), 1509 - 16 Enoyl-CoA hydratase deficiency: identification of a new type of D-bifunctional protein deficiency; van Grunsven EG et al.; D-bifunctional protein is involved in the peroxisomal beta-oxidation of very long chain fatty acids, branched chain fatty acids and bile acid intermediates . In line with the central role of D-bifunctional protein in the beta-oxidation of these three types of fatty acids, all patients with D-bifunctional protein deficiency so far reported in the literature show elevated levels of very long chain fatty acids, branched chain fatty acids and bile acid inter-mediates . In contrast, we now report two novel patients with D-bifunctional protein deficiency who both have normal levels of bile acid intermediates . Complementation analysis and D-bifunctional protein activity measurements revealed that both patients had an isolated defect in the enoyl-CoA hydratase domain of D-bifunctional protein . Subsequent mutation analysis showed that both patients are homozygous for a missense mutation (N457Y), which is located in the enoyl-CoA hydratase coding part of the D-bifunctional protein gene . Expression of the mutant protein in the yeast Saccharomyces cerevisiae confirmed that the N457Y mutation is the disease-causing mutation . Immunoblot analysis of patient fibroblast homogenates showed that the protein levels of full-length D-bifunctional protein were strongly reduced while the enoyl-CoA hydratase component produced after processing within the peroxisome was undetectable, which indicates that the mutation leads to an unstable protein. Hum Mol Genet, 1999 Aug, 8(8), 1397 - 407 MID2, a homologue of the Opitz syndrome gene MID1: similarities in subcellular localization and differences in expression during development; Buchner G et al.; The B-box family is an expanding new family of genes encoding proteins involved in diverse cellular functions such as developmental patterning and oncogenesis . A member of this protein family, MID1, is the gene responsible for the X-linked form of Opitz G/BBB syndrome, a developmental disorder characterized by defects of the midline structures . We now report the identification of MID2, a new transcript closely related to MID1 . MID2 maps to Xq22 in human and to the syntenic region on the mouse X chromosome . The two X-linked genes share the same domains, the same exon-intron organization, a high degree of similarity at the protein level and the same subcellular localization, both being confined to the cytoplasm in association to micro-tubular structures . The expression pattern studied by RNA in situ hybridization in mouse revealed that Mid2 is expressed early in development and the highest level of expression is detected in the heart, unlike Mid1 for which no expression was detected in the developing heart . Together, these data suggest that midin and MID2 have a similar biochemical function but a different physiological role during development. J Virol, 1999 Aug, 73(8), 6680 - 90 Patterns of chemokine receptor fusion cofactor utilization by human immunodeficiency virus type 1 variants from the lungs and blood; Singh A et al.; Human immunodeficiency virus type 1 (HIV-1) infection is highly compartmentalized, with distinct viral genotypes being found in the lungs, brain, and other organs compared with blood . CCR5 and CXCR4 are the principal HIV-1 coreceptors, and a number of other molecules support entry in vitro but their roles in vivo are uncertain . To address the relationship between tissue compartmentalization and the selective use of entry coreceptors, we generated functional env clones from primary isolates derived from the lungs and blood of three infected individuals and analyzed their use of the principal, secondary, orphan, and virus-encoded coreceptors for fusion . All Env proteins from lung viruses used CCR5 but not CXCR4, while those from blood viruses used CCR5 or CXCR4 or both . The orphan receptor APJ was widely used for fusion by Env proteins from both blood and lung viruses, but none used the cytomegalovirus-encoded receptor US28 . Fusion mediated by the secondary coreceptors CCR2b, CCR3, CCR8, and CX3CR1 and orphan receptors GPR1, GPR15, and STRL33 was variable and heterogeneous, with relatively broad utilization by env clones from isolates of one subject but limited use by env clones from the other two subjects . However, there was no clear distinction between blood and lung viruses in secondary or orphan coreceptor fusion patterns . In contrast to fusion, none of the secondary or orphan receptors enabled efficient productive infection . These results confirm, at the level of cofactor utilization, previous observations that HIV-1 populations in the lungs and blood are biologically distinct and demonstrate diversity within lung-derived as well as blood-derived quasispecies . However, the heterogeneity in coreceptor utilization among clones from each isolate and the lack of clear distinction between lung- and blood-derived Env proteins argue against selective coreceptor utilization as a major determinant of compartmentalization. J Virol, 1999 Aug, 73(8), 6551 - 8 The Epstein-Barr virus BZLF1 protein interacts physically and functionally with the histone acetylase CREB-binding protein; Adamson AL et al.; The Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) is a key regulator of the EBV latent-to-lytic switch . Z is a transcriptional activator which induces EBV early gene expression . We demonstrate here that Z interacts with CREB-binding protein (CBP), a histone acetylase and transcriptional coactivator . This interaction requires the amino-terminal region of CBP as well as the transactivation and leucine zipper domains of Z . We show that CBP enhances Z-mediated transactivation of EBV early promoters, in reporter gene assays and in the context of the endogenous genome . We also demonstrate that Z decreases CREB transactivation function and that this inhibitory effect is reversed by overexpression of CBP . We show that Z also interacts directly with CREB . However, mutational analysis indicates that Z inhibition of CREB activity requires the direct interaction between Z and CBP but not the direct interaction between Z and CREB . We propose that Z interacts with CBP to enhance viral early gene transcription . In addition, the Z-CBP interaction may control host cellular transcription factor activity through competition for limiting amounts of cellular CBP. J Biol Chem, 1999 Jul 16, 274(29), 20569 - 77 Involvement of PITPnm, a mammalian homologue of Drosophila rdgB, in phosphoinositide synthesis on Golgi membranes; Aikawa Y et al.; Phosphatidylinositol transfer protein (PITP) is involved in phospholipase C-mediated signaling and membrane trafficking . We previously reported cloning and characterization of a gene encoding for membrane-bound PITP, named PITPnm, that is a mammalian homologue of the Drosophila retinal degeneration B (rdgB) gene (Aikawa, Y., Hara, H., and Watanabe, T . (1997) Biochem . Biophys . Res . Commun . 236, 559-564) . Here we report the subcellular localization of PITPnm protein and provide evidence for its involvement in phosphatidylinositol 4-phosphate (PtdIns 4-P) synthesis . PITPnm is an integral membrane protein that largely localized in close association with membranes of Golgi vacuoles and the endoplasmic reticulum (ER) . The amino terminus region of PITPnm was exposed to cytoplasmic side . Interaction with various phosphoinositides was observed in the amino terminus region spanning from 196 amino acids to 257 amino acids of PITPnm . At the amino terminus regions of 1-372 amino acids, PITPnm formed a complex with type III PtdIns 4-kinase . The transmembrane and carboxyl-terminal portions (residues 418-1242) functioned to retain the PITPnm in the Golgi vacuole . These results suggest that PITPnm plays a role in phosphoinositide synthesis on the Golgi vacuoles and possibly in the PtdIns signaling pathway in mammalian cells. J Biol Chem, 1999 Jul 16, 274(29), 20521 - 8 Poly(ADP-ribose) polymerase and Ku autoantigen form a complex and synergistically bind to matrix attachment sequences; Galande S et al.; Genomic sequences with a cluster of ATC sequence stretches where one strand consists exclusively of well mixed As, Ts, and Cs confer high base unpairing propensity under negative superhelical strain . Such base unpairing regions (BURs) are typically found in scaffold or matrix attachment regions (SARs/MARs) that are thought to contribute to the formation of the loop domain structure of chromatin . Several proteins, including cell type-specific proteins, have been identified that bind specifically to double-stranded BURs either in vitro or in vivo . By using BUR-affinity chromatography to isolate BUR-binding proteins from breast cancer SK-BR-3 cells, we almost exclusively obtained a complex of poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) . Both PARP and DNA-PK are activated by DNA strand breaks and are implicated in DNA repair, recombination, DNA replication, and transcription . In contrast to the previous notion that PARP and Ku autoantigen, the DNA-binding subunit of DNA-PK, mainly bind to free ends of DNA, here we show that both proteins individually bind BURs with high affinity and specificity in an end-independent manner using closed circular BUR-containing DNA substrates . We further demonstrate that PARP and Ku autoantigen form a molecular complex in vivo and in vitro in the absence of DNA, and as a functional consequence, their affinity to the BURs are synergistically enhanced . ADP-ribosylation of the nuclear extract abrogated the BUR binding activity of this complex . These results provide a mechanistic link toward understanding the functional overlap of PARP and DNA-PK and suggest a novel role for these proteins in the regulation of chromatin structure and function. J Biol Chem, 1999 Jul 16, 274(29), 20336 - 43 Kinetic analysis of human serine/threonine protein phosphatase 2Calpha; Fjeld CC et al.; The PPM family of Ser/Thr protein phosphatases have recently been shown to down-regulate the stress response pathways in eukaryotes . Within the stress pathway, key signaling kinases, which are activated by protein phosphorylation, have been proposed as the in vivo substrates of PP2C, the prototypical member of the PPM family . Although it is known that these phosphatases require metal cations for activity, the molecular details of these important reactions have not been established . Therefore, here we report a detailed biochemical study to elucidate the kinetic and chemical mechanism of PP2Calpha . Steady-state kinetic and product inhibition studies revealed that PP2Calpha employs an ordered sequential mechanism, where the metal cations bind before phosphorylated substrate, and phosphate is the last product to be released . The metal-dependent activity of PP2C (as reflected in kcat and kcat/Km), indicated that Fe2+ was 1000-fold better than Mg2+ . The pH rate profiles revealed two ionizations critical for catalytic activity . An enzyme ionization with a pKa value of 7 must be unprotonated for catalysis, and an enzyme ionization with a pKa of 9 must be protonated for substrate binding . Bronsted analysis of substrate leaving group pKa indicated that phosphomonoester hydrolysis is rate-limiting at pH 7 . 0, but not at pH 8.5 where a common step independent of the nature of the substrate and alcohol product limits turnover (kcat) . Rapid reaction kinetics between phosphomonoester and PP2C yielded exponential "bursts" of product formation, consistent with phosphate release being the slow catalytic step at pH 8.5 . Dephosphorylation of synthetic phosphopeptides corresponding to several protein kinases revealed that PP2C displays a strong preference for diphosphorylated peptides in which the phosphorylated residues are in close proximity. J Biol Chem, 1999 Jul 16, 274(29), 20235 - 43 A small region in HMG I(Y) is critical for cooperation with NF-kappaB on DNA; Zhang XM et al.; The high mobility group HMG I(Y) protein has been reported to promote the expression of several NF-kappaB-dependent genes by enhancing the binding of NF-kappaB to DNA . The molecular origins of cooperativity in the binding of NF-kappaB and HMG I(Y) to DNA are not well understood . Here we have examined the determinants of specificity in the binding of HMG I(Y), both alone and in cooperation with NF-kappaB, to two different DNA elements, PRDII from the interferon-beta enhancer and IgkappaB from the immunoglobulin kappa light chain enhancer . Of particular interest was the influence of a flanking AT-rich sequence on binding by HMG I(Y) . Utilizing yeast one-hybrid screening assays together with alanine-scanning mutagenesis, we have identified mutations of residues in HMG I(Y) that decrease cooperative binding of NF-kappaB to PRDII and IgkappaB sites . These same mutations similarly decreased the binding of HMG I(Y) alone to DNA, and paradoxically, decreased the strength of protein-protein interactions between HMG I(Y) and NF-kappaB . Of the three tandemly repeated basic regions that represent putative DNA-binding motifs in HMG I(Y), the residues within the second repeat are most important for recognition of core NF-kappaB sites, whereas the second and third repeats both appear to be involved in binding to sites that are flanked by AT-rich sequences . Overall, the second repeat of HMG I(Y) is primarily responsible for the stimulatory effect of this protein on the binding of NF-kappaB to PRDII and IgkappaB elements. J Biol Chem, 1999 Jul 16, 274(29), 20110 - 5 Interaction of BiP with the J-domain of the Sec63p component of the endoplasmic reticulum protein translocation complex; Misselwitz B et al.; Proteins of the Hsp70 family of ATPases interact with a conserved domain of their J-protein partners, the J-domain, to function in numerous cellular processes . We have studied the interaction of BiP, an Hsp70 family member in the lumen of the endoplasmic reticulum, with the J-domain of Sec63p, a component of the Sec complex involved in post-translational protein translocation across the endoplasmic reticulum membrane . In a real-time solid phase binding assay, BiP binds to the immobilized Sec complex or to a fusion protein of the J-domain and glutathione S-transferase in a reaction that requires ATP hydrolysis . In the final complex, BiP is bound in the ADP form with its peptide binding pocket occupied . An intact peptide binding pocket is required for this interaction . Our experiments suggest that the activation of BiP by the J-domain involves a transient contact between these components, and that in the absence of physiological substrates, J-activated BiP binds even to the J-proteins themselves. Biol Cell, 1999 Mar, 91(2), 99 - 108 Drosophila centrosomes are unable to trigger parthenogenetic development of Xenopus eggs; Tournier F et al.; Centrosomes are powerful and exclusive parthenogenetic agents in the Xenopus egg . We have previously shown that heterologous centrosomes from various vertebrate species were able to promote egg cleavage in Xenopus and that human centrosome activity was associated with an insoluble proteinacious structure that is not significantly simpler than the native centrosome . In this work, we have investigated the parthenogenetic capacity of more evolutionary distant centrosomes . We show that centrosomes devoid of centrioles, such as SPBs isolated from Saccharomyces cerevisiae, do not form asters of microtubules in cytoplasmic extracts from Xenopus eggs, and are inactive in the parthenogenetic test . We further show that Drosophila centrosomes which possess a typical centriole architecture, and are quite active to nucleate microtubules in Xenopus cytoplasmic extracts, are unable to trigger egg cleavage . This was observed both with centrosomes isolated from Drosophila syncytial embryos and nucleus-centrosome complexes from the Drosophila Kc23 cell line . We demonstrate that this inability could not be restored after pre-incubation of Drosophila centrosomes in the egg cytoplasm before injection . We conclude that the parthenogenetic activity of a centrosome is not directly linked to its capacity to nucleate microtubules from the egg tubulin, and that the evolutionary conserved nine-fold symmetrical structure of the centriole cannot be considered as sufficient for triggering procentriole assembly. Klin Lab Diagn, 1999 May, (5), 14 - 6 {The optimization of a method for determining RNAse activity by using high-polymer RNA}; Kolpakov AI et al.; Different methods for precipitation of free nucleotides are compared with the aim of improving the method for estimating RNAse activity . The precipitators were 0.75% uranyl acetate in 0.25% HClO4, 96% ethanol with MgCl2 in different concentrations, pure ethanol, and chlorine acid . Measurements of RNAse activity by means of 0.02M MgCl2 in 96% ethanol whose volume is equal to the reaction mixture were the optimal. Genes Dev, 1999 Jul 1, 13(13), 1729 - 41 Metal ion catalysis during group II intron self-splicing: parallels with the spliceosome; Sontheimer EJ et al.; The identical reaction pathway executed by the spliceosome and self-splicing group II intron ribozymes has prompted the idea that both may be derived from a common molecular ancestor . The minimal sequence and structural similarities between group II introns and the spliceosomal small nuclear RNAs, however, have left this proposal in question . Mechanistic comparisons between group II self-splicing introns and the spliceosome are therefore important in determining whether these two splicing machineries may be related . Here we show that 3'-sulfur substitution at the 5' splice site of a group II intron causes a metal specificity switch during the first step of splicing . In contrast, 3'-sulfur substitution has no significant effect on the metal specificity of the second step of cis-splicing . Isolation of the second step uncovers a metal specificity switch that is masked during the cis-splicing reaction . These results demonstrate that group II intron ribozymes are metalloenzymes that use a catalytic metal ion for leaving group stabilization during both steps of self-splicing . Furthermore, because 3'-sulfur substitution of a spliceosomal intron has precisely the same effects as were observed during cis-splicing of the group II intron, these results provide striking parallels between the catalytic mechanisms employed by these two systems. Genes Dev, 1999 Jul 1, 13(13), 1664 - 77 Proper metaphase spindle length is determined by centromere proteins Mis12 and Mis6 required for faithful chromosome segregation; Goshima G et al.; High-fidelity chromosome transmission is fundamental in controlling the quality of the cell division cycle . The spindle pole-to-pole distance remains constant from metaphase to anaphase A . We show that fission yeast sister centromere-connecting proteins, Mis6 and Mis12, are required for correct spindle morphogenesis, determining metaphase spindle length . Thirty-five to sixty percent extension of metaphase spindle length takes place in mis6 and mis12 mutants . This may be due to incorrect spindle morphogenesis containing impaired sister centromeres or force unbalance between pulling by the linked sister kinetochores and kinetochore-independent pushing . The mutant spindle fully extends in anaphase, although it is accompanied by drastic missegregation by aberrant sister centromere separation . Hence, metaphase spindle length may be crucial for segregation fidelity . Suppressors of mis12 partly restore normal metaphase spindle length . In mis4 that is defective in sister chromatid cohesion, metaphase spindle length is also long, but anaphase spindle extension is blocked, probably due to the activated spindle checkpoint . Extensive missegregation is caused in mis12 only when Mis12 is inactivated from the previous M through to the following M, an effective way to avoid missegregation in the cell cycle . Mis12 has conserved homologs in budding yeast and filamentous fungi. Genes Dev, 1999 Jul 1, 13(13), 1653 - 63 Heat-shock-induced activation of stress MAP kinase is regulated by threonine- and tyrosine-specific phosphatases; Nguyen AN et al.; In eukaryotic species from yeast to human, stress-activated protein kinases (SAPKs), members of a MAP kinase (MAPK) subfamily, regulate the transcriptional response to various environmental stress . It is poorly understood how diverse forms of stress are sensed and transmitted to SAPKs . Here, we report the heat shock regulation of the fission yeast Spc1 SAPK, a homolog of human p38 and budding yeast Hog1p . Although osmostress and oxidative stress induce strong activation of the Wis1 MAPK kinase (MEK), which activates Spc1 through Thr-171/Tyr-173 phosphorylation, activation of Wis1 upon heat shock is relatively weak and transient . However, in heat-shocked cells, Pyp1, the major tyrosine phosphatase that dephosphorylates and inactivates Spc1, is inhibited for its interaction with Spc1, which leads to strong activation of Spc1 . Subsequently, Spc1 activity is rapidly attenuated by Thr-171 dephosphorylation, whereas Tyr-173 remains phosphorylated . Thr-171 dephosphorylation is compromised in a strain lacking functional type 2C serine/threonine phosphatases (PP2C), Ptc1 and Ptc3 . Moreover, Ptc1 and Ptc3 can dephosphorylate Thr-171 of Spc1 both in vivo and in vitro . These observations strongly suggest that PP2C enzymes play an important role in the attenuation of Spc1 activity in heat-shocked cells . Thus, transient activation of Spc1 upon heat shock is ensured by differential regulation of threonine and tyrosine phosphorylation. Science, 1999 Jul 9, 285(5425), 263 - 5 hRAD30 mutations in the variant form of xeroderma pigmentosum; Johnson RE et al.; Xeroderma pigmentosum (XP) is an autosomal recessive disease characterized by a high incidence of skin cancers . Yeast RAD30 encodes a DNA polymerase involved in the error-free bypass of ultraviolet (UV) damage . Here it is shown that XP variant (XP-V) cell lines harbor nonsense or frameshift mutations in hRAD30, the human counterpart of yeast RAD30 . Of the eight mutations identified, seven would result in a severely truncated hRad30 protein . These results indicate that defects in hRAD30 cause XP-V, and they suggest that error-free replication of UV lesions by hRad30 plays an important role in minimizing the incidence of sunlight-induced skin cancers. Science, 1999 Jul 9, 285(5425), 254 - 7 A functional assay for centromere-associated sister chromatid cohesion; Megee PC et al.; Cohesion of sister chromatids occurs along the entire length of chromosomes, including the centromere where it plays essential roles in chromosome segregation . Here, minichromosomes in the budding yeast Saccharomyces cerevisiae are exploited to generate a functional assay for DNA sequences involved in cohesion . The centromeric DNA element CDEIII was found to be necessary but not sufficient for cohesion . This element was shown previously to be required for assembly of the kinetochore, the centromere-associated protein complex that attaches chromosomes to the spindle . These observations establish a link between centromere-proximal cohesion and kinetochore assembly. Science, 1999 Jul 9, 285(5425), 251 - 4 Ploidy regulation of gene expression; Galitski T et al.; Microarray-based gene expression analysis identified genes showing ploidy-dependent expression in isogenic Saccharomyces cerevisiae strains that varied in ploidy from haploid to tetraploid . These genes were induced or repressed in proportion to the number of chromosome sets, regardless of the mating type . Ploidy-dependent repression of some G1 cyclins can explain the greater cell size associated with higher ploidies, and suggests ploidy-dependent modifications of cell cycle progression . Moreover, ploidy regulation of the FLO11 gene had direct consequences for yeast development. Science, 1999 Jul 9, 285(5425), 242 - 4 A nucleoside transporter from Trypanosoma brucei involved in drug resistance; Maser P et al.; Drug resistance of pathogens is an increasing problem whose underlying mechanisms are not fully understood . Cellular uptake of the major drugs against Trypanosoma brucei spp., the causative agents of sleeping sickness, is thought to occur through an unusual, so far unidentified adenosine transporter . Saccharomyces cerevisiae was used in a functional screen to clone a gene (TbAT1) from Trypanosoma brucei brucei that encodes a nucleoside transporter . When expressed in yeast, TbAT1 enabled adenosine uptake and conferred susceptibility to melaminophenyl arsenicals . Drug-resistant trypanosomes harbor a defective TbAT1 variant . The molecular identification of the entry route of trypanocides opens the way to approaches for diagnosis and treatment of drug-resistant sleeping sickness. Biotechnol Bioeng, 1999 Jun 20, 63(6), 684 - 93 Near-infrared spectroscopy for bioprocess monitoring and control; Yeung KS et al.; This article describes the calibration of a spectroscopic scanning instrument for the measurement of selected contaminants in a complex biological process stream . Its use is for the monitoring of a process in which contaminants are to be removed selectively by flocculation from yeast cell homogenate . The main contaminants are cell debris, protein, and RNA . A low-cost instrument has been developed for sensitivity in the region of the NIR spectrum (from 1900 to 2500 nm) where preliminary work found NIR signatures from cell debris, protein, and RNA . Calibration models have been derived using a multivariate method for concentrations of these contaminants, such as would be found after the flocculation process . Two strategies were compared for calibrating the NIR instrument . In one case, samples were prepared by adding materials representative of the contaminants to clarified yeast homogenate so the contaminant levels were well known but outside the range of interest . In the other case, where samples were like those from the process stream after flocculation and floc removal, there was uncertainty of analysis of contaminant level, but the calibration was in the range of interest . Calibration using process stream samples gave results close to those derived from traditional assays . When the calibration models were used to predict the contaminant concentrations in previously unseen samples, the correlation coefficients between measurements and predictions were above 90% in all cases but one . The prediction errors were similar to the errors in the traditional assays . Mol Biol Cell, 1999 Jul, 10(7), 2461 - 74 Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway; Kurz M et al.; Two major routes of preprotein targeting into mitochondria are known . Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23-Tim17 complex . Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22-Tim54 of the inner membrane . Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway . We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route . In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23-Tim17 pathway . The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70 . In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial . We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP. Biochim Biophys Acta, 1999 Jul 9, 1439(1), 89 - 94 Identification and characterization of the mouse cDNA encoding acyl-CoA:dihydroxyacetone phosphate acyltransferase; Ofman R et al.; We used the amino acid sequence of human acyl-CoA:dihydroxyacetone phosphate acyltransferase (DHAPAT) as bait to screen the database of expressed sequence tags (dbEST) and identified several partial mouse cDNA clones showing high identity . Primers were selected based on the dbEST sequences and used for amplification of this transcript from cDNA prepared from mouse skin fibroblasts . The complete nucleotide sequence was then determined and revealed an open reading frame (ORF) of 2034 bp encoding a protein consisting of 678 amino acids with a calculated molecular mass of 76870 . The deduced amino acid sequence showed high identity (80%) with that of human DHAPAT and also revealed a typical peroxisomal targeting signal type 1 (PTS1) at its extreme carboxy-terminus (alanine-lysine-leucine, AKL) . Definitive evidence that this cDNA indeed codes for DHAPAT was obtained by heterologous expression in the yeast Saccharomyces cerevisiae . Northern blot analysis revealed high expression of DHAPAT especially in mouse heart, liver and testis. Biochim Biophys Acta, 1999 Jul 7, 1446(1-2), 149 - 55 Molecular cloning and expression of the Na+/H+ exchanger gene in Oryza sativa; Fukuda A et al.; Na+/H+ exchanger catalyzes the countertransport of Na+ and H+ across membranes . We isolated a rice cDNA clone the deduced amino acid sequence of which had homology with a putative Na+/H+ exchanger in Saccharomyces cerevisiae, NHX1 . The sequence contains 2330 bp with an open reading frame of 1608 bp . The deduced amino acid sequence is similar to that of NHX1 and NHE isoforms in mammals, and shares high similarity with the sequences within predicted transmembrane segments and an amiloride-binding domain . The expression of the gene was increased by salt stress . These results suggest that the product of the novel gene, OsNHX1, functions as a Na+/H+ exchanger, and plays important roles in salt tolerance of rice. Gene, 1999 Jul 8, 234(2), 403 - 9 Characterization of mouse Trip6: a putative intracellular signaling protein; Wang Y et al.; Trip6 is a human LIM domain-containing protein that has been identified in yeast two-hybrid screens as interacting with a variety of proteins . Trip6 has been proposed to transport signals from the cell surface to the nucleus . In this report, we have characterized a mouse cDNA encoding Trip6 . Mouse Trip6 is highly similar to human Trip6, especially in the C-terminal LIM domain region, and the in vitro and in vivo mouse Trip6 cDNA directs the synthesis of a polypeptide with a relative mobility of approx . 57kDa on SDS-polyacrylomide gels . Full-length Trip6 localizes to discrete cytoplasmic patches when overexpressed in chicken embryo fibroblasts, consistent with localization to focal adhesion plaques . However, deletion of the N-terminal 115 amino acids allows Trip6 to enter the nucleus of CEF . A GAL4 fusion protein containing the LIM domain region of mouse Trip6 can activate transcription in yeast and chicken fibroblasts . Our results indicate that the functional domains and properties of mouse Trip6 are highly conserved between humans and mice, and are consistent with a model in which Trip6 relays signals from the cell surface to the nucleus. Gene, 1999 Jul 8, 234(2), 381 - 94 A variety of RNA polymerases II and III-dependent promoter classes is repressed by factors containing the Krüppel-associated/finger preceding box of zinc finger proteins; Senatore B et al.; KRAB/FPB (Kruppel-associated/finger preceding box) domains are small, portable transcriptional repression motifs, encoded by hundreds of vertebrates C2-H2-type zinc finger genes . We report that KRAB/FPB domains feature an unprecedented, highly promiscuous DNA-binding dependent transcriptional repressing activity . Indeed, template bound chimeric factors containing KRAB/FPB modules actively repress in vivo the transcription of distinct promoter classes that depend on different core elements, recruit distinct basal transcriptional apparatuses and are transcribed either by RNA polymerase II or III . The promoter types repressed in transient assays in a dose- and DNA-binding dependent, but position- and orientation-independent manner, by GAL4-KRAB/FPB fusions include an RNA polymerase II-dependent small nuclear RNA promoter (U1) as well as RNA polymerase III-dependent class 2 (adenovirus VA1), class 3 (human U6) and atypical (human 7SL) promoters . Down-modulation of all of these templates depended on factors containing the A module of the KRAB/FPB domain . Data provide further insights into the properties and mode of action of this widespread repression motif, and support the notion that genes belonging to distinct classes may be repressed in vivo by KRAB/FPB containing zinc finger proteins . The exquisitely DNA-binding dependent transcriptional promiscuity exhibited by KRAB/FPB domains may provide a unique model system for studying the mechanism by which a promoter recruited repression motif can down-modulate a large variety of promoter types. Curr Opin Neurobiol, 1999 Jun, 9(3), 336 - 42 Brain protein serine/threonine phosphatases; Price NE et al.; All of the known protein serine/threonine phosphatases are expressed in the brain . These enzymes participate in a variety of signaling pathways that modulate neuronal activity . The multifunctional activity of many serine/threonine phosphatases is achieved through their association with targeting proteins . Identification and analysis of targeting molecules has led to new insights into the functions of protein phosphatases in neuronal signaling . The recent use of transgenic mice has also increased our understanding of the physiological roles of these enzymes in the brain. Hum Gene Ther, 1999 Jun 10, 10(9), 1499 - 507 Ligand-dependent regulation of plasmid-based transgene expression in vivo; Abruzzese RV et al.; As gene therapy advances, the ability to regulate transgene expression will become paramount for safety and efficacy . In this study, we investigate the ability of the mifepristone-dependent GeneSwitch system to regulate the expression of trangenes delivered to mice by nonviral methods . Two plasmids, one encoding the chimeric GeneSwitch protein, the other an inducible transgene for secreted human placental alkaline phosphatase (SEAP), were delivered to the hind-limb muscles of adult mice . Modulation of the level of secretion of the transgene product into serum was achieved by intraperitoneal administration of low doses of the drug mifepristone (MFP) . The EC50 for induction of transgene expression by MFP was 0.03 +/- 0.005 mg/kg . The maximal level of transgene expression after induction was equal to or higher than that displayed by a plasmid driven by the CMV enhancer/promoter . The average magnitude of induction was 14- to 19-fold . Multiple rounds of drug-dependent regulation of transgene expression in vivo were demonstrated . In BALB/c mice, the ability to regulate transgene expression persisted for approximately 3 weeks, until the appearance of neutralizing antibodies to the secreted transgene product . In immune-deficient mice, the ability to repetitively regulate transgene expression persisted for at least 5 weeks . Although the dynamic range of regulation needs improvement, the plasmid-based GeneSwitch system has features that are attractive for gene therapy applications. Cancer Lett, 1999 May 24, 139(2), 183 - 7 Mutation analysis of hBUB1 in aneuploid HNSCC and lung cancer cell lines; Yamaguchi K et al.; Aneuploidy is frequently observed in many types of human cancer cells, suggesting that mutations of genes required for chromosomal stability may occur in human tumors . The BUB gene is a component of the mitotic checkpoint in budding yeast that delays anaphase in the presence of spindle damage thus increasing the probability of successful delivery of a euploid genome to each daughter cell . Recently, human homologues of the BUB gene were identified and mutant alleles of hBUB1 were detected in two colorectal tumor cell lines . Transfection of one mutant allele led to dominant disruption of the mitotic checkpoint control in a euploid cell, suggesting that aneuploidy in some tumors could be due to defects in the mitotic checkpoint . We analyzed the entire coding sequence of hBUB1 for mutation in 31 head and neck squamous cell carcinoma (HNSCC) and lung cancer cell lines, most with severe aneuploidy . We found expression of the hBUB1 gene in all cell lines and only a single nucleotide substitution in one cell line without a resultant change in amino acid sequence . Our study demonstrates that hBUB1 is rarely a target for genetic alterations in tumors of the respiratory tract. Plant Mol Biol, 1999 May, 40(1), 37 - 44 The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range; Korshunova YO et al.; The molecular basis for the transport of manganese across membranes in plant cells is poorly understood . We have found that IRT1, an Arabidopsis thaliana metal ion transporter, can complement a mutant Saccharomyces cerevisiae strain defective in high-affinity manganese uptake (smf1 delta) . The IRT1 protein has previously been identified as an iron transporter . The current studies demonstrated that IRT1, when expressed in yeast, can transport manganese as well . This manganese uptake activity was inhibited by cadmium, iron(II) and zinc, suggesting that IRT1 can transport these metals . The IRT1 cDNA also complements a zinc uptake-deficient yeast mutant strain (zrt1zrt2), and IRT1-dependent zinc transport in yeast cells is inhibited by cadmium, copper, cobalt and iron(III) . However, IRT1 did not complement a copper uptake-deficient yeast mutant (ctr1), implying that this transporter is not involved in the uptake of copper in plant cells . The expression of IRT1 is enhanced in A . thaliana plants grown under iron deficiency . Under these conditions, there were increased levels of root-associated manganese, zinc and cobalt, suggesting that, in addition to iron, IRT1 mediates uptake of these metals into plant cells . Taken together, these data indicate that the IRT1 protein is a broad-range metal ion transporter in plants. Mol Gen Genet, 1999 Jun, 261(4-5), 871 - 82 Double-strand break repair can lead to high frequencies of deletions within short CAG/CTG trinucleotide repeats; Richard GF et al.; Trinucleotide repeats undergo contractions and expansions in humans, leading in some cases to fatal neurological disorders . The mechanism responsible for these large size variations is unknown, but replication-slippage events are often suggested as a possible source of instability . We constructed a genetic screen that allowed us to detect spontaneous expansions/contractions of a short trinucleotide repeat in yeast . We show that deletion of RAD27, a gene involved in the processing of Okazaki fragments, increases the frequency of contractions tenfold . Repair of a chromosomal double-strand break (DSB) using a trinucleotide repeat-containing template induces rearrangements of the repeat with a frequency 60 times higher than the natural rate of instability of the same repeat . Our data suggest that both gene conversion and single-strand annealing are major sources of trinucleotide repeat rearrangements. Mol Cell, 1999 Jun, 3(6), 717 - 27 A role for TBP dimerization in preventing unregulated gene expression; Jackson-Fisher AJ et al.; The recruitment of the TATA box-binding protein (TBP) to promoters in vivo is often rate limiting in gene expression . We present evidence that TBP negatively autoregulates its accessibility to promoter DNA in yeast through dimerization . The crystal structure of TBP dimers was used to design point mutations in the dimer interface . These mutants are impaired for dimerization in vitro, and in vivo they generate large increases in activator-independent gene expression . Overexpression of wild-type TBP suppresses these mutants, possibly by heterodimerizing with them . In addition to loss of autorepression, dimerization-defective TBPs are rapidly degraded in vivo . Direct detection of TBP dimers in vivo was achieved through chemical cross-linking . Taken together, the data suggest that TBP dimerization prevents unregulated gene expression and its own degradation. Mol Cell, 1999 Jun, 3(6), 687 - 95 The 19S regulatory complex of the proteasome functions independently of proteolysis in nucleotide excision repair; Russell SJ et al.; The 26S proteasome degrades proteins targeted by the ubiquitin pathway, a function thought to explain its role in cellular processes . The proteasome interacts with the ubiquitin-like N terminus of Rad23, a nucleotide excision repair (NER) protein, in Saccharomyces cerevisiae . Deletion of the ubiquitin-like domain causes UV radiation sensitivity . Here, we show that the ubiquitin-like domain of Rad23 is required for optimal activity of an in vitro NER system . Inhibition of proteasomal ATPases diminishes NER activity in vitro and increases UV sensitivity in vivo . Surprisingly, blockage of protein degradation by the proteasome has no effect on the efficiency of NER . This establishes that the regulatory complex of the proteasome has a function independent of protein degradation. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 7968 - 73 p200 ARF-GEP1: a Golgi-localized guanine nucleotide exchange protein whose Sec7 domain is targeted by the drug brefeldin A; Mansour SJ et al.; The drug brefeldin A (BFA) disrupts protein traffic and Golgi morphology by blocking activation of ADP ribosylation factors (ARFs) through an unknown mechanism . Here, we investigated the cellular localization and BFA sensitivity of human p200 ARF-GEP1 (p200), a ubiquitously expressed guanine nucleotide exchange factor of the Sec7 domain family . Multiple tagged forms of the full-length polypeptide localized to tight ribbon-like perinuclear structures that overlapped with the Golgi marker mannosidase II and were distinct from the pattern observed with ERGIC53/58 . Analysis of several truncated forms mapped the Golgi-localization signal to the N-terminal third of p200 . BFA treatment of transiently or stably transfected cells resulted in the redistribution of Golgi markers and in loss of cell viability, thereby indicating that overproduction of p200 may not be sufficient to overcome the toxic effect . A 39-kDa fragment spanning the Sec7 domain catalyzed loading of guanosine 5'-{gamma-thio}triphosphate onto class I ARFs and displayed clear sensitivity to BFA . Kinetic analysis established that BFA did not compete with ARF for interaction with p200 but, rather, acted as an uncompetitive inhibitor that only targeted the p200-ARF complex with an inhibition constant of 7 microM . On the basis of these results, we propose that accumulation of an abortive p200-ARF complex in the presence of BFA likely leads to disruption of Golgi morphology . p200 mapped to chromosome 8q13, 3.56 centirays from WI-6151, and database searches revealed the presence of putative isoforms whose inhibition may account for the effects of BFA on various organelles. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 7791 - 6 Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription; Bieniasz PD et al.; Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR) promoter element by the viral Tat protein is an essential step in the HIV-1 life cycle . Tat function is mediated by the TAR RNA target element encoded within the LTR and is known to require the recruitment of a complex consisting of Tat and the cyclin T1 (CycT1) component of positive transcription elongation factor b (P-TEFb) to TAR . Here, we demonstrate that both TAR and Tat become entirely dispensable for activation of the HIV-1 LTR promoter when CycT1/P-TEFb is artificially recruited to a heterologous promoter proximal RNA target . The level of activation observed was indistinguishable from the level induced by Tat and was neither inhibited nor increased when Tat was expressed in trans . Activation by artificially recruited CycT1 depended on the ability to bind the CDK9 component of P-TEFb . In contrast, although binding to both Tat and TAR was essential for the ability of CycT1 to act as a Tat cofactor, these interactions became dispensable when CycT1 was directly recruited to the LTR . Importantly, activation of the LTR both by Tat and by directly recruited CycT1 was found to be at the level of transcription elongation . Together, these data demonstrate that recruitment of CycT1/P-TEFb to the HIV-1 LTR is fully sufficient to activate this promoter element and imply that the sole role of the Tat/TAR axis in viral transcription is to permit the recruitment of CycT1/P-TEFb. J Cell Sci, 1999 Aug, 112 ( Pt 15), 2485 - 92 LAP2 binding protein 1 (L2BP1/BAF) is a candidate mediator of LAP2-chromatin interaction; Furukawa K; Lamina-associated polypeptide (LAP) 2, which directly interacts with B-type lamins and chromosomes, is an integral membrane protein specifically distributed along the inner nuclear membrane of the nuclear envelope . The chromatin- and lamin-binding activity of LAP2 suggests that LAP2 plays an important role in targeting mitotic vesicles to chromosomes and reorganizing the nuclear structure at the end of mitosis . Here I identified a LAP2 interacting protein, termed L2BP1 (LAP2 binding protein 1) . The rat L2BP1 cDNA sequence is predicted to encode a protein of 89 amino acids which turns out to be a rat homolog of mouse and human BAF (Barrier-to-Autointegration Factor) . L2BP1 is distributed diffusely throughout the nucleus in interphase cells . It is, however, highly concentrated at the chromosomes during the M-phase . Further, the L2BP1 binding domain of LAP2 overlaps its chromosome-binding region . These findings suggest that L2BP1 is a candidate mediator of LAP2-chromosome interaction at the end of mitosis. Biochemistry, 1999 Jul 6, 38(27), 8826 - 30 Control of the histone-acetyltransferase activity of Tip60 by the HIV-1 transactivator protein, Tat; Creaven M et al.; Tip60, a cellular histone-acetyltransferase, is known to interact with the HIV-1-encoded transactivator protein, Tat . In this work, we show that the interaction of Tat with Tip60 efficiently inhibits the Tip60 histone-acetyltransferase activity . Besides its histone-acetyltransferase activity, Tip60 can undergo an autoacetylation which is not affected by Tat interaction . Our data show that Tip60 does not significantly influence Tat-dependent transcriptional activation of the 5'-LTR of HIV, suggesting that its interaction with Tat affects some intrinsic cellular process . We were then able to identify a cellular gene, Mn-dependent superoxide dismutase (Mn-SOD), that has a Tip60-dependent transcriptional activity . Interestingly, the simultaneous expression of Tat and Tip60 abolishes the effect of Tip60 on the activity of the Mn-SOD promoter . We postulate that the HIV-1 transactivator, Tat, in targeting Tip60 hinders the expression of cellular genes (such as Mn-SOD) which normally interfere with the efficient replication and propagation of the virus. Cytogenet Cell Genet, 1999, 84(3-4), 199 - 205 Human and mouse GPAA1 (Glycosylphosphatidylinositol anchor attachment 1) genes: genomic structures, chromosome loci and the presence of a minor class intron; Inoue N et al.; Many eukaryotic cell surface proteins are anchored to the membrane with glycosylphosphatidylinositol (GPI) that is covalently linked to the carboxyl-terminus . A Saccharomyces cerevisiae gaa1 mutant is defective in posttranslational attachment of GPI to proteins . A recent report demonstrated that the GPAA1 gene encodes a component of a transamidase that mediates GPI-anchor attachment . Here, we report structures and chromosome loci of human and mouse GPAA1 genes . Both genes consist of twelve exons that span about 4 kb . Human and mouse GPAA1s are located at 8q24.3 and 15E, respectively . There is a human pseudo GPAA1 gene (GPAA1P1) that is located at 2q12-->q14 . Introns 8 of human and mouse GPAA1s were minor class introns bearing AT at the 5' splice sites and AC and AT at the 3' splice sites, respectively . The 3' splice sites of corresponding introns of African green monkey, Chinese hamster, dog and rat were AC, AT, AT and AA, respectively . The mouse GPAA1 gene (Gpaa1) bearing AG at the 3' splice site prepared by site-directed mutagenesis was functional, indicating that any nucleotide is allowed at the 3' end of a minor class intron. Gene, 1999 Jun 24, 234(1), 109 - 17 Isolation and characterization of PDE10A, a novel human 3', 5'-cyclic nucleotide phosphodiesterase; Loughney K et al.; A gene encoding a novel human 3', 5'-cyclic nucleotide phosphodiesterase (PDE) was identified and characterized . PDE10A1 encodes a protein that is 779 amino acids in length . An incomplete cDNA for a second 5'-splice variant, PDE10A2, was isolated . The proteins encoded by the two variants share 766 amino acids in common . This common region includes an amino-terminal domain with partial homology to the cGMP-binding domains of PDE2, PDE5 and PDE6 as well as a carboxy-terminal region with homology to the catalytic regions of mammalian PDEs . Northern analysis revealed that PDE10A is widely expressed . The PDE10A gene was mapped to three yeast artificial chromosomes (YACs) that contain human DNA from chromosome 6q26-27 . A recombinant protein corresponding to the 766 amino acid region common to PDE10A1 and PDE10A2 was expressed in yeast . It hydrolyzed both cAMP and cGMP . Inhibitors that are selective for other PDE families are poor inhibitors of PDE10A; however, PDE10A is inhibited by the non-specific PDE inhibitor, IBMX. EMBO J, 1999 Jul 1, 18(13), 3808 - 19 A role for a replicator dominance mechanism in silencing; Palacios DeBeer MA et al.; The role of the natural HMR-E silencer in modulating replication initiation and silencing by the origin recognition complex (ORC) was examined . When natural HMR-E was the only silencer controlling HMR, the silencer's ORC-binding site (ACS) was dispensable for replication initiation but essential for silencing, indicating that a non-silencer chromosomal replicator(s) existed in close proximity to the silencer . Further analysis revealed that regions flanking both sides of HMR-E contained replicators . In contrast to replication initiation by the intact silencer, initiation by the non-silencer replicator(s) was abolished in an orc2-1 mutant, indicating that these replicators were extremely sensitive to defects in ORC . Remarkably, the activity of one of the non-silencer replicators correlated with reduced silencing; inactivation of these replicators caused by either the orc2-1 mutation or the deletion of flanking sequences enhanced silencing . These data were consistent with a role for the ORC bound to the HMR-E silencer ACS in suppressing the function of neighboring ORC molecules capable of inhibiting silencing, and indicated that differences in ORC-binding sites within HMR itself had profound effects on ORC function . Moreover, replication initiation by natural HMR-E was inefficient, suggesting that closely spaced replicators within HMR contributed to an inhibition of replication initiation. EMBO J, 1999 Jul 1, 18(13), 3667 - 75 The TIM17.23 preprotein translocase of mitochondria: composition and function in protein transport into the matrix; Moro F et al.; We have analysed the structural organization of the TIM17.23 complex, the preprotein translocase of the mitochondrial inner membrane specific for protein targeting to the matrix . The components Tim17, Tim23 and Tim44 are present in this complex in equimolar amounts . A sub-complex containing Tim23 and Tim44 but no Tim17, or a sub-complex containing Tim23 and Tim17 but no Tim44 was not detected . Tim44 is peripherally associated at the matrix side . Tim44 forms dimers which recruit two molecules of mt-Hsp70 to the sites of protein import . A sequential, hand-over-hand mode of interaction of these two mt-Hsp70.Tim44 complexes with a translocating polypeptide chain is proposed. EMBO J, 1999 Jul 1, 18(13), 3575 - 85 Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly; Arendt CS et al.; Proteins targeted for degradation by the ubiquitin-proteasome system are degraded by the 26S proteasome . The core of this large protease is the 20S proteasome, a barrel-shaped structure made of a stack of four heptameric rings . Of the 14 different subunits that make up the yeast 20S proteasome, three have proteolytic active sites: Doa3/beta5, Pup1/beta2 and Pre3/beta1 . Each of these subunits is synthesized with an N-terminal propeptide that is autocatalytically cleaved during particle assembly . We show here that the propeptides have both common and distinct functions in proteasome biogenesis . Unlike the Doa3 propeptide, which is crucial for proteasome assembly, the Pre3 and Pup1 propeptides are dispensable for cell viability and proteasome formation . However, mutants lacking these propeptide-encoding elements are defective for specific peptidase activities, are more sensitive to environmental stresses and have subtle defects in proteasome assembly . Unexpectedly, a critical function of the propeptide is the protection of the N-terminal catalytic threonine residue against Nalpha-acetylation . For all three propeptide-deleted subunits, activity of the affected catalytic center is fully restored when the Nat1-Ard1 Nalpha-acetyltransferase is mutated . In addition to delineating a novel function for proteasome propeptides, these data provide the first biochemical evidence for the postulated participation of the alpha-amino group in the proteasome catalytic mechanism. Nat Genet, 1999 Jul, 22(3), 281 - 5 Systematic determination of genetic network architecture; Tavazoie S et al.; Technologies to measure whole-genome mRNA abundances and methods to organize and display such data are emerging as valuable tools for systems-level exploration of transcriptional regulatory networks . For instance, it has been shown that mRNA data from 118 genes, measured at several time points in the developing hindbrain of mice, can be hierarchically clustered into various patterns (or 'waves') whose members tend to participate in common processes . We have previously shown that hierarchical clustering can group together genes whose cis-regulatory elements are bound by the same proteins in vivo . Hierarchical clustering has also been used to organize genes into hierarchical dendograms on the basis of their expression across multiple growth conditions . The application of Fourier analysis to synchronized yeast mRNA expression data has identified cell-cycle periodic genes, many of which have expected cis-regulatory elements . Here we apply a systematic set of statistical algorithms, based on whole-genome mRNA data, partitional clustering and motif discovery, to identify transcriptional regulatory sub-networks in yeast-without any a priori knowledge of their structure or any assumptions about their dynamics . This approach uncovered new regulons (sets of co-regulated genes) and their putative cis-regulatory elements . We used statistical characterization of known regulons and motifs to derive criteria by which we infer the biological significance of newly discovered regulons and motifs . Our approach holds promise for the rapid elucidation of genetic network architecture in sequenced organisms in which little biology is known. Nucleic Acids Res, 1999 Jul 15, 27(14), 2825 - 31 Construction of a variability map for eukaryotic large subunit ribosomal RNA; Ben Ali A et al.; In this paper, we present a variability map of the eukaryotic large subunit ribosomal RNA, showing the distribution of variable and conserved sites in this molecule . The variability of each site in this map is indicated by means of a colored dot . Construction of the variability map was based on the substitution rate calibration (SRC) method, in which the substitution rate of each nucleotide site is computed by looking at the frequency with which sequence pairs differ at that site as a function of their evolutionary distance . Variability maps constructed by this method provide a much more accurate and objective description of site-to-site variability than visual inspection of sequence alignments. Science, 1999 Jul 2, 285(5424), 107 - 10 Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein; Taylor DR et al.; Most isolates of hepatitis C virus (HCV) infections are resistant to interferon, the only available therapy, but the mechanism underlying this resistance has not been defined . Here it is shown that the HCV envelope protein E2 contains a sequence identical with phosphorylation sites of the interferon-inducible protein kinase PKR and the translation initiation factor eIF2alpha, a target of PKR . E2 inhibited the kinase activity of PKR and blocked its inhibitory effect on protein synthesis and cell growth . This interaction of E2 and PKR may be one mechanism by which HCV circumvents the antiviral effect of interferon. J Biol Chem, 1999 Jul 9, 274(28), 20034 - 9 Photocross-linking of an oriented DNA repair complex . Ku bound at a single DNA end; Yoo S et al.; Ku protein binds broken DNA ends, triggering a double-strand DNA break repair pathway . The spatial arrangement of the two Ku subunits in the initial Ku-DNA complex, when the Ku protein first approaches the broken DNA end, is not well defined . We have investigated the geometry of the complex using a novel set of photocross-linking probes that force Ku protein to be constrained in position and orientation, relative to a single free DNA end . Results suggest that this complex is roughly symmetric and that both Ku subunits make contact with an approximately equal area of the DNA . The complex has a strongly preferred orientation, with Ku70-DNA backbone contacts located proximal and Ku80-DNA backbone contacts located distal to the free end . Ku70 also contacts functional groups in the major groove proximal to the free end . Ku80 apparently does not make major groove contacts . Results are consistent with a model where the Ku70 and Ku80 subunits contact the major and minor grooves of DNA, respectively. J Biol Chem, 1999 Jul 9, 274(28), 19985 - 91 A cytoskeletal localizing domain in the cyclase-associated protein, CAP/Srv2p, regulates access to a distant SH3-binding site; Yu J et al.; In the yeast, Saccharomyces cerevisiae, adenylyl cyclase consists of a 200-kDa catalytic subunit (CYR1) and a 70-kDa subunit (CAP/SRV2) . CAP/Srv2p assists the small G protein Ras to activate adenylyl cyclase . CAP also regulates the cytoskeleton through an actin sequestering activity and is directed to cortical actin patches by a proline-rich SH3-binding site (P2) . In this report we analyze the role of the actin cytoskeleton in Ras/cAMP signaling . Two alleles of CAP, L16P(Srv2) and R19T (SupC), first isolated in genetic screens for mutants that attenuate cAMP levels, reduced adenylyl cyclase binding, and cortical actin patch localization . A third mutation, L27F, also failed to localize but showed no loss of either cAMP signaling or adenylyl cyclase binding . However, all three N-terminal mutations reduced CAP-CAP multimer formation and SH3 domain binding, although the SH3-binding site is about 350 amino acids away . Finally, disruption of the actin cytoskeleton with latrunculin-A did not affect the cAMP phenotypes of the hyperactive Ras2(Val19) allele . These data identify a novel region of CAP that controls access to the SH3-binding site and demonstrate that cytoskeletal localization of CAP or an intact cytoskeleton per se is not necessary for cAMP signaling. J Biol Chem, 1999 Jul 9, 274(28), 19731 - 7 Activation of the phagocyte NADPH oxidase protein p47(phox) . Phosphorylation controls SH3 domain-dependent binding to p22(phox); Huang J et al.; Activation of phagocyte NADPH oxidase requires interaction between p47(phox) and p22(phox) . p47(phox) in resting phagocytes does not bind p22(phox) . Phosphorylation of serines in the p47(phox) C terminus enables binding to the p22(phox) C terminus by inducing a conformational change in p47(phox) that unmasks the SH3A domain . We report that an arginine/lysine-rich region in the p47(phox) C terminus binds the p47(phox) SH3 domains expressed in tandem (SH3AB) but does not bind the individual N-terminal SH3A and C-terminal SH3B domains . Peptides matching amino acids 301-320 and 314-335 of the p47(phox) arginine/lysine-rich region block the p47(phox) SH3AB/p22(phox) C-terminal and p47(phox) SH3AB/p47(phox) C-terminal binding and inhibit NADPH oxidase activity in vitro . Peptides with phosphoserines substituted for serines 310 and 328 do not block binding and are poor inhibitors of oxidase activity . Mutated full-length p47(phox) with aspartic acid substitutions to mimic the effects of phosphorylations at serines 310 and 328 bind the p22(phox) proline-rich region in contrast to wild-type p47(phox) . We conclude that the p47(phox) SH3A domain-binding site is blocked by an interaction between the p47(phox) SH3AB domains and the C-terminal arginine/lysine-rich region . Phosphorylation of serines in the p47(phox) C terminus disrupts this interaction leading to exposure of the SH3A domain, binding to p22(phox), and activation of the NADPH oxidase. J Biol Chem, 1999 Jul 9, 274(28), 19677 - 85 Activation of the cAMP-specific phosphodiesterase PDE4D3 by phosphorylation . Identification and function of an inhibitory domain; Lim J et al.; Splicing variants of type 4 phosphodiesterases (PDE4) are regulated by phosphorylation . In these proteins, a conserved region is located between the amino-terminal domain, which is the target for phosphorylation, and the catalytic domain . Previous studies have indicated that nested deletions encompassing this region cause an increase in catalytic activity, suggesting this domain exerts an inhibitory constraint on catalysis . Here, we have further investigated the presence and function of this domain . A time-dependent increase in hydrolytic activity was observed when PDE4D3 from FRTL-5 cells was incubated with the endoproteinase Lys-C . The activation was abolished by protease inhibitors and was absent when a phosphorylated enzyme was used . Western blot analysis with PDE4D-specific antibodies indicated the Lys-C treatment separates the catalytic domain of PDE4D3 from the inhibitory domain . Incubation with antibodies recognizing an epitope within this domain caused a 3- to 4-fold increase in activity of native or recombinant PDE4D3 . Again, PDE activation by these antibodies had properties similar to, and not additive with, the activation by protein kinase A phosphorylation . An interaction between the inhibitory domain and both regulatory and catalytic domains of PDE4D3 was detected by the yeast two-hybrid system . Mutations of Ser54 to Ala in the regulatory domain decreased or abolished this interaction, whereas mutations of Ser54 to the negatively charged Asp strengthened it . These data strongly support the hypothesis that an inhibitory domain is present in PDE4D and that phosphorylation of the regulatory domain causes activation of the enzyme by modulating the interaction between inhibitory and catalytic domains. J Biol Chem, 1999 Jul 9, 274(28), 19513 - 6 The Nijmegen breakage syndrome protein is essential for Mre11 phosphorylation upon DNA damage; Dong Z et al.; The Nijmegen breakage syndrome (NBS), a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation . Repair of DNA double-strand breaks by radiation is dependent on a multifunctional complex containing Rad50, Mre11, and the NBS1 gene product, p95 (NBS protein, nibrin) . The role of p95 in these repair processes is unknown . Here it is demonstrated that Mre11 is hyperphosphorylated in a cell cycle-independent manner in response to treatment of cells with genotoxic agents including gamma irradiation . This response is abrogated in two independently established NBS cell lines that have undetectable levels of the p95 protein . NBS cells are also deficient for radiation-induced nuclear foci containing Mre11, while those with Rad51 are unaffected . An analysis of the kinetic relationship between Mre11 phosphorylation and the appearance of its radiation-induced foci indicates that the former precedes the latter . Together, these data suggest that specific phosphorylation of Mre11 is induced by DNA damage, and p95 is essential in this process, perhaps by recruiting specific kinases. Oncogene, 1999 Jun 24, 18(25), 3761 - 5 Mutational analysis of p51A/TAp63gamma, a p53 homolog, in non-small cell lung cancer and breast cancer; Sunahara M et al.; p51, a novel family member of human p53, is a recently identified candidate tumor suppressor gene mapped at chromosome 3q28 . Like p53, p51 was found to activate p21Waf1/Cip1 and to induce apoptosis . Since the DNA loss at 3q is reported in several cancers including non-small cell lung cancer (NSCLC), we screened for mutations in p51A (TAp63gamma), an isoform of p51 with short C-terminal region, in 80 NSCLCs as well as 85 breast cancers by RT-PCR single strand conformation polymorphism (SSCP) analysis and DNA sequencing . In NSCLCs, p51 was expressed in most tumors at variable levels and we found three missense and one silent mutations: Gln31His (transactivation domain) in two tumors, Ala148Pro (DNA-binding domain) and Leu248Leu (DNA-binding domain) . In the tumor with Ala148Pro or the silent mutation, only the mutant gene appeared to be expressed . The modified FASAY method to test the ability of yeast expressing p51A cDNA to grow in medium lacking histidine has revealed that Ala148Pro results in a loss of function, while Gln31His does not . In contrast to NSCLC, no mutation was observed in all 85 breast cancers by the similar method . Our results suggest that, because of infrequent mutation, p51 may not be a Knudson type tumor suppressor in most NSCLCs and breast cancers . Nevertheless, in at least a part of NSCLC, p51 may play a certain role in carcinogenesis in a tissue-specific manner. Mol Cell Biochem, 1999 Apr, 194(1-2), 227 - 34 Increased peroxisomal fatty acid beta-oxidation and enhanced expression of peroxisome proliferator-activated receptor-alpha in diabetic rat liver; Asayama K et al.; To determine whether the increased fatty acid beta-oxidation in the peroxisomes of diabetic rat liver is mediated by a common peroxisome proliferation mechanism, we measured the activation of long-chain (LC) and very long chain (VLC) fatty acids catalyzed by palmitoyl CoA ligase (PAL) and lignoceryl CoA ligase and oxidation of LC (palmitic acid) and VLC (lignoceric acid) fatty acids by isotopic methods . Immunoblot analysis of acyl-CoA oxidase (ACO), and Northern blot analysis of peroxisome proliferator-activated receptor (PPAR-alpha), ACO, and PAL were also performed . The PAL activity increased in peroxisomes and mitochondria from the liver of diabetic rats by 2.6-fold and 2.1 -fold, respectively . The lignoceroyl-CoA ligase activity increased by 2.6-fold in diabetic peroxisomes . Palmitic acid oxidation increased in the diabetic peroxisomes and mitochondria by 2.5-fold and 2.7-fold, respectively, while lignoceric acid oxidation increased by 2.0-fold in the peroxisomes . Immunoreactive ACO protein increased by 2-fold in the diabetic group . The mRNA levels for PPAR-alpha, ACO and PAL increased 2.9-, 2.8- and 1.6-fold, respectively, in the diabetic group . These results suggest that the increased supply of fatty acids to liver in diabetic state stimulates the expression of PPAR-alpha and its target genes responsible for the metabolism of fatty acids. Appl Biochem Biotechnol, 1999 Mar, 76(3), 217 - 27 Stabilization and translation of immobilized mRNA on latex beads for cell-free protein synthesis system; Kobatake E et al.; The stability of immobilized mRNA against ribonucleases was investigated in a cell-free protein synthesis system . The plasmid-encoding protein A with the 20-mer poly(A) tail under the control of T7 promoter was constructed, and the corresponding mRNA was synthesized by T7 RNA polymerase reaction . The resulting mRNA was immobilized on oligo(dT)-immobilized latex beads by hybridization utilizing the poly(A) tail of mRNA at the 3'-terminus . The mRNA was stabilized against three types of nucleases (3'-OH exonuclease, 5'-OH exonuclease, and endonuclease) by immobilization . Translation of immobilized mRNA with a continuous-flow cell-free protein-synthesizing system from Saccharomyces cerevisiae was ascertained . Reusability of the immobilized mRNA as genetic information was also examined. Nucleic Acids Res, 1999 Jul 15, 27(14), 2848 - 51 Codon usage as a tool to predict the cellular location of eukaryotic ribosomal proteins and aminoacyl-tRNA synthetases; Chiapello H et al.; In spite of many efforts, the prediction of the location of proteins in eukaryotic cells (cytoplasm, mitochondrion or chloroplast) is still far from straightforward . In some cases (e.g . ribosomal proteins and aminoacyl-tRNA synthetases) both the cytoplasmic proteins and their organellar counterparts are encoded by the nuclear genome . A factorial correspondence analysis of the codon usage in yeast and Caenorhabditis elegans shows that the codon usage of those nuclear genes encoding ribosomal proteins or aminoacyl-tRNA synthetases is markedly different, depending on the final location of the proteins (cytoplasmic or mitochondrial) . As a consequence, the location of such proteins-whose sequences are now frequently determined by systematic genomic sequencing-can be easily and quickly predicted . A WWW interface has been developed, aimed at providing a user-friendly tool for codon usage pattern analysis . It is available from http://www.genetique.uvsq.fr/afc.html Science, 1999 Jul 2, 285(5424), 96 - 100 A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export; York JD et al.; In order to identify additional factors required for nuclear export of messenger RNA, a genetic screen was conducted with a yeast mutant deficient in a factor Gle1p, which associates with the nuclear pore complex (NPC) . The three genes identified encode phospholipase C and two potential inositol polyphosphate kinases . Together, these constitute a signaling pathway from phosphatidylinositol 4, 5-bisphosphate to inositol hexakisphosphate (IP6) . The common downstream effects of mutations in each component were deficiencies in IP6 synthesis and messenger RNA export, indicating a role for IP6 in GLE1 function and messenger RNA export. DNA Cell Biol, 1999 Jun, 18(6), 503 - 12 Cloning and characterization of rat BAT3 cDNA; Ozaki T et al.; HLA-B-associated transcript 3 (BAT3) was originally identified as one of the genes located within human major histocompatibility complex . It encodes a large proline-rich protein with unknown function . In this study, we found that a fragment of the BAT3 gene product interacts with a candidate tumor suppressor, DAN, in the yeast-based two-hybrid system . We cloned the full-length rat BAT3 cDNA from a fibroblast 3Y1 cDNA library . Our sequence analysis has demonstrated that rat BAT3 cDNA is 3617 nucleotides in length and encodes a full-length BAT3 (1098 amino acids) with an estimated molecular mass of 114,801 daltons, which displays an 87.4% identity with human BAT3 . The deletion experiment revealed that the N-terminal region (amino acid residues 1-80) of DAN was required for the interaction with BAT3 . Green fluorescent protein-tagged BAT3 was largely localized in the cytoplasm of COS cells . Northern hybridization showed that BAT3 mRNA was expressed in all the adult rat tissues examined but predominantly in testis . In addition, the level of BAT3 mRNA expression was more downregulated in some of the transformed cells, including v-mos- and v-Ha-ras-transformed 3Y1 cells, than in the parental cells. DNA Cell Biol, 1999 Jun, 18(6), 471 - 9 Regulation of the insulin gene by glucose: stimulation of trans-activation potency of human PDX-1 N-terminal domain; Shushan EB et al.; The beta cells in pancreatic islets of Langerhans increase insulin gene transcription in response to glucose . The pancreatic and duodenal homeobox-1 (PDX-1) plays a major role in glucose-induced insulin transcription . We studied the functional regions of the human PDX-1 protein fused to the DNA-binding domain of the transcription factor Gal4 . The results indicate that the N-terminal domain of the hPDX-1, required for transactivation (amino acids 1-120) in transfected betaTC6 and HeLa cells, is also regulated by extracellular glucose concentrations in transfected rat islets . Deletion analyses have led to the mapping of two regions within the N terminus that are essential for its trans-activation properties . One sequence spans amino acids 97-120 in transfected islet and HeLa cells or amino acids 77-120 in betaTC6 cells; the other includes the highly conserved B box (amino acids 31-41) . We thus present evidence of a glucose effect on hPDX-1 trans-activation activity, in addition to the previously described regulatory effect on its DNA-binding activity. Cytopathology, 1999 Jun, 10(3), 180 - 5 Cytological and immunocytochemical evaluation of thyroid and breast masses in patients with a previous neoplasm: case reports; Pisani T et al.; The diagnosis of secondary tumours represents one of the most important fields in the application of fine needle aspiration cytology (FNAC) . We studied two patients, one with a history of breast cancer and one with a previous tumour of the thyroid, who showed a second mass, in the thyroid and in the breast, respectively, during follow up . The aim of our study was to evaluate if cytology, performed on FNAC smears, may distinguish a metastatic lesion from a second primary tumour, or if further immunocytochemistry should be performed . Our data demonstrate that, while cytology may be indicative of a second primary tumour, the histotype should be confirmed by immunocytochemical staining. Phytochemistry, 1999 Jul, 51(6), 781 - 5 Iridoid and seco-iridoid glucosides from Chioccoca alba (Rubiaceae); Carbonezi CA et al.; Phytochemical investigation of Chioccoca alba afforded three new iridoids, alboside I, alboside II and alboside III, and a new seco-iridoid alboside V . Alboside IV showed moderate activity towards the DNA repair-deficient mutant RS321 of Saccharomyces cerevisiae . The structural elucidation of the new compounds was performed by ES-MS and by 1D and 2D NMR spectroscopic methods. Biophys J, 1999 Jul, 77(1), 54 - 69 De novo simulations of the folding thermodynamics of the GCN4 leucine zipper; Mohanty D et al.; Entropy Sampling Monte Carlo (ESMC) simulations were carried out to study the thermodynamics of the folding transition in the GCN4 leucine zipper (GCN4-lz) in the context of a reduced model . Using the calculated partition functions for the monomer and dimer, and taking into account the equilibrium between the monomer and dimer, the average helix content of the GCN4-lz was computed over a range of temperatures and chain concentrations . The predicted helix contents for the native and denatured states of GCN4-lz agree with the experimental values . Similar to experimental results, our helix content versus temperature curves show a small linear decline in helix content with an increase in temperature in the native region . This is followed by a sharp transition to the denatured state . van't Hoff analysis of the helix content versus temperature curves indicates that the folding transition can be described using a two-state model . This indicates that knowledge-based potentials can be used to describe the properties of the folded and unfolded states of proteins. Biochemistry, 1999 Jun 22, 38(25), 8138 - 49 Thermal versus guanidine-induced unfolding of ubiquitin . An analysis in terms of the contributions from charge-charge interactions to protein stability; Ibarra-Molero B et al.; We have characterized the guanidine-induced unfolding of both yeast and bovine ubiquitin at 25 degrees C and in the acidic pH range on the basis of fluorescence and circular dichroism measurements . Unfolding Gibbs energy changes calculated by linear extrapolation from high guanidine unfolding data are found to depend very weakly on pH . A simple explanation for this result involves the two following assumptions: (1) charged atoms of ionizable groups are exposed to the solvent in native ubiquitin (as supported by accessible surface area calculations), and Gibbs energy contributions associated with charge desolvation upon folding (a source of pK shifts) are small; (2) charge-charge interactions (another source of pK shifts upon folding) are screened out in concentrated guanidinium chloride solutions . We have also characterized the thermal unfolding of both proteins using differential scanning calorimetry . Unfolding Gibbs energy changes calculated from the calorimetric data do depend strongly on pH, a result that we attribute to the pH dependence of charge-charge interactions (not eliminated in the absence of guanidine) . In fact, we find good agreement between the difference between the two series of experimental unfolding Gibbs energy changes (determined from high guanidine unfolding data by linear extrapolation and from thermal denaturation data in the absence of guanidine) and the theoretical estimates of the contribution from charge-charge interactions to the Gibbs energy change for ubiquitin unfolding obtained by using the solvent-accessibility-corrected Tanford-Kirkwood model, together with the Bashford-Karplus (reduced-set-of-sites) approximation . This contribution is found to be stabilizing at neutral pH, because most charged groups on the native protein interact mainly with groups of the opposite charge, a fact that, together with the absence of large charge-desolvation contributions, may explain the high stability of ubiquitin at neutral pH . In general, our analysis suggests the possibility of enhancing protein thermal stability by adequately redesigning the distribution of solvent-exposed, charged residues on the native protein surface. Bioorg Med Chem Lett, 1999 Jun 7, 9(11), 1533 - 6 Isolation and characterization of an active-site peptide from a sterol methyl transferase with a mechanism-based inhibitor; Marshall JA et al.; Chemical affinity labeling of pure sterol methyl transferase (SMT) from Saccharomyces cerevisiae using the mechanism-based irreversible inhibitor, {3-3H}26,27-dehydrozymosterol, inhibited the SMT with an apparent Ki of 1.1 microM and k(inact) of 1.52 min(-1) . The protein-inhibitor adduct was subjected to cleavage with trypsin and the resulting covalently modified peptide was analyzed by Edman sequencing from the N-terminus . The radiochemically labeled ca . 5.0 kDa peptide fragment of the cleavage mixture was shown to be contiguous through 17 residues to a segment that includes a highly conserved hydrophobic motif (Region I, stretching between T78 and F91) characteristic of SMT enzymes . The results confirm that Region I is the sterol binding/active site. Protein Sci, 1999 Jun, 8(6), 1358 - 61 Simple sequence is abundant in eukaryotic proteins; Golding GB; All proteins of Saccharomyces cerevisiae have been compared to determine how frequently segments from one protein are present in other proteins . Proteins that are recently evolutionarily related were excluded . The most frequently present protein segments are long, tandem repetitions of a single amino acid . For some of these segments, up to 14% of all proteins in the genome were found to have similar peptides within them . These peptide segments may not be functional protein domains . Although they are the most common shared feature of yeast proteins, their ubiquity and simplicity argue that their probable function may be to simply serve as spacers between other protein motifs. FEBS Lett, 1999 Jun 11, 452(3), 170 - 6 Cloning and characterization of hGMEB1, a novel glucocorticoid modulatory element binding protein; Theriault JR et al.; A 21-bp element called glucocorticoid modulatory element (GME) modulates the glucocorticoid receptor-mediated responses via the binding of an as yet poorly characterized transacting complex of proteins containing the 88-kDa GMEB1 and the 67-kDa GMEB2 . Using heat shock protein 27 (HSP27) as bait in the yeast two-hybrid assay, we cloned a 1.83-kb cDNA encoding a novel 573-amino acid protein called human GMEB1 (hGMEB1) . hGMEB1 possesses a KDWK domain, contains sequences almost identical (36/38) to three tryptic peptides of rat GMEB1 and shares 38% identity with rat GMEB2 . hGMEB1 is ubiquitously expressed as a 85-kDa protein in all cell lines and tissues examined . In vitro translated hGMEB1 bound specifically to GME oligonucleotides yielding a complex of similar size to the complex obtained using rat liver nuclear extracts . Both complexes were supershifted with an antibody specific to hGMEB1 . Co-immunoprecipitation experiments confirmed the in vivo interaction of HSP27 with hGMEB1. Hindustan Antibiot Bull, 1997 Feb-Nov, 39(1-4), 16 - 9 Affinity chromatography of maltase on aliphatic amines as ligands; Kulkarni MV et al.; The primary amino group, the competitive inhibitor of maltase, was used as ligand and the enzyme was purified to homogeneity in a single step . The amino group affiants of ethylene (C2-NH2) and hexamethylene (C6-NH2) diamines were prepared by coupling to cyanogen bromide activated Sepharose CL-4B . The enzyme was quantitatively adsorbed at alkaline pH (pH 8.2), while the elution could be effected only in presence of maltose at acidic pH . The elution of enzyme by maltose was independent of spacer arms (C2 and C6) which suggests specific binding of the enzyme through inhibitor site. Phytochemistry, 1999 Mar, 50(6), 925 - 30 Cytochrome P450 monooxygenases of DIBOA biosynthesis: specificity and conservation among grasses; Glawischnig E et al.; DIBOA and DIMBOA are secondary metabolites of grasses which function as natural pesticides . The four maize genes BX2 through BX5 encode cytochrome P450-dependent monooxygenases that catalyse four consecutive reactions in the biosynthesis of these secondary products . Although BX2-BX5 share significant sequence homology, the four enzymes have evolved into specific enzymes each catalysing predominantly only one reaction in the pathway . In addition to these natural reactions, BX3 hydroxylates 1,4-benzoxazin-3-one and BX2 shows pCMA demethylase activity . With respect to DIBOA biosynthesis, identical enzymatic reactions have been found in rye as compared to maize, indicating early evolution of the P450 enzymes in the grasses. Mol Pharmacol, 1999 Jul, 56(1), 141 - 6 Cross-resistance to ionizing radiation in a murine leukemic cell line resistant to cis-dichlorodiammineplatinum(II): role of Ku autoantigen; Frit P et al.; cis-Dichlorodiammineplatinum(II) (CDDP; cisplatin) is commonly used in combination with ionizing radiation (IR) in the treatment of various malignancies . In vitro, many observations suggest that acquisition of CDDP resistance in cell lines confers cross-resistance to IR, but the molecular mechanisms involved have not been well documented yet . We report here the selection and characterization of a murine CDDP-resistant L1210 cell line (L1210/3R) that exhibits cross-resistance to IR because of an increased capacity to repair double-strand breaks compared with parental cells (L1210/P) . In resistant cells, electrophoretic mobility shift assays revealed an increased DNA-end binding activity that could be ascribed, by supershifting the retardation complexes with antibodies, to the autoantigen Ku . The heterodimeric Ku protein, composed of 86-kDa (Ku80) and 70-kDa (Ku70) subunits, is the DNA-targeting component of DNA-dependent protein kinase (DNA-PK), which plays a critical role in mammalian DNA double-strand breaks repair . The increased Ku-binding activity in resistant cells was associated with an overexpression affecting specifically the Ku80 subunit . These data strongly suggest that the increase in Ku activity is responsible for the phenotype of cross-resistance to IR . In addition, these observations, along with previous results from DNA-PK- mutant cells, provide evidence in favor of a role of Ku/DNA-PK in resistance to CDDP . These results suggest that Ku activity may be an important molecular target in cancer therapy at the crossroad between cellular responses to CDDP and IR. Mol Pharmacol, 1999 Jul, 56(1), 77 - 84 Topoisomerase poisoning activity of novel disaccharide anthracyclines; Guano F et al.; Doxorubicin and idarubicin are very effective anticancer drugs in the treatment of human hematological malignancies and solid tumors . These agents are well known topoisomerase II poisons; however, some anthracycline analogs recently have been shown to poison topoisomerase I . In the present work, we assayed novel disaccharide analogs and the parent drug, idarubicin, for their poisoning effects of human topoisomerase I and topoisomerases IIalpha and IIbeta . Drugs were evaluated with a DNA cleavage assay in vitro and with a yeast system to test whether the agents were able to poison the enzymes in vivo . We have found that the test agents are potent poisons of both topoisomerases IIalpha and IIbeta . The axial orientation of the second sugar relative to the first one of the novel disaccharide analogs was shown to be required for poisoning activity and cytotoxicity . Interestingly, idarubicin and the new analogs stimulated topoisomerase I-mediated DNA cleavage at low levels in vitro . As expected, the cytotoxic level of the drug was highly affected by the content of topoisomerase II; nevertheless, the test agents had a yeast cell-killing activity that also was weakly dependent on cellular topoisomerase I content . The results are relevant for the full understanding of the molecular mechanism of topoisomerase poisoning by anticancer drugs, and they define structural determinants of anthracyclines that may help in the rational design of new compounds directed against topoisomerase I. Genes Dev, 1999 Jun 15, 13(12), 1614 - 26 Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34; Seol JH et al.; SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development . Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF . Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex . Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S . Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1 . Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1 . Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol . The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1 . We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases. J Cell Biol, 1999 Jun 28, 145(7), 1435 - 42 Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion; Ungermann C et al.; Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles . We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex . Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex . After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking . Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction . Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p. J Cell Biol, 1999 Jun 28, 145(7), 1407 - 17 Nuclear import of the TATA-binding protein: mediation by the karyopherin Kap114p and a possible mechanism for intranuclear targeting; Pemberton LF et al.; Binding of the TATA-binding protein (TBP) to the promoter is the first and rate limiting step in the formation of transcriptional complexes . We show here that nuclear import of TBP is mediated by a new karyopherin (Kap) (importin) family member, Kap114p . Kap114p is localized to the cytoplasm and nucleus . A complex of Kap114p and TBP was detected in the cytosol and could be reconstituted using recombinant proteins, suggesting that the interaction was direct . Deletion of the KAP114 gene led to specific mislocalization of TBP to the cytoplasm . We also describe two other potential minor import pathways for TBP . Consistent with other Kaps, the dissociation of TBP from Kap114p is dependent on RanGTP . However, we could show that double stranded, TATA-containing DNA stimulates this RanGTP-mediated dissociation of TBP, and is necessary at lower RanGTP concentrations . This suggests a mechanism where, once in the nucleus, TBP is preferentially released from Kap114p at the promoter of genes to be transcribed . In this fashion Kap114p may play a role in the intranuclear targeting of TBP. J Cell Biol, 1999 Jun 28, 145(7), 1369 - 80 Assembly of 5S ribosomal RNA is required at a specific step of the pre-rRNA processing pathway; Dechampesme AM et al.; A collection of yeast strains surviving with mutant 5S RNA has been constructed . The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm . The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits . The shortage of 60S subunits was due to a specific defect in the processing of the 27SB precursor RNA that gives rise to the mature 25S and 5.8S rRNA . The processing rate of the 27SB pre-rRNA was specifically delayed, whereas the 27SA and 20S pre-rRNA were processed at a normal rate . The defect was partially corrected by increasing the amount of mutant 5S RNA . We propose that the 5S RNA is recruited by the pre-60S particle and that its recruitment is necessary for the efficient processing of the 27SB RNA precursor . Such a mechanism could ensure that all newly formed mature 60S subunits contain stoichiometric amounts of the three rRNA components. Cold Spring Harb Symp Quant Biol, 1998, 63, 233 - 41 Functional and structural analysis of the subunits of human transcription factor TFIID; Davidson I et al.; The past few years have brought many new insights concerning the structure and function of TAFII proteins . In the future, further biochemical and structural studies will no doubt lead to a greater understanding of the molecular organization of TFIID complexes . A better understanding of the function of metazoan, in particular, mammalian, TAFIIs in cell cycle progression and gene activation will, however, require the use of novel genetic techniques in addition to the biochemical analyses. J Immunol, 1999 Jul 1, 163(1), 412 - 9 The murine antiapoptotic protein A1 is induced in inflammatory macrophages and constitutively expressed in neutrophils; Orlofsky A et al.; Myeloid leukocytes are thought to regulate their susceptibility to apoptosis upon migration to a site of inflammation . However, factors that determine survival have not been well characterized in these cells . We have examined the expression of murine A1, an antiapoptotic Bcl-2 relative found in activated myeloid cells, during the course of an acute inflammatory response . Intraperitoneal infection of mice with the virulent RH strain of Toxoplasma gondii led to a 5- to 10-fold increase in A1 mRNA levels in peritoneal cells after several days . Bcl-2 expression was unchanged . The increase in A1 expression depended on the dose of the organism and coincided with a sharp increase in peritoneal cellularity . A1 protein levels were also increased as determined by Western blot analysis and immunohistochemical studies . All neutrophils and approximately half of the macrophages in the inflammatory exudate contained high levels of A1 in cytoplasm . A1 expression did not correlate with intracellular parasitization . Peripheral blood neutrophils from normal mice strongly expressed A1 protein, whereas normal monocytes showed only weak staining . Bax mRNA was induced in parallel with A1 in macrophages . Exudate macrophages and granulocytes that were apoptotic by TUNEL staining occasionally appeared to display A1 throughout the cell nucleus . These studies identify A1 as a potential regulator of apoptosis during acute inflammation. Proteins, 1999 Jun 1, 35(4), 447 - 52 Correlation between knowledge-based and detailed atomic potentials: application to the unfolding of the GCN4 leucine zipper; Mohanty D et al.; The relationship between the unfolding pseudo free energies of reduced and detailed atomic models of the GCN4 leucine zipper is examined . Starting from the native crystal structure, a large number of conformations ranging from folded to unfolded were generated by all-atom molecular dynamics unfolding simulations in an aqueous environment at elevated temperatures . For the detailed atomic model, the pseudo free energies are obtained by combining the CHARMM all-atom potential with a solvation component from the generalized Born, surface accessibility, GB/SA, model . Reduced model energies were evaluated using a knowledge-based potential . Both energies are highly correlated . In addition, both show a good correlation with the root mean square deviation, RMSD, of the backbone from native . These results suggest that knowledge-based potentials are capable of describing at least some of the properties of the folded as well as the unfolded states of proteins, even though they are derived from a database of native protein structures . Since only conformations generated from an unfolding simulation are used, we cannot assess whether these potentials can discriminate the native conformation from the manifold of alternative, low-energy misfolded states . Nevertheless, these results also have significant implications for the development of a methodology for multiscale modeling of proteins that combines reduced and detailed atomic models. J Biol Chem, 1999 Jul 2, 274(27), 19286 - 93 Eukaryotic molybdopterin synthase . Biochemical and molecular studies of Aspergillus nidulans cnxG and cnxH mutants; Unkles SE et al.; We describe the primary structure of eukaryotic molybdopterin synthase small and large subunits and compare the sequences of the lower eukaryote, Aspergillus nidulans, and a higher eukaryote, Homo sapiens . Mutants in the A . nidulans cnxG (encoding small subunit) and cnxH (large subunit) genes have been analyzed at the biochemical and molecular level . Chlorate-sensitive mutants, all the result of amino acid substitutions, were shown to produce low levels of molybdopterin, and growth tests suggest that they have low levels of molybdoenzymes . In contrast, chlorate-resistant cnx strains have undetectable levels of molybdopterin, lack the ability to utilize nitrate or hypoxanthine as sole nitrogen sources, and are probably null mutations . Thus on the basis of chlorate toxicity, it is possible to distinguish between amino acid substitutions that permit a low level of molybdopterin production and those mutations that completely abolish molybdopterin synthesis, most likely reflecting molybdopterin synthase activity per se . Residues have been identified that are essential for function including the C-terminal Gly of the small subunit (CnxG), which is thought to be crucial for the sulfur transfer process during the formation of molybdopterin . Two independent alterations at residue Gly-148 in the large subunit, CnxH, result in temperature sensitivity suggesting that this residue resides in a region important for correct folding of the fungal protein . Many years ago it was proposed, from data showing that temperature-sensitive cnxH mutants had thermolabile nitrate reductase, that CnxH is an integral part of the molybdoenzyme nitrate reductase (MacDonald, D . W., and Cove, D . J . (1974) Eur . J . Biochem . 47, 107-110) . Studies of temperature-sensitive cnxH mutants isolated in the course of this study do not support this hypothesis . Homologues of both molybdopterin synthase subunits are evident in diverse eukaryotic sources such as worm, rat, mouse, rice, and fruit fly as well as humans as discussed in this article . In contrast, molybdopterin synthase homologues are absent in the yeast Saccharomyces cerevisiae . Precursor Z and molybdopterin are undetectable in this organism nor do there appear to be homologues of molybdoenzymes. Cancer Res, 1999 Jun 15, 59(12), 2810 - 4 Identification of a transactivation activity in the COOH-terminal region of p73 which is impaired in the naturally occurring mutants found in human neuroblastomas; Takada N et al.; p73 is a recently cloned tumor suppressor gene that is highly homologous to p53, and the products of both possess similar functions in inhibiting cell growth and inducing apoptosis . Interestingly, the COOH-terminal region of p53 displays no significant homology with that of p73 . Moreover, p73 has an additional segment at its COOH terminus . Recently, we have found two mutations of p73 with amino acid substitution (P405R and P425L) in primary neuroblastomas . Because the region (amino acid residues 382-491) contains a glutamine- and proline-rich domain, we hypothesized that it has a transactivation function, and the mutations found in tumors result in loss of function . To test it, we used the yeast GAL4 DNA-binding fusion system . Yeast transformants expressing a GAL4-p73(1-112) or a GAL4-p73alpha(380-513) fusion protein were grown in SD medium lacking histidine and tryptophan and exhibited a significant induction of beta-galactosidase activity . Transient transfection experiments revealed that both of fusion proteins could induce the chloramphenicol acetyltransferase activity in mammalian cells, indicating that the COOH-terminal as well as NH2-terminal regions of p73 had significantly high levels of transactivation activity . Furthermore, the former activity was severely impaired in two naturally occurring mutant forms found in neuroblastomas . These suggest that, unlike p53, p73 has two domains with transactivation function, one in the NH2-terminal region and the other in the COOH-terminal region . Loss of function mutation in the latter might be involved in tumorigenesis and/or tumor progression. Cancer Res, 1999 Jun 15, 59(12), 2781 - 6 The transcriptional activities of p53 and its homologue p51/p63: similarities and differences; Shimada A et al.; p51/p63 is a novel p53 homologue that has been shown to act as a transcriptional activator through the p53-binding sequence of the p21/WAF1 promoter and to induce apoptosis when it is expressed transiently in a human tumor cell line . We developed transcription assay systems for these two related genes in both Saccharomyces cerevisiae and mammalian cells and used them to investigate the functional similarities and differences of these genes . We found that p51/p63 trans-activated the previously identified p53 target genes, but the degree of the transactivation by p51/p63 differed from that by p53 . These results suggest that the cellular signal on p51/p63 cross-talks partially but not completely with that of the p53 pathway. Cell Death Differ, 1999 Jun, 6(6), 525 - 32 Survival activity of Bcl-2 homologs Bcl-w and A1 only partially correlates with their ability to bind pro-apoptotic family members; Holmgreen SP et al.; Certain Bcl-2 family members promote cell survival, whereas others promote apoptosis . To explore further how heterodimerization of opposing members affects survival activity, we have compared the abilities of the anti-apoptotic Bcl-w and A1 to bind to the pro-apoptotic Bax, Bak, Bad and Bik and to protect cells from their cytotoxic action . Bcl-w co-immunoprecipitated from cell lysates with Bax, Bak, Bad and Bik, but A1 bound only Bak and Bik . Mutation of A1 at a highly conserved glycine within the BH1 domain prevented binding, but the comparable Bcl-w mutant still bound Bak, Bad and Bik, indicating that the glycine is not essential for all heterodimerization . Bcl-w and A1 protected against apoptosis induced by over-expression of Bax or Bad but not that induced by Bak or Bik . With several gene pairs, binding and protection were discordant . The results may reflect critical threshold affinities but also suggest that certain pro-apoptotic proteins may also contribute to apoptosis by a mechanism independent of binding pro-survival proteins. Cell Death Differ, 1999 May, 6(5), 454 - 62 Etoposide-induced activation of c-jun N-terminal kinase (JNK) correlates with drug-induced apoptosis in salivary gland acinar cells; Anderson SM et al.; We have examined the ability of etoposide to induce apoptosis in two recently established rat salivary acinar cell lines . Etoposide induced apoptosis in the parotid C5 cell line as evidenced by the appearance of cytoplasmic blebbing and nuclear condensation, DNA fragmentation and cleavage of PARP . Etoposide also induced activation of c-jun N-terminal kinase (JNK) in parotid C5 cells by 4 h after treatment, with maximal activation at 8 - 10 h . Coincident with activation of JNK, the amount of activated ERK1 and ERK2 decreased in etoposide-treated parotid C5 cells . In contrast to the parotid C5 cells, the vast majority of submandibular C6 cells appeared to be resistant to etoposide-induced apoptosis . Likewise, activation of JNKs was not observed in etoposide-treated submandibular C6 cells, and the amount of activated ERK1 and ERK2 decreased only slightly . Etoposide treatment of either cell line had no effect upon the activation of p38 . Treatment of the parotid C5 cells with Z-VAD-FMK, a caspase inhibitor, inhibited etoposide-induced activation of JNK and DNA fragmentation . These data suggest that etoposide may induce apoptosis in parotid C5 cells by activating JNKs and suppressing the activation of ERKs, thus creating an imbalance in these two signaling pathways. J Physiol, 1999 Jul 15, 518 ( Pt 2), 325 - 36 Involvement of the n-terminus of Kir6.2 in coupling to the sulphonylurea receptor; Reimann F et al.; 1 . ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits . ATP inhibits the channel by interacting with Kir6.2, while sulphonylureas block channel activity by interaction with a high-affinity site on SUR1 and a low-affinity site on Kir6.2 . MgADP and diazoxide interact with SUR1 to promote channel activity . 2 . We examined the effect of N-terminal deletions of Kir6.2 on the channel open probability, ATP sensitivity and sulphonylurea sensitivity by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2/SUR1 . 3 . A 14 amino acid N-terminal deletion (DeltaN14) did not affect the gating, ATP sensitivity or tolbutamide block of a truncated isoform of Kir6.2, Kir6.2DeltaC26, expressed in the absence of SUR1 . Thus, the N-terminal deletion does not alter the intrinsic properties of Kir6.2 . 4 . When Kir6.2DeltaN14 was coexpressed with SUR1, the resulting KATP channels had a higher open probability (Po = 0.7) and a lower ATP sensitivity (Ki = 196 microM) than wild-type (Kir6.2/SUR1) channels (Po = 0.32, Ki = 28 microM) . High-affinity tolbutamide block was also abolished . 5 . Truncation of five or nine amino acids from the N-terminus of Kir6.2 also enhanced the open probability, and reduced both the ATP sensitivity and the fraction of high-affinity tolbutamide block, although to a lesser extent than for the DeltaN14 deletion . Site-directed mutagenesis suggests that hydrophobic residues in Kir6 . 2 may be involved in this effect . 6 . The reduced ATP sensitivity of Kir6.2DeltaN14 may be explained by the increased Po . However, when the Po was decreased (by ATP), tolbutamide was unable to block Kir6 . 2DeltaN14/SUR1-K719A,K1385M currents, despite the fact that the drug inhibited Kir6.2-C166S/SUR1-K719A,K1385M currents (which in the absence of ATP have a Po of > 0.8 and are not blocked by tolbutamide) . Thus the N-terminus of Kir6.2 may be involved in coupling sulphonylurea binding to SUR1 to closure of the Kir6.2 pore. Brain Res Mol Brain Res, 1999 Jun 18, 70(1), 9 - 17 Differential expression of the small GTP-binding proteins RhoA, RhoB, Cdc42u and Cdc42b in developing rat neocortex; Olenik C et al.; Studies with cultured cells indicate that small GTPases of the Rho family may be involved in cell proliferation, differentiation, as well as migration . Therefore, we have studied the expression of four members of this protein family, i.e., RhoA, RhoB, the ubiquitous Cdc42u, and brain specific Cdc42b, during the embryonic and early postnatal development of rat neocortex . A clear isoform specificity of expression was found during the prenatal development . Thus, RhoA and Cdc42u were present in the proliferation zone while RhoB and Cdc42b were expressed only in the cortical plate where neural cells settle and differentiate . After birth, this isoform specificity quickly disappeared so that already at postnatal day 8 the adult pattern of expression was present . Our findings of a differential expression of the small GTP-binding proteins RhoA, RhoB, Cdc42u and Cdc42b in developing brain neocortex suggest isoform specific functions during neurogenesis and differentiation . Biochem Biophys Res Commun, 1999 Jun 24, 260(1), 193 - 8 Phosphorylation of the cyclosome is required for its stimulation by Fizzy/cdc20; Shteinberg M et al.; Exit from mitosis in eukaryotic cells is regulated by the cyclosome (also called anaphase promoting complex or APC), a multisubunit ubiquitin ligase that acts on mitotic cyclins . Previous studies in a cell-free system from clam oocytes have shown that the activation of the cyclosome at the end of mitosis involves its phosphorylation by protein kinase Cdk1/cyclin B . Genetic and biochemical studies have furthermore indicated that cyclosome activity also requires a WD-40 repeat containing protein called Fizzy (FZY) or Cdc20 . It has been suggested {Fang et al . (1998) Mol . Cell 2, 163-171} that in the presence of FZY, the phosphorylation of the cyclosome is not critical for its activation . By contrast, we find that the activity of the interphase, non-phosphorylated form of the cyclosome from clam embryos is not stimulated by FZY to a significant extent . However, when interphase cyclosome is first incubated with protein kinase Cdk1/cyclin B, the subsequent supplementation of FZY greatly stimulates its cyclin-ubiquitin ligase activity . Furthermore, phosphatase treatment of purified mitotic cyclosome prevents its stimulation by FZY, a process that can be reversed by the action of protein kinase Cdk1/cyclin B . We conclude that in the early embryonic cell cycles, the primary event in the activation of the cyclosome at the end of mitosis is its Cdk1-dependent phosphorylation and activation by FZY takes place in a subsequent process . Neuroreport, 1999 May 14, 10(7), 1409 - 15 A novel candidate presenilin-1 interacting protein containing tetratricopeptide repeats; Prihar G et al.; The yeast two-hybrid system, immunofluorescence and co-immunoprecipitation techniques were used to identify a novel candidate protein with which presenilin-1 (PS-1) interacts . This interacting protein, the gene of which is encoded on chromosome 16, contains two tetratricopeptide repeats (TPR) that are known to mediate interactions between proteins, appears to be primarily localized to the cytoplasm of transfected HEK293 cells, and is expressed in brain . Preliminary yeast two-hybrid data suggests this candidate may interact with both heat shock protein-90 and heat shock protein-70 and thus may be a novel member of TPR-containing proteins which interact with this complex. Biosci Biotechnol Biochem, 1999 May, 63(5), 820 - 6 Molecular cloning and expression of the gene encoding a phospholipase A1 from Aspergillus oryzae; Watanabe I et al.; Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes removal of the acyl group from position 1 of lecithin to form lysolecithin . The genomic DNA and cDNA encoding PLA1 from Aspergillus oryzae were cloned with the mixed deoxyribonucleotide-primed polymerase chain reaction . The PLA1 gene is composed of 1,056 bp and has four exons and three short introns (63, 54, and 51 bp) . The deduced amino acid sequence of PLA1 contained the N-terminal sequence of the mature PLA1 analyzed by Edman degradation . PLA1 cDNA has an open reading frame of 885 bp encoding the PLA1 precursor of 295 amino acid residues . The mature PLA1 is composed of 269 amino acid residues, and a prepro-sequence of 26 amino acid residues is at the N-terminal region of the PLA1 precursor . PLA1 has two possible N-glycosylation sites (Asn27 and Asn55) . PLA1 has a consensus pentapeptide (-Gly-His-Ser-Xaa-Gly-), which is conserved in lipases . The amino acid sequence of PLA1 showed 47% identity with that of mono- and diacylglycerol lipase from Penicillium camembertii . The PLA1 cDNA was expressed in Saccharomyces cerevisiae KS58-2D, indicating the cloned gene to be functional. Pac Symp Biocomput . 1999;:578-89. The hydrophilic, protease-sensitive terminal domains of eucaryotic DNA topoisomerases have essential intracellular functions; Shaiu WL et al.; The amino-terminus of eucaryotic DNA topoisomerase I and the carboxy-terminus of eucaryotic DNA topoisomerase II contain sequences that are enriched in charged amino acid residues, hyper-sensitive to protease digestion, not required for the in vitro topoisomerase activities, able to tolerate insertion and deletion mutations, and thus may have a disordered structure . In an interesting contrast to the catalytically essential core domain, the sequences in these terminal hydrophilic domains are not conserved among the topoisomerases from different species . However, many lines of evidence, including those presented here, demonstrate that the topoisomerase tail domains have critical intracellular functions . The biological functions of the amino-terminus of topoisomerase I include the nuclear import and targeting to the transcriptionally active loci . The carboxy-terminus of topoisomerase II also contains the sequences necessary for nuclear localization and possibly sequences necessary for other critical functions. Pac Symp Biocomput . 1999;:138-49. A comparative analysis of computational motif-detection methods; Hudak J et al.; The detection of motifs within and among families of protein sequences can provide useful information regarding the function, structure and evolution of a protein . With the increasing number of computer programs available for motif detection, a comparative evaluation of the programs from a biological perspective is warranted . This study uses a set of 20 reverse transcriptase (RT) protein sequences to test and compare the ability of 7 different computational methods to locate the ordered-series-of-motifs that are well characterized in the RT sequences . The results provide insight to biologists as to the usage, value, and reliability of the numerous methods available. Bioelectrochem Bioenerg, 1999 May, 48(2), 261 - 6 Microelectronic sensors for measurement of electromagnetic fields of living cells and experimental results; Jelinek F et al.; Microelectronic sensors are used for measurements of electromagnetic fields generated by synchronized cultures of yeast cells . Cold sensitive mutant tub2-401 of Saccharomyces cerevisiae is used . The measured electromagnetic signals in the frequency range from 8 to 9 MHz are compared with evolution of the reassembled microtubules . The detected signals peak in the time interval 25-30 min and 45-60 min after the release of the cells from the restrictive to the permissive temperature . The first maximum corresponds to the stage when the mitotic spindle is formed and binds chromatids . The second maximum is measured when the processes of anaphase A and of anaphase B take place. Mutat Res, 1999 May 14, 434(1), 3 - 15 Ku, a DNA repair protein with multiple cellular functions? Featherstone C, Jackson SP. The Ku protein binds to DNA ends and other types of discontinuity in double-stranded DNA . It is a tightly associated heterodimer of approximately 70 kDa and approximately 80 kDa subunits that together with the approximately 470 kDa catalytic subunit, DNA-PKcs, form the DNA-dependent protein kinase . This enzyme is involved in repairing DNA double-strand breaks (DSBs) caused, for example, by physiological oxidation reactions, V(D)J recombination, ionizing radiation and certain chemotherapeutic drugs . The Ku-dependent repair process, called illegitimate recombination or nonhomologous end joining (NHEJ), appears to be the main DNA DSB repair mechanism in mammalian cells . Ku itself is probably involved in stabilizing broken DNA ends, bringing them together and preparing them for ligation . Ku also recruits DNA-PKcs to the DSB, activating its kinase function . Targeted disruption of the genes encoding Ku70 and Ku80 has identified significant differences between Ku-deficient mice and DNA-PKcs-deficient mice . Although all three gene products are clearly involved in repairing ionizing radiation-induced damage and in V(D)J recombination, Ku-knockout mice are small, and their cells fail to proliferate in culture and show signs of premature senescence . Recent findings have implicated yeast Ku in telomeric structure in addition to NHEJ . Some of the phenotypes of the Ku-knockout mice may indicate a similar role for Ku at mammalian telomeres. Proc Natl Acad Sci U S A, 1999 Jun 22, 96(13), 7415 - 20 Efficient export of the glucose transporter Hxt1p from the endoplasmic reticulum requires Gsf2p; Sherwood PW et al.; Mutations in the GSF2 gene cause glucose starvation phenotypes in Saccharomyces cerevisiae . We have isolated the HXT1 gene, which encodes a low-affinity, high-capacity glucose transporter, as a multicopy suppressor of a gsf2 mutation . We show that gsf2 mutants accumulate Hxt1p in the endoplasmic reticulum (ER) and that Gsf2p is a 46-kDa integral membrane protein localized to the ER . gsf2 mutants also display a galactose growth defect and abnormal localization of the galactose transporter Gal2p but are not defective in function or localization of the high-affinity glucose transporter Hxt2p . These findings suggest that Gsf2p functions in the ER to promote the secretion of certain hexose transporters. Proc Natl Acad Sci U S A, 1999 Jun 22, 96(13), 7376 - 81 Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation; Luo G et al.; The Mre11/Rad50 protein complex functions in diverse aspects of the cellular response to double-strand breaks (DSBs), including the detection of DNA damage, the activation of cell cycle checkpoints, and DSB repair . Whereas genetic analyses in Saccharomyces cerevisiae have provided insight regarding DSB repair functions of this highly conserved complex, the implication of the human complex in Nijmegen breakage syndrome reveals its role in cell cycle checkpoint functions . We established mRad50 mutant mice to examine the role of the mammalian Mre11/Rad50 protein complex in the DNA damage response . Early embryonic cells deficient in mRad50 are hypersensitive to ionizing radiation, consistent with a role for this complex in the repair of ionizing radiation-induced DSBs . However, the null mrad50 mutation is lethal in cultured embryonic stem cells and in early developing embryos, indicating that the mammalian Mre11/Rad50 protein complex mediates functions in normally growing cells that are essential for viability. Proc Natl Acad Sci U S A, 1999 Jun 22, 96(13), 7306 - 11 Cdc20 associates with the kinase aurora2/Aik; Farruggio DC et al.; Cdc20/fizzy family proteins are involved in activation of the anaphase-promoting complex/cyclosome, which catalyzes the ubiquitin-dependent proteolysis of cell cycle regulatory proteins such as anaphase inhibitors and mitotic cyclins, leading to chromosome segregation and exit from mitosis . Previous work has shown that human Cdc20 (hCdc20/p55CDC) associates with one or more kinases . We report here that Cdc20-associated myelin basic protein kinase activity peaks sharply in early M phase (embryonic cells) or in G2 phase (somatic cells) . In HeLa cells, Cdc20 is associated with the kinase aurora2/Aik . Aurora2/Aik is a member of the aurora/Ipl1 family of kinases that, like Cdc20, previously has been shown to be localized at mitotic spindle poles and is involved in regulating chromosome segregation and maintaining genomic stability . The demonstration that Cdc20 is associated with aurora2/Aik suggests that some function of Cdc20 is carried out or regulated through its association with aurora2/Aik. Nature, 1999 Jun 10, 399(6736), 609 - 13 Binding of TBP to promoters in vivo is stimulated by activators and requires Pol II holoenzyme; Kuras L et al.; In eukaryotes, transcriptional activators have been proposed to function by recruiting the RNA polymerase II (Pol II) machinery, by altering the conformation of this machinery, or by affecting steps after initiation, but the evidence is not definitive . Genomic footprinting of yeast TATA-box elements reveals activator-dependent alterations of chromatin structure and activator-independent protection, but little is known about the association of specific components of the Pol II machinery with promoters in vivo . Here we analyse TATA-box-binding-protein (TBP) occupancy of 30 yeast promoters in vivo . We find that TBP association with promoters is stimulated by activators and inhibited by the Cyc8-Tup1 repressor, and that transcriptional activity correlates strongly with the degree of TBP occupancy . In a small subset of promoters, TBP occupancy is higher than expected when gene activity is low, and the activator-dependent increase is modest . TBP association depends on the Pol II holoenzyme component Srb4, but not on the Kin28 subunit of the transcription factor TFIIH, even though both proteins are generally required for transcription . Thus in yeast cells, TBP association with promoters occurs in concert with the Pol II holoenzyme, activator-dependent recruitment of the Pol II machinery occurs at the vast majority of promoters, and Kin28 acts after the initial recruitment. Nature, 1999 Jun 10, 399(6736), 605 - 9 Enhancement of TBP binding by activators and general transcription factors; Li XY et al.; Eukaryotic transcriptional activators function, at least in part, by promoting assembly of the preinitiation complex, which comprises RNA polymerase II and its general transcription factors (GTFs) . Activator-mediated stimulation of the assembly of the preinitiation complex has been studied in vitro but has been relatively refractory to in vivo analysis . Here we use a DNA-crosslinking/immunoprecipitation assay to study in living cells the first step in the assembly of the preinitiation complex, the interaction between the TATA-box-binding protein (TBP) and its binding site, the TATA box . Analysis of a variety of endogenous yeast genes, and of a series of activators of differing strength, reveals a general correlation between TBP binding and transcriptional activity . Using mutant yeast strains, we show that Mot1 prevents the binding of TBP to inactive promoters and that activator-mediated stimulation of TBP binding requires additional GTFs, including TFIIB and Srb4 . Taken together, our results indicate that TBP binding in vivo is stringently controlled, and that the ability of activators to stimulate this step in the assembly of the preinitiation complex is a highly cooperative process involving multiple transcription factors. RNA, 1999 Jun, 5(6), 819 - 31 Transient interaction of BBP/ScSF1 and Mud2 with the splicing machinery affects the kinetics of spliceosome assembly; Rutz B et al.; Removal of introns from pre-mRNA is an essential step of gene expression . The splicing reaction is catalyzed in a large complex termed the spliceosome . Introns are recognized during the early steps of spliceosome assembly with the formation of commitment complexes . Intron recognition is mediated by the interaction of splicing factors with conserved sequences present in the pre-mRNA . BBP/SF1 participates in this recognition by interacting with the pre-mRNA branch point in both yeast and mammals . This protein, which is essential in yeast, also interacts with the U2AF65/Mud2 splicing factor . However, its precise role in splicing complex formation is still unclear . We have now analyzed the presence of BBP and Mud2 in yeast splicing complexes using supershift and coprecipitation assays . We found that BBP is present together with Mud2 in commitment complex 2 (CC2), but is not detectable in commitment complex 1 (CC1) . Furthermore, genetic and biochemical depletion of BBP demonstrated that it is required for CC2 formation . In addition we observed that BBP and Mud2 are not detectable in pre-spliceosomes . These are the first commitment complex components that are shown to be released during or immediately after pre-spliceosome formation . Interestingly, depletion of BBP or disruption of MUD2 had no significant effect on pre-spliceosome formation and splicing in vitro but led to a transient accumulation of CC1 . These observations support a model in which BBP and Mud2 are recycled during transition from CC2 to pre-spliceosome. RNA, 1999 Jun, 5(6), 794 - 804 Mutations in the MOF2/SUI1 gene affect both translation and nonsense-mediated mRNA decay; Cui Y et al.; Recent studies have demonstrated that cells have evolved elaborate mechanisms to rid themselves of aberrant proteins and transcripts . The nonsense-mediated mRNA decay pathway (NMD) is an example of a pathway that eliminates aberrant mRNAs . In yeast, a transcript is recognized as aberrant and is rapidly degraded if a specific sequence, called the DSE, is present 3' of a premature termination codon . Results presented here show that strains harboring the mof2-1, mof4-1, mof5-1, and mof8-1 alleles, previously demonstrated to increase the efficiency of programmed -1 ribosomal frameshifting, decrease the activity of the NMD pathway . The effect of the mof2-1 allele on NMD was characterized in more detail . Previous results demonstrated that the wild-type MOF2 gene is identical to the SUI1 gene . Studies on the mof2-1 allele of the SUI1 gene indicate that in addition to its role in recognition of the AUG codon during translation initiation and maintenance of the appropriate reading frame during translation elongation, the Mof2 protein plays a role in the NMD pathway . The Mof2p/Sui1 p is conserved throughout nature and the human homolog of the Mof2p/Sui1p functions in yeast cells to activate NMD . These results suggest that factors involved in NMD are general modulators that act in several aspects of translation and mRNA turnover. RNA, 1999 Jun, 5(6), 711 - 9 mRNA surveillance in eukaryotes: kinetic proofreading of proper translation termination as assessed by mRNP domain organization? Hilleren P, Parker R. In the last few years it has become clear that a conserved mRNA degradation system, referred to as mRNA surveillance, exists in eukaryotic cells to degrade aberrant mRNAs . This process plays an important role in checking that mRNAs have been properly synthesized and functions, at least in part, to increase the fidelity of gene expression by degrading aberrant mRNAs that, if translated, would produce truncated proteins . A critical issue is how normal and aberrant mRNAs are distinguished and how that distinction leads to differences in mRNA stability . Recent results suggest a model with three main points . First, mRNPs have a domain organization that is, in part, a reflection of the completion of nuclear pre-mRNA processing events . Second, the critical aspect of distinguishing a normal from an aberrant mRNA is the environment of the translation termination codon as determined by the organization of the mRNP domains . Third, the cell distinguishes proper from improper termination through an internal clock that is the rate of ATP hydrolysis by Upf1p . If termination is completed before ATP hydrolysis, the mRNA is protected from mRNA degradation . Conversely, if termination is slow, then ATP hydrolysis and a structural rearrangement occurs before termination is completed, which affects the fate of the terminating ribosome in a manner that fails to stabilize the mRNA . This proposed system of distinguishing normal from aberrant transcripts is similar to, but distinct from other systems of kinetic proofreading that affect the accuracy of other biogenic processes such as translation accuracy and spliceosome assembly. Nucleic Acids Res, 1999 Jul 1, 27(13), 2578 - 84 Dissection of the NF-Y transcriptional activation potential; de Silvio A et al.; NF-Y is a trimeric CCAAT-binding factor with histone fold subunits (NF-YB/NF-YC) and bipartite activation domains located on NF-YA and NF-YC . We reconstituted the NF-Y activation potential in vivo with GAL4 DBD fusions . In the GAL4-YA configuration, activation requires co-expression of the three subunits; with GAL4-YB and GAL4-YC, transfections of the histone fold partners are sufficient, provided that the Q-rich domain of NF-YC is present . Combinations of mutants indicate that the Q-rich domains of NF-YA and NF-YC are redundant in the trimeric complex . Glutamines 101 and 102 of NF-YA are required for activity . We assayed NF-Y on different promoter targets, containing single or multiple GAL4 sites: whereas on a single site NF-Y is nearly as powerful as VP16, on multiple sites neither synergistic nor additive effects are observed . NF-Y activates TATA and Inr core elements and the overall potency is in the same range as other Q-rich and Pro-rich activation domains . These results represent the first in vivo evidence of subunit interactions studies and further support the hypothesis that NF-Y is a general promoter organizer rather than a brute activator. Mol Cell Biol, 1999 Jul, 19(7), 5179 - 88 The disabled 1 phosphotyrosine-binding domain binds to the internalization signals of transmembrane glycoproteins and to phospholipids; Howell BW et al.; Disabled gene products are important for nervous system development in drosophila and mammals . In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning . The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions . Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship . Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons . Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 microM affinity . Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands . The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding . We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal. Mol Cell Biol, 1999 Jul, 19(7), 5166 - 9 Sister chromatid exchanges are mediated by homologous recombination in vertebrate cells; Sonoda E et al.; Sister chromatid exchange (SCE) frequency is a commonly used index of chromosomal stability in response to environmental or genetic mutagens . However, the mechanism generating cytologically detectable SCEs and, therefore, their prognostic value for chromosomal stability in mitotic cells remain unclear . We examined the role of the highly conserved homologous recombination (HR) pathway in SCE by measuring SCE levels in HR-defective vertebrate cells . Spontaneous and mitomycin C-induced SCE levels were significantly reduced for chicken DT40 B cells lacking the key HR genes RAD51 and RAD54 but not for nonhomologous DNA end-joining (NHEJ)-defective KU70(-/-) cells . As measured by targeted integration efficiency, reconstitution of HR activity by expression of a human RAD51 transgene restored SCE levels to normal, confirming that HR is the mechanism responsible for SCE . Our findings show that HR uses the nascent sister chromatid to repair potentially lethal DNA lesions accompanying replication, which might explain the lethality or tumorigenic potential associated with defects in HR or HR-associated proteins. Mol Cell Biol, 1999 Jul, 19(7), 4888 - 96 Cell cycle control of Cdc7p kinase activity through regulation of Dbf4p stability; Oshiro G et al.; In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase . We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate . Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis . These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle . However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p . The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G1 cells . This G1-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p . Overexpression of Dbf4p in the G1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity . Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle. Mol Cell Biol, 1999 Jul, 19(7), 4582 - 91 Dominant negative murine serum response factor: alternative splicing within the activation domain inhibits transactivation of serum response factor binding targets; Belaguli NS et al.; Primary transcripts encoding the MADS box superfamily of proteins, such as MEF2 in animals and ZEMa in plants, are alternatively spliced, producing several isoformic species . We show here that murine serum response factor (SRF) primary RNA transcripts are alternatively spliced at the fifth exon, deleting approximately one-third of the C-terminal activation domain . Among the different muscle types examined, visceral smooth muscles have a very low ratio of SRFDelta5 to SRF . Increased levels of SRFDelta5 correlates well with reduced smooth muscle contractile gene activity within the elastic aortic arch, suggesting important biological roles for differential expression of SRFDelta5 variant relative to wild-type SRF . SRFDelta5 forms DNA binding-competent homodimers and heterodimers . SRFDelta5 acts as a naturally occurring dominant negative regulatory mutant that blocks SRF-dependent skeletal alpha-actin, cardiac alpha-actin, smooth alpha-actin, SM22alpha, and SRF promoter-luciferase reporter activities . Expression of SRFDelta5 interferes with differentiation of myogenic C2C12 cells and the appearance of skeletal alpha-actin and myogenin mRNAs . SRFDelta5 repressed the serum-induced activity of the c-fos serum response element . SRFDelta5 fused to the yeast Gal4 DNA binding domain displayed low transcriptional activity, which was complemented by overexpression of the coactivator ATF6 . These results indicate that the absence of exon 5 might be bypassed through recruitment of transcription factors that interact with extra-exon 5 regions in the transcriptional activating domain . The novel alternatively spliced isoform of SRF, SRFDelta5, may play an important regulatory role in modulating SRF-dependent gene expression. J Biol Chem, 1999 Jun 25, 274(26), 18543 - 51 Identification of a novel region critical for calcineurin function in vivo and in vitro; Jiang B et al.; Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase that plays critical functional roles in T-cell activation and other Ca2+-mediated signal transduction pathways in mammalian cells . In Saccharomyces cerevisiae, calcineurin regulates the transcription of several genes involved in maintaining ion homeostasis (PMC1, PMR1, and PMR2) and cell wall synthesis (FKS2) . In this paper, we report the identification and characterization of 11 single amino acid substitutions in the yeast calcineurin catalytic subunit Cna1p . We show that six substitutions (R177G, F211S, S232F, D258V, L259P, and A262P) affect the stability of calcineurin and that two substitutions (V385D and M400R) disrupt the interaction between Cna1p and the calcineurin regulatory subunit Cnb1p . We also identify three mutations (S373P, H375L, and L379S) that are clustered between the catalytic and the calcineurin B subunit-binding domains . These mutations do not significantly affect the ability of Cna1p to interact with Cnb1p, calmodulin, or Fkb1p (FK506-binding protein) . However, these residue substitutions dramatically affect calcineurin activity both in vitro and in vivo . Thus, by using a random mutagenesis approach, we have shown for the first time that the linker region of the calcineurin catalytic subunit, as defined by the Ser373, His375, and Leu379 residues, is crucial for its function as a phosphatase. J Biol Chem, 1999 Jun 25, 274(26), 18285 - 9 Identification of TATA-binding protein-free TAFII-containing complex subunits suggests a role in nucleosome acetylation and signal transduction; Brand M et al.; Recently we identified a novel human (h) multiprotein complex, called TATA-binding protein (TBP)-free TAFII-containing complex (TFTC), which is able to nucleate RNA polymerase II transcription and can mediate transcriptional activation . Here we demonstrate that TFTC, similar to other TBP-free TAFII complexes (yeast SAGA, hSTAGA, and hPCAF) contains the acetyltransferase hGCN5 and is able to acetylate histones in both a free and a nucleosomal context . The recently described TRRAP cofactor for oncogenic transcription factor pathways was also characterized as a TFTC subunit . Furthermore, we identified four other previously uncharacterized subunits of TFTC: hADA3, hTAFII150, hSPT3, and hPAF65beta . Thus, the polypeptide composition of TFTC suggests that TFTC is recruited to chromatin templates by activators to acetylate histones and thus may potentiate initiation and activation of transcription. J Biol Chem, 1999 Jun 25, 274(26), 18157 - 60 Catalytic mechanism and function of invariant glutamic acid 173 from the histone acetyltransferase GCN5 transcriptional coactivator; Tanner KG et al.; Within chromatin, reversible acetylation of core histones is critical for transcriptional activation of eukaryotic target genes . The recent identification of intrinsic histone acetyltransferase (HAT) catalytic activity from a number of transcriptional co-activators (including yeast GCN5, p300/CBP, P/CAF, and TAFII250), has underscored the importance of protein acetylation in transcriptional control . The GCN5 family is the prototype for a diverse group of at least four distinct human HATs families . Although there is now a clear link between in vivo HAT catalytic activity and gene activation, little is known about the molecular mechanisms of histone acetylation . Herein, we report the first detailed biochemical study that probes the catalytic mechanism and the function of invariant glutamic acid 173 within the GCN5 family of HATs . Our results suggest that the HAT reaction involves the formation of a ternary complex (histones, acetyl-CoA, and enzyme) where the epsilon-amino group of histone lysine residues directly attacks the bound acetyl-CoA . The acetylation reaction requires deprotonation of the epsilon-amino group prior to nucleophilic attack . Employing site-directed mutagenesis, chemical modification, steady-state, and pH-dependent rate analysis, it is demonstrated that glutamic acid 173 is an essential catalytic residue, acting as a general base catalyst by deprotonating the histone substrate. J Mol Biol, 1999 Jun 25, 289(5), 1327 - 41 Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex; Parkhurst KM et al.; Binding of the TATA-binding protein (TBP) to the "TATA" sequences present in the promoters of eukaryotic class II genes is the first step in the sequential assembly of transcription pre-initiation complexes . Myriad structural changes, including severe bending of the DNA, accompany TBP-TATA complex formation . A detailed kinetic study has been conducted to elucidate the mechanistic details of TBP binding and DNA bending . The binding of Saccharomyces cerevisiae TBP to the adenovirus major late promoter (AdMLP) was followed in real-time through a range of temperatures and TBP concentrations using fluorescence resonance energy transfer (FRET) and stopped-flow mixing . The results of association and relaxation kinetics and equilibrium binding experiments were analyzed globally to obtain the complete kinetic and energetic profile of the reaction . This analysis reveals a complex mechanism with two intermediate species, with the DNA in the intermediates apparently bent similarly to the DNA in the final complex . TBP binding and DNA bending occur simultaneously through the multiple steps of the reaction . The first and third steps in this sequential process show nearly identical large increases in both enthalpy and entropy, whereas the middle step is highly exothermic and proceeds with a large decrease in entropy . The first intermediate is significantly populated at equilibrium and resembles the final complex both structurally and energetically . It is postulated that both this intermediate and the final complex bind transcription factor IIB in the second step of pol II pre-initiation complex assembly . A consequence of such a reactive intermediate is that the rate of assembly of transcriptionally competent pre-initiation complexes from bi-directionally bound TBP is greatly increased . Genomics, 1999 Jun 15, 58(3), 270 - 80 Identification and chromosomal location of two human genes encoding enzymes potentially involved in proteolytic maturation of farnesylated proteins; Freije JM et al.; Two human cDNAs encoding proteins similar to yeast enzymes involved in proteolytic processing of farnesylated proteins like a-factor mating pheromone and Ras2p have been cloned from an ovary cDNA library . These proteins have been tentatively called Face-1 and Face-2 (farnesylated protein-converting enzymes 1 and 2), respectively, and are integral membrane proteins, belonging to distinct families of metalloproteinases . Northern blot analysis of poly(A)+ RNAs isolated from a wide variety of human tissues demonstrated that both genes are expressed in all examined tissues, which suggests that these enzymes play housekeeping roles in normal processes . Fluorescence in situ hybridization experiments showed that the human FACE-1 gene maps to 1p34, whereas FACE-2 is located at 11q13, a region frequently amplified in human carcinomas and lymphomas . On the basis of these results, we suggest that inhibition of Face-1 and/or Face-2 could be part of strategies directed to block the functioning of prenylated proteins activated in oncogenic processes, including Ras proteins . Science, 1999 Jun 18, 284(5422), 1985 - 8 Preventing neurodegeneration in the Drosophila mutant bubblegum; Min KT et al.; The Drosophila melanogaster recessive mutant bubblegum (bgm) exhibits adult neurodegeneration, with marked dilation of photoreceptor axons . The bubblegum mutant shows elevated levels of very long chain fatty acids (VLCFAs), as seen in the human disease adrenoleukodystrophy (ALD) . In ALD, the excess can be lowered by dietary treatment with "Lorenzo's oil," a mixture of unsaturated fatty acids . Feeding the fly mutant one of the components, glyceryl trioleate oil, blocked the accumulation of excess VLCFAs as well as development of the pathology . Mutant flies thus provide a potential model system for studying mechanisms of neurodegenerative disease and screening drugs for treatment. Methods Enzymol, 1999, 304, 76 - 99 Preparation/analysis of chromatin replicated in vivo and in isolated nuclei; Chang L et al.; This article outlined biochemical methodologies for the labeling, detection, and analysis of newly replicated and newly assembled nucleosomes . The isolation of specific vertebrate factors that may be involved in chromatin assembly in vivo, such as nucleoplasmin, CAF-1, and NAP-1 and their counterparts in Drosophila and yeast add a further dimension to the study of nucleosome assembly in living cells . In particular, the ability to genetically manipulate the yeast system, together with the identification of yeast enzymes that acetylate newly synthesized H4, will certainly provide exciting new avenues for the investigation of chromatin assembly in vivo. FEBS Lett, 1999 May 28, 451(3), 220 - 6 Characterisation of a plant 3-phosphoinositide-dependent protein kinase-1 homologue which contains a pleckstrin homology domain; Deak M et al.; A plant homologue of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1) has been identified in Arabidopsis and rice which displays 40% overall identity with human 3-phosphoinositide-dependent protein kinase-1 . Like the mammalian 3-phosphoinositide-dependent protein kinase-1, Arabidopsis 3-phosphoinositide-dependent protein kinase-1 and rice 3-phosphoinositide-dependent protein kinase-1 possess a kinase domain at N-termini and a pleckstrin homology domain at their C-termini . Arabidopsis 3-phosphoinositide-dependent protein kinase-1 can rescue lethality in Saccharomyces cerevisiae caused by disruption of the genes encoding yeast 3-phosphoinositide-dependent protein kinase-1 homologues . Arabidopsis 3-phosphoinositide-dependent protein kinase-1 interacts via its pleckstrin homology domain with phosphatidic acid, PtdIns3P, PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and to a lesser extent with PtdIns(4,5)P2 and PtdIns4P . Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is able to activate human protein kinase B alpha (PKB/AKT) in the presence of PtdIns(3,4,5)P3 . Arabidopsis 3-phosphoinositide-dependent protein kinase-1 is only the second plant protein reported to possess a pleckstrin homology domain and the first plant protein shown to bind 3-phosphoinositides. FEMS Microbiol Rev, 1999 Jun, 23(3), 277 - 95 3'-End processing of pre-mRNA in eukaryotes; Wahle E et al.; 3'-Ends of almost all eukaryotic mRNAs are generated by endonucleolytic cleavage and addition of a poly(A) tail . In mammalian cells, the reaction depends on the sequence AAUAAA upstream of the cleavage site, a degenerate GU-rich sequence element downstream of the cleavage site and stimulatory sequences upstream of AAUAAA . Six factors have been identified that carry out the two reactions . With a single exception, they have been purified to homogeneity and cDNAs for 11 subunits have been cloned . Some of the cooperative RNA-protein and protein-protein interactions within the processing complex have been analyzed, but many details, including the identity of the endonuclease, remain unknown . Several examples of regulated polyadenylation are being analyzed at the molecular level . In the yeast Saccharomyces cerevisiae, sequences directing cleavage and polyadenylation are more degenerate than in metazoans, and a downstream element has not been identified . The list of processing factors may be complete now with approximately a dozen polypeptides, but their functions in the reaction are largely unknown . 3'-Processing is known to be coupled to transcription . This connection is thought to involve interactions of processing factors with the mRNA cap as well as with RNA polymerase II. Trends Cell Biol, 1999 Jul, 9(7), 249 - 52 First the CDKs, now the DDKs; Johnston LH et al.; In budding yeast, Dbf4p and Cdc7p control initiation of DNA synthesis . They form a protein kinase - Cdc7p being the catalytic subunit and Dbf4p a cyclin-like molecule that activates the kinase in late G1 phase . Dbf4p also targets Cdc7p to origins of replication, where probable substrates include certain Mcm proteins . Recent studies have identified Dbf4p- and Cdc7p-related proteins in fission yeast and metazoans . These homologues also phosphorylate Mcm proteins and could have a similar function to that of Dbf4p-Cdc7p in budding yeast . Thus, it seems likely that, like the cyclin-dependent kinases (CDKs), the Dbf4p-Cdc7p activity is conserved in all eukaryotes. Arch Microbiol, 1999 May-Jun, 171(6), 386 - 96 The acuH gene of Aspergillus nidulans, required for growth on acetate and long-chain fatty acids, encodes a putative homologue of the mammalian carnitine/acylcarnitine carrier; De Lucas JR et al.; The Aspergillus nidulans acuH gene, required for growth on acetate and long-chain fatty acids, was cloned by complementation of the acuH13 mutation . Northern blotting analysis showed that transcription of the acuH gene occurs in acetate-grown mycelium and at higher levels in oleate-grown mycelium, but not during growth on glucose minimal medium . The acuH gene encodes a protein of 326 amino acids that belongs to the mitochondrial carrier family . The ACUH protein contains three related segments of approximately 100 amino acids in length, each segment comprising two hydrophobic domains that are probably folded into two transmembrane alpha-helices linked by an extensive polar region . Sequence comparisons suggest that the acuH gene of A . nidulans encodes the homologue of the carnitine/acylcarnitine carrier of rat and man . The uncharacterised proteins YOR100C of Saccharomyces cerevisiae, COLT of Drosophila melanogaster, and DIF-1 of Caenorhabditis elegans also seem to be homologues of ACUH . In addition to the motifs present in all members of the mitochondrial carrier family, we propose the highly conserved motif R(A,S)(V,F)PANAA(T,C)F within the sixth hydrophobic domain of these proteins as the characteristic feature of the carnitine carrier subfamily . The proposed function of the ACUH protein is the transport of acetylcarnitine molecules from the cytosol to the mitochondrial matrix, a process required during growth on acetate or on long-chain fatty acids. Hum Mol Genet, 1999 Jul, 8(7), 1263 - 9 Presenilins interact with Rab11, a small GTPase involved in the regulation of vesicular transport; Dumanchin C et al.; Presenilin 1 (PS1) mutations account for the majority of early-onset dominant cases of familial Alzheimer's disease . Presenilins (PSs) are located in many intra-cellular compartments such as the endoplasmic reticulum, Golgi apparatus, nuclear region and vesicular structures . These proteins include from seven to nine putative transmembrane domains, with the N- and C-terminal ends and a large hydrophilic loop orientated towards the cytoplasm . We report an interaction between the human PS1 or PS2 hydrophilic loop and Rab11, a small GTPase belonging to the Ras-related superfamily . Interaction domains were mapped to codons 374-400 for PS1 and to codons 106-179 for Rab11, a region including the fourth GTP-binding domain . Considering the implication of Rab proteins in vesicular transport pathways, the PS-Rab11 inter-action suggests that PSs might be involved in amyloid precursor protein vesicular routing. Nat Genet, 1999 Jun, 22(2), 151 - 8 Hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial ornithine transporter; Camacho JA et al.; Neurospora crassa ARG13 and Saccharomyces cerevisiae ARG11 encode mitochondrial carrier family (MCF) proteins that transport ornithine across the mitochondrial inner membrane . We used their sequences to identify EST candidates that partially encode orthologous mammalian transporters . We thereby identified such a gene (ORNT1) that maps to 13q14 and whose expression, similar to that of other urea cycle (UC) components, was high in liver and varied with changes in dietary protein . ORNT1 expression restores ornithine metabolism in fibroblasts from patients with hyperammonaemia-hyperornithinaemia-homocitrullinuria (HHH) syndrome . In a survey of 11 HHH probands, we identified 3 ORNT1 mutant alleles that account for 21 of 22 possible mutant ORNT1 genes in our patients: F188delta, which is common in French-Canadian HHH patients and encodes an unstable protein; E180K, which encodes a stable, properly targeted protein that is inactive; and a 13q14 microdeletion . Our results show that ORNT1 encodes the mitochondrial ornithine transporter involved in UC function and is defective in HHH syndrome. EMBO J, 1999 Jun 15, 18(12), 3509 - 19 Progressive cis-inhibition of telomerase upon telomere elongation; Marcand S et al.; In yeast, the constant length of telomeric DNA results from a negative regulation of telomerase by the telomere itself . Here we follow the return to equilibrium of an abnormally shortened telomere . We observe that telomere elongation is restricted to a few base pairs per generation and that its rate decreases progressively with increasing telomere length . In contrast, in the absence of telomerase or in the presence of an over-elongated telomere, the degradation rate linked to the succession of generations appears to be constant, i.e . independent of telomere length . Together, these results indicate that telomerase is gradually inhibited at its site of action by the elongating telomere . The implications of this finding for the dynamics of telomere length regulation are discussed in this study. EMBO J, 1999 Jun 15, 18(12), 3451 - 62 Sm and Sm-like proteins assemble in two related complexes of deep evolutionary origin; Salgado-Garrido J et al.; A group of seven Sm proteins forms a complex that binds to several RNAs in metazoans . All Sm proteins contain a sequence signature, the Sm domain, also found in two yeast Sm-like proteins associated with the U6 snRNA . We have performed database searches revealing the presence of 16 proteins carrying an Sm domain in the yeast genome . Analysis of this protein family confirmed that seven of its members, encoded by essential genes, are homologues of metazoan Sm proteins . Immunoprecipitation revealed that an evolutionarily related subgroup of seven Sm-like proteins is directly associated with the nuclear U6 and pre-RNase P RNAs . The corresponding genes are essential or required for normal vegetative growth . These proteins appear functionally important to stabilize U6 snRNA . The two last yeast Sm-like proteins were not found associated with RNA, and neither was essential for vegetative growth . To investigate whether U6-associated Sm-like protein function is widespread, we cloned several cDNAs encoding homologous human proteins . Two representative human proteins were shown to associate with U6 snRNA-containing complexes . We also identified archaeal proteins related to Sm and Sm-like proteins . Our results demonstrate that Sm and Sm-like proteins assemble in at least two functionally conserved complexes of deep evolutionary origin. EMBO J, 1999 Jun 15, 18(12), 3214 - 21 Transport of the ADP/ATP carrier of mitochondria from the TOM complex to the TIM22.54 complex; Endres M et al.; Members of the mitochondrial carrier family such as the ADP/ATP carrier (AAC) are composed of three structurally related modules . Here we show that each of the modules contains a mitochondrial import signal recognized by Tim10 and Tim12 in the intermembrane space . The first and the second module are translocated across the outer membrane independently of the membrane potential, DeltaDeltapsipsi, but they are not inserted into the inner membrane . The third module interacts tightly with the TOM complex and thereby prevents complete translocation of the precursor across the outer membrane . At this stage, binding of a TIM9.10 complex confers a topology to the translocation intermediate which reflects the modular structure of the AAC . The precursor is then transferred to the TIM9.10.12 complex, still interacting with the TOM complex . Release of the precursor from the TOM complex and insertion into the inner membrane by the TIM22.54 complex requires a DeltaDeltapsipsi-responsive signal in the third module. J Steroid Biochem Mol Biol, 1999 Feb, 68(3-4), 163 - 9 EcR interacts with corepressors and harbours an autonomous silencing domain functional in both Drosophila and vertebrate cells; Thormeyer D et al.; The ecdysone receptor (EcR) is a member of the large family of nuclear hormone receptors, which are ligand regulated transcription factors . In general, ligand converts these receptors into a transcriptional activator . Some vertebrate nuclear hormone receptors, such as the thyroid hormone and retinoic acid receptors, silence gene expression in the absence of ligand . EcR is involved in fly metamorphosis and is used in vertebrates as an inducible system for expression of transgenes . Here, we show that a Drosophila receptor, the EcR, harbours an autonomous silencing function in its carboxy-terminus . Interestingly, EcR mediates also silencing in vertebrate cells . In concordance with this EcR interacts with the corepressors SMRT and N-CoR, while addition of ligand reduces this interaction . Conversely, the v-erbA oncogene product, a thyroid hormone receptor derivative, mediates silencing in Drosophila cells . Thus, our data suggest the involvement of an evolutionarily conserved mechanism by which nuclear hormone receptors mediate gene silencing in multicellular organisms. Cell, 1999 May 28, 97(5), 657 - 66 Crystal structure of a phosphatidylinositol 3-phosphate-specific membrane-targeting motif, the FYVE domain of Vps27p; Misra S et al.; Phosphatidylinositol 3-phosphate regulates membrane trafficking and signaling pathways by interacting with the FYVE domains of target proteins . The 1.15 A structure of the Vps27p FYVE domain reveals two antiparallel beta sheets and an alpha helix stabilized by two Zn2+-binding clusters . The core secondary structures are similar to a rabphilin-3A Zn2+-binding domain and to the C1 and LIM domains . Phosphatidylinositol 3-phosphate binds to a pocket formed by the (R/K)(R/K)HHCR motif . A lattice contact shows how anionic ligands can interact with the phosphatidylinositol 3-phosphate-binding site . The tip of the FYVE domain has basic and hydrophobic surfaces positioned so that nonspecific interactions with the phospholipid bilayer can abet specific binding to phosphatidylinositol 3-phosphate. Cell, 1999 May 28, 97(5), 635 - 46 Structural view of the Ran-Importin beta interaction at 2.3 A resolution; Vetter IR et al.; Transport receptors of the Importin beta family shuttle between the nucleus and cytoplasm and mediate transport of macromolecules through nuclear pore complexes . They interact specifically with the GTP-binding protein Ran, which in turn regulates their interaction with cargo . Here, we report the three-dimensional structure of a complex between Ran bound to the nonhydrolyzable GTP analog GppNHp and a 462-residue fragment from Importin beta . The structure of Importin beta shows 10 tandem repeats resembling HEAT and Armadillo motifs . They form an irregular crescent, the concave site of which forms the interface with Ran-triphosphate . The importin-binding site of Ran does not overlap with that of the Ran-binding domain of RanBP2. Cell, 1999 May 28, 97(5), 609 - 20 MEC1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-strand breaks; Mills KD et al.; The yeast Sir2/3/4p complex is found in abundance at telomeres, where it participates in the formation of silent heterochromatin and telomere maintenance . Here, we show that Sir3p is released from telomeres in response to DNA double-strand breaks (DSBs), binds to DSBs, and mediates their repair, independent of cell mating type . Sir3p relocalization is S phase specific and, importantly, requires the DNA damage checkpoint genes MEC1 and RAD9 . MEC1 is a homolog of ATM, mutations in which cause ataxia telangiectasia (A-T), a disease characterized by various neurologic and immunologic abnormalities, a predisposition for cancer, and a cellular defect in repair of DSBs . This novel mode by which preformed DNA repair machinery is mobilized by DNA damage sensors may have implications for human diseases resulting from defective DSB repair. Cell, 1999 May 28, 97(5), 565 - 74 The protein import motor of mitochondria: unfolding and trapping of preproteins are distinct and separable functions of matrix Hsp70; Voisine C et al.; Mitochondrial heat shock protein 70 (mtHsp70) functions in unfolding, translocation, and folding of imported proteins . Controversial models of mtHsp70 action have been discussed: (1) physical trapping of preproteins is sufficient to explain the various mtHsp70 functions, and (2) unfolding of preproteins requires an active motor function of mtHsp70 ("pulling") . Intragenic suppressors of a mutant mtHsp70 separate two functions: a nonlethal folding defect caused by enhanced trapping of preproteins, and a conditionally lethal unfolding defect caused by an impaired interaction of mtHsp70 with the membrane anchor Tim44 . Even enhanced trapping in wild-type mitochondria does not generate a pulling force . The motor function of mtHsp70 cannot be explained by passive trapping alone but includes an essential ATP-dependent interaction with Tim44 to generate a pulling force and unfold preproteins. J Neurosci, 1999 Jun 15, 19(12), 4727 - 38 Changes in expression of the DNA repair protein complex DNA-dependent protein kinase after ischemia and reperfusion; Shackelford DA et al.; Reperfusion of ischemic tissue causes an immediate increase in DNA damage, including base lesions and strand breaks . Damage is reversible in surviving regions indicating that repair mechanisms are operable . DNA strand breaks are repaired by nonhomologous end joining in mammalian cells . This process requires DNA-dependent protein kinase (DNA-PK), composed of heterodimeric Ku antigen and a 460,000 Da catalytic subunit (DNA-PKcs) . In this study, a rabbit spinal cord model of reversible ischemia was used to demonstrate the effect of acute CNS injury on the activity and expression of DNA-dependent protein kinase . The DNA-binding activity of Ku antigen, analyzed by an electrophoretic mobility shift assay, increased during reperfusion after a short ischemic insult (15 min of occlusion), from which the animals recover neurological function . After severe ischemic injury (60 min of occlusion) and reperfusion that results in permanent paraplegia, Ku DNA binding was reduced . Protein levels of the DNA-PK components-Ku70, Ku80, and DNA-PKcs-were monitored by immunoblotting . After 60 min of occlusion, the amount of DNA-PKcs and the enzyme poly(ADP-ribose) polymerase (PARP) decreased with the same time course during reperfusion . Concurrently 150 and 120 kDa fragments were immunostained by an anti-DNA-PKcs monoclonal antibody . This antibody was shown to cross-react with alpha-fodrin breakdown products . The 120 kDa fodrin peptide is associated with caspase-3 activation during apoptosis . Both DNA-PKcs and PARP are also substrates for caspase-3-like activities . The results are consistent with a model in which after a short ischemic insult, DNA repair proteins such as DNA-PK are activated . After severe ischemic injury, DNA damage overwhelms repair capabilities, and cell death programs are initiated. J Cell Biol, 1999 Jun 14, 145(6), 1251 - 64 Aip1p interacts with cofilin to disassemble actin filaments; Rodal AA et al.; Actin interacting protein 1 (Aip1) is a conserved component of the actin cytoskeleton first identified in a two-hybrid screen against yeast actin . Here, we report that Aip1p also interacts with the ubiquitous actin depolymerizing factor cofilin . A two-hybrid-based approach using cofilin and actin mutants identified residues necessary for the interaction of actin, cofilin, and Aip1p in an apparent ternary complex . Deletion of the AIP1 gene is lethal in combination with cofilin mutants or act1-159, an actin mutation that slows the rate of actin filament disassembly in vivo . Aip1p localizes to cortical actin patches in yeast cells, and this localization is disrupted by specific actin and cofilin mutations . Further, Aip1p is required to restrict cofilin localization to cortical patches . Finally, biochemical analyses show that Aip1p causes net depolymerization of actin filaments only in the presence of cofilin and that cofilin enhances binding of Aip1p to actin filaments . We conclude that Aip1p is a cofilin-associated protein that enhances the filament disassembly activity of cofilin and restricts cofilin localization to cortical actin patches. J Cell Biol, 1999 Jun 14, 145(6), 1177 - 88 O-Glycosylation of Axl2/Bud10p by Pmt4p is required for its stability, localization, and function in daughter cells; Sanders SL et al.; Cells of the yeast Saccharomyces cerevisiae choose bud sites in a manner that is dependent upon cell type: a and alpha cells select axial sites; a/alpha cells utilize bipolar sites . Mutants specifically defective in axial budding were isolated from an alpha strain using pseudohyphal growth as an assay . We found that a and alpha mutants defective in the previously identified PMT4 gene exhibit unipolar, rather than axial budding: mother cells choose axial bud sites, but daughter cells do not . PMT4 encodes a protein mannosyl transferase (pmt) required for O-linked glycosylation of some secretory and cell surface proteins (Immervoll, T., M . Gentzsch, and W . Tanner . 1995 . Yeast . 11:1345-1351) . We demonstrate that Axl2/Bud10p, which is required for the axial budding pattern, is an O-linked glycoprotein and is incompletely glycosylated, unstable, and mislocalized in cells lacking PMT4 . Overexpression of AXL2 can partially restore proper bud-site selection to pmt4 mutants . These data indicate that Axl2/Bud10p is glycosylated by Pmt4p and that O-linked glycosylation increases Axl2/ Bud10p activity in daughter cells, apparently by enhancing its stability and promoting its localization to the plasma membrane. J Mol Biol, 1999 Jun 4, 289(2), 205 - 9 Reinterpretation of GCN4-p1 folding kinetics: partial helix formation precedes dimerization in coiled coil folding; Myers JK et al.; The folding of coiled coil peptides has traditionally been interpreted in terms of native dimer and unfolded monomers . Calculations using AGADIR and experimental studies of fragments suggest that the monomers of the coiled coil peptide, GCN4-p1, contain significant residual helical structure . A simple model based on diffusion-collision theory predicts not only the measured folding rate within an order of magnitude, but also predicts remarkably well the effect of alanine to glyXcine mutations . We suggest that intrinsic helix stability is a major determinant of the folding rate of the GCN4 coiled coil . Nature, 1999 Jun 3, 399(6735), 487 - 91 Interaction of E1 and hSNF5 proteins stimulates replication of human papillomavirus DNA; Lee D et al.; Mammalian viruses often use components of the host's cellular DNA replication machinery to carry out replication of their genomes, which enables these viruses to be used as tools for characterizing factors that are involved in cellular DNA replication . The human papillomavirus (HPV) E1 protein is essential for replication of the virus DNA . Here we identify the cellular factor that participates in viral DNA replication by using a two-hybrid assay in the yeast Saccharomyces cerevisiae and E1 protein as bait . Using this assay, we isolated Inil/hSNF5, a component of the SWI/SNF complex which facilitates transcription by altering the structure of chromatin . In vitro binding and immunoprecipitation confirmed that E1 interacts directly with Ini1/hSNF5 . Transient DNA-replication assay revealed that HPV DNA replication is stimulated in a dose-dependent manner by addition of Ini1/hSNF5, and that Ini1/hSNF5 antisense RNA blocks the replication of HPV DNA . Amino-acid substitution at residues that are conserved among E1 proteins prevented the E1-Ini1/hSNF5 interaction and reduced DNA replication of HPV in vivo . Our results indicate that Ini1/hSNF5 is required for the efficient replication of papillomavirus DNA and is therefore needed, either alone or in complex with SWI/SNF complex, for mammalian DNA replication as well. Am J Hum Genet, 1999 Jul, 65(1), 50 - 8 X-linked dyskeratosis congenita is predominantly caused by missense mutations in the DKC1 gene; Knight SW et al.; Dyskeratosis congenita is a rare inherited bone marrow-failure syndrome characterized by abnormal skin pigmentation, nail dystrophy, and mucosal leukoplakia . More than 80% of patients develop bone-marrow failure, and this is the major cause of premature death . The X-linked form of the disease (MIM 305000) has been shown to be caused by mutations in the DKC1 gene . The gene encodes a 514-amino-acid protein, dyskerin, that is homologous to Saccharomyces cerevisiae Cbf5p and rat Nap57 proteins . By analogy to the homologues in other species, dyskerin is predicted to be a nucleolar protein with a role in both the biogenesis of ribosomes and, in particular, the pseudouridylation of rRNA precursors . We have determined the genomic structure of the DKC1 gene; it consists of 15 exons spanning a region of 15 kb . This has enabled us to screen for mutations in the genomic DNA, by using SSCP analysis . Mutations were detected in 21 of 37 additional families with dyskeratosis congenita that were analyzed . These mutations consisted of 11 different single-nucleotide substitutions, which resulted in 10 missense mutations and 1 putative splicing mutation within an intron . The missense change A353V was observed in 10 different families and was shown to be a recurring de novo event . Two polymorphisms were also detected, one of which resulted in the insertion of an additional lysine in the carboxy-terminal polylysine domain . It is apparent that X-linked dyskeratosis congenita is predominantly caused by missense mutations; the precise effect on the function of dyskerin remains to be determined. Biochem Mol Biol Int, 1999 May, 47(5), 873 - 80 The involvement of hexokinases in trehalose synthesis; Peixoto DN et al.; Yeast cells harboring a MAL2-8c gene accumulate trehalose during the transition phase of growth on glucose due to the presence of the ADPG-dependent trehalose 6-phosphate synthase . Under these conditions, glucokinase appeared not to provide G-6-P for trehalose synthesis and the two hexokinases seemed to act synergistically . After incubation in d-xylose, trehalose levels in these cells dropped almost in 90%, confirming the involvement of both hexokinases in the accumulation of this carbohydrate . Nevertheless, G-6-P levels appeared to be similar in all strains . Some explanations for this paradox are discussed . In stationary phase, neither of the three isoenzymes were involved in trehalose synthesis . Possibly, gluconeogenesis provides the substrate for trehalose synthesis at that stage. Biochem Mol Biol Int, 1999 May, 47(5), 757 - 63 Nucleoid proteins of pea chloroplasts: detection of a protein homologous to ribosomal protein; Oleskina YuP et al.; Basic proteins were isolated from purified pea chloroplast nucleoids by acid extraction . Using RP-HPLC, the component composition of the basic proteins was studied . SDS-PAGE of major HPLC-fractions showed that the basic nucleoid proteins are heterogeneous with mol . masses of components from 17 to 30 kDa . One polypeptide with mol . mass of 28 kDa (P28) was obtained by RP-HPLC . The sequencing of three tryptic peptides of P28 (T6, T17, and T19) showed that they are homologous to the ribosomal protein L19 of Saccharomyces cerevisiae . The possible functional role of ribosomal proteins in chloroplast nucleoids is discussed. J Cell Physiol, 1999 Jul, 180(1), 105 - 13 Differential metal response and regulation of human heavy metal-inducible genes; Murata M et al.; A number of heavy metal-inducible genes have been reported, but their ranges of response to various metal species are not well known . It is also unclear if these genes are regulated through common mechanisms . To answer these questions, we compared induction kinetics of human metal-inducible genes including the MT-IIA (coding for a metallothionein isoform), hsp70 (coding for the 70-kDa heat-shock protein), and c-fos genes in HeLa cells exposed to Zn, Cd, Ag, Hg, Cu(II), Co, or Ni ions . Transcripts from these three genes were increased after exposure to wide ranges of metals, but each gene was unique in its induction kinetics . Generally, induction was observed at lower metal concentrations in the order of MT-IIA, hsp70, and c-fos . These genes also showed differential responses in time course: more rapid induction was observed in the order of c-fos, hsp70, and MT-IIA after exposure to Zn or Cd . Since the metal-responsive element (MRE) and heat shock element (HSE) of the MT-IIA and hsp70 genes, respectively, are thought to be the cis-acting DNA elements that mediate metal response, we compared the properties of proteins that specifically bind to these elements . MRE-binding activity was detected only in the extract from cells exposed to Zn . By contrast, HSE-binding activity was detected in extracts from cells treated with Zn, Cd, Ag, and Cu . The former was also activated by Zn in vitro, while the latter was not . Each of these DNA-binding activities showed no affinity to the recognition sequence of the other . These results demonstrate that the human metal-inducible genes have broad ranges of response to a variety of heavy metals, but suggest that they are probably regulated through independent mechanisms. J Virol, 1999 Jul, 73(7), 5282 - 93 Role of the ATP-binding domain of the human papillomavirus type 11 E1 helicase in E2-dependent binding to the origin; Titolo S et al.; Replication of the genome of human papillomaviruses (HPV) is initiated by the recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin . We determined, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yeast two-hybrid system and in vitro . This region of E1 encompasses the ATP-binding domain . Here we have examined the role of this ATP-binding domain, and of ATP, on E2-dependent binding of E1 to the origin . Several amino acid substitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating that the structural integrity of this domain is essential for the interaction . The structural constraints imposed on the E1 P loop may differ between HPV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitution that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme . Other substitutions in the E1 P loop, or in two other conserved motifs of the ATP-binding domain, were tolerated, indicating that ATP binding is not essential for interaction with E2 . Nevertheless, ATP-Mg stimulated the E2-dependent binding of E1 to the origin in vitro . This stimulation was maximal at the physiological temperature (37 degrees C) and did not require ATP hydrolysis . In contrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that of mutant E1 proteins with alterations in the DNA-binding domain . These results are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin. J Biol Chem, 1999 Jun 18, 274(25), 18017 - 25 Functional characterization of multiple transactivating elements in beta-catenin, some of which interact with the TATA-binding protein in vitro; Hecht A et al.; beta-Catenin, a member of the family of Armadillo repeat proteins, plays a dual role in cadherin-mediated cell adhesion and in signaling by Wnt growth factors . Upon Wnt stimulation beta-catenin undergoes nuclear translocation and serves as transcriptional coactivator of T cell factor DNA-binding proteins . Previously the transactivation potential of different portions of beta-catenin has been demonstrated, but the precise location of transactivating elements has not been established . Also, the mechanism of transactivation by beta-catenin and the molecular basis for functional differences between beta-catenin and the closely related proteins Armadillo and Plakoglobin are poorly understood . Here we have used a yeast system for the detailed characterization of the transactivation properties of beta-catenin . We show that its transactivation domains possess a modular structure, consist of multiple subelements that cover broad regions at its N and C termini, and extend considerably into the Armadillo repeat region . Compared with beta-catenin the N termini of Plakoglobin and Armadillo have different transactivation capacities that may explain their distinct signaling properties . Furthermore, transactivating elements of beta-catenin interact specifically and directly with the TATA-binding protein in vitro providing further evidence that a major function of beta-catenin during Wnt signaling is to recruit the basal transcription machinery to promoter regions of Wnt target genes. J Biol Chem, 1999 Jun 18, 274(25), 17837 - 44 Identification of the stef gene that encodes a novel guanine nucleotide exchange factor specific for Rac1; Hoshino M et al.; The Rho family GTPases are involved in a variety of cellular events by changing the organization of actin cytoskeletal networks in response to extracellular signals . However, it is not clearly known how their activities are spatially and temporally regulated . Here we report the identification of a novel guanine nucleotide exchange factor for Rac1, STEF, which is related in overall amino acid sequence and modular structure to mouse Tiam1 and Drosophila SIF proteins . STEF protein contains two pleckstrin homology domains, a PDZ domain and a Dbl homology domain . The in vitro assay showed that STEF protein specifically enhanced the dissociation of GDP from Rac1 but not that from either RhoA or Cdc42 . Expression of a truncated STEF protein in culture cells induced membrane ruffling with altered actin localization, which implies that this protein also activates Rac1 in vivo . The stef transcript was observed in restricted parts of mice, including cartilaginous tissues and the cortical plate of the central nervous system during embryogenesis . These findings suggested that STEF protein participates in the control of cellular events in several developing tissues, possibly changing the actin cytoskeletal network by activating Rac1. J Biol Chem, 1999 Jun 18, 274(25), 17718 - 24 Structural basis for the selectivity of the RGS protein, GAIP, for Galphai family members . Identification of a single amino acid determinant for selective interaction of Galphai subunits with GAIP; Woulfe DS et al.; GAIP is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by some G protein alpha subunits . In the present studies, we have examined the structural basis for the ability of GAIP to discriminate among members of the Galphai family . Galphai1, Galphai3, and Galphao interacted strongly with GAIP, whereas Galphai2 interacted weakly and Galphas did not interact at all . A chimeric G protein composed of a Galphai2 N terminus and a Galphai1 C terminus interacted as strongly with GAIP as native Galphai1, whereas a chimeric N-terminal Galphai1 with a Galphai2 C terminus did not interact . These results suggest that the determinants responsible for GAIP selectivity between these two Galphais reside within the C-terminal GTPase domain of the G protein . To further localize residues contributing to G protein-GAIP selectivity, a panel of 15 site-directed Galphai1 and Galphai2 mutants were assayed . Of the Galphai1 mutants tested, only that containing a mutation at aspartate 229 located at the N terminus of Switch 3 did not interact with GAIP . Furthermore, the only Galphai2 variant that interacted strongly with GAIP contained a replacement of the corresponding Galphai2 Switch 3 residue (Ala230) with aspartate . To determine whether GAIP showed functional preferences for Galpha subunits that correlate with the binding data, the ability of GAIP to enhance the GTPase activity of purified alpha subunits was tested . GAIP catalyzed a 3-5-fold increase in the rate of GTP hydrolysis by Galphai1 and Galphai2(A230D) but no increase in the rate of Galphai2 and less than a 2-fold increase in the rate of Galphai1(D229A) under the same conditions . Thus, GAIP was able to discriminate between Galphai1 and Galphai2 in both binding and functional assays, and in both cases residue 229/230 played a critical role in selective recognition. J Biol Chem, 1999 Jun 18, 274(25), 17643 - 8 D-site binding protein transactivation requires the proline- and acid-rich domain and involves the coactivator p300; Lamprecht C et al.; The D-site binding protein (DBP) is a member of the proline- and acid-rich (PAR) domain subfamily of basic/leucine zipper proteins and is involved in transcriptional regulation in the liver . Deletion analysis of the DBP protein was carried out in an effort to define the function of the conserved PAR domain . Internal deletions of the protein, i.e . removing portions of the PAR domain, resulted in a substantial loss in transactivation of a high affinity DBP reporter construct when assayed in Hep G2 cells . These same sequences conferred significant transactivation to GAL4 DNA binding domain fusion proteins, indicating that this region acts as part of an independent activation domain comprised of sequences in both the amino terminus and in the PAR domain of DBP . The coexpression of full-length expression constructs for both DBP and hepatic leukemia factor resulted in a dramatic increase in activation mediated by the GAL4-DBP fusion proteins, suggesting the involvement of a regulated coactivator in this process . DBP transactivation appears to be a p300-dependent process, as a 12 S E1A expression construct disrupted DBP-mediated transactivation, and a p300 expression vector, but not a CREB binding protein vector, was able to restore DBP transactivation . These results suggest that the PAR domain is required for DBP activation, which occurs through a regulated, p300-dependent process. J Biol Chem, 1999 Jun 18, 274(25), 17431 - 6 Identification of an intramolecular interaction between the SH3 and guanylate kinase domains of PSD-95; McGee AW et al.; Postsynaptic density-95 (PSD-95/SAP-90) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins that assemble protein complexes at synapses and other cell junctions . MAGUKs comprise multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GK) domains, and these binding sites mediate the scaffolding function of MAGUK proteins . Synaptic binding partners for the PDZ and GK domains of PSD-95 have been identified, but the role of the SH3 domain remains elusive . We now report that the SH3 domain of PSD-95 mediates a specific interaction with the GK domain . The GK domain lacks a poly-proline motif that typically binds to SH3 domains; instead, SH3/GK binding is a bi-domain interaction that requires both intact motifs . Although isolated SH3 and GK domains can bind in trans, experiments with intact PSD-95 molecules indicate that intramolecular SH3/GK binding dominates and prevents intermolecular associations . SH3/GK binding is conserved in the related Drosophila MAGUK protein DLG but is not detectable for Caenorhabditis elegans LIN-2 . Many previously identified genetic mutations of MAGUKs in invertebrates occur in the SH3 or GK domains, and all of these mutations disrupt intramolecular SH3/GK binding. Genes Dev, 1999 Jun 1, 13(11), 1453 - 63 Negative regulation of male development in Caenorhabditis elegans by a protein-protein interaction between TRA-2A and FEM-3; Mehra A et al.; The tra-2 gene of the nematode Caenorhabditis elegans encodes a predicted membrane protein, TRA-2A, that promotes XX hermaphrodite development . Genetic analysis suggests that tra-2 is a negative regulator of three genes that are required for male development: fem-1, fem-2, and fem-3 . We report that the carboxy-terminal region of TRA-2A interacts specifically with FEM-3 in the yeast two-hybrid system and in vitro . Consistent with the idea that FEM-3 is a target of negative regulation, we find that excess FEM-3 can overcome the feminizing effect of tra-2 and cause widespread masculinization of XX somatic tissues . In turn, we show that the masculinizing effects of excess FEM-3 can be suppressed by overproduction of the carboxy-terminal domain of TRA-2A . A FEM-3 fragment that retains TRA-2A-binding activity can masculinize fem-3(+) animals, but not fem-3 mutants, suggesting that it is possible to release and to activate endogenous FEM-3 by titrating TRA-2A . We propose that TRA-2A prevents male development by interacting directly with FEM-3 and that a balance between the opposing activities of TRA-2A and FEM-3 determines sex-specific cell fates in somatic tissues . When the balance favors FEM-3, it acts through or with the other FEM proteins to promote male cell fates. Mol Biol Rep, 1999 Apr, 26(1-2), 125 - 30 Endoplasmic reticulum degradation . Reverse protein transport and its end in the proteasome; Plemper RK et al.; Degradation of misfolded or unassembled proteins of the secretory pathway is an essential function of the quality control system of the Endoplasmic Reticulum (ER) . Using yeast as a model organism we show that a mutated and therefore misfolded soluble lumenal protein carboxypeptidase yscY (CPY), and a polytopic membrane protein, the ATP-binding cassette transporter Pdr5 (Pdr5), are retrograde transported out of the ER and degraded via the cytoplasmic ubiquitin-proteasome system . Retrograde transport depends on an intact Sec61 translocon . Complete import of CPY into the lumen of the ER requests a new targeting mechanism for retrograde transport of the malfolded enzyme through the Sec61 channel to occur . For soluble CPY, but not for the polytopic membrane protein Pdr5 action of the ER-lumenal Hsp70 chaperone Kar2 is necessary to deliver the protein to the ubiquitin-proteasome machinery . Polyubiquitination of CPY and Pdr5 by the ubiquitin conjugating enzymes Ubc6 and Ubc7 is crucial for degradation to occur . Also transport of CPY out of the ER-lumen depends on ubiquitination . Newly discovered proteins of the ER membrane, Derl, Der3/Hrd1, and Hrd3 are specifically involved in the retrograde transport processes. Mol Biol Rep, 1999 Apr, 26(1-2), 35 - 8 Activator complexes containing the proteasomal regulatory ATPases S10b (SUG2) and S6 (TBP1) in different tissues and organisms; Hastings R et al.; Each 19S regulator of the 26S proteasome contains six ATPase subunits as well as many (>14) non-ATPase protein subunits . The ATPase subunits have been detected in other complexes which may regulate transcription and possibly other cellular processes . The S10b (yeast SUG2 or human p42) and the S6' (TBP1) ATPases have been found in an activator complex (modulator) prepared from bovine red cells . We have identified and partially characterised a similar activator from different human tissues (from soluble extracts of human brain, placenta and human embryonic kidney cells) and an insect: an activator is present in soluble extracts of abdominal intersegmental muscle from Manduca sexta . Activation is ATP and concentration dependent . There is no stimulation of human red cell-derived 20S proteasome by the Manduca activator ruling out 11S regulator in the preparations . Additionally, cross-species activation occurs: the Manduca activator increases the activity of rat skeletal muscle 26S proteasomes and the human placental activator similarly increases the activity of 26S proteasomes prepared from muscles from Manduca sexta . Finally, there is no evidence for other ATPases in the activator complex. Oncogene, 1999 Jun 3, 18(22), 3422 - 6 Mutations of a novel human RAD54 homologue, RAD54B, in primary cancer; Hiramoto T et al.; Association of breast tumor susceptibility gene products BRCA1 and BRCA2 with the RAD51 recombination protein suggested that cancer could arise through defects in recombination . The identification of NBS1, responsible for Nijmegen breakage syndrome, from the MRE11/RAD50 recombination protein complex also supports this hypothesis . However, our mutation analysis revealed that known members of the RAD52 epistasis group are rarely mutated in human primary cancer . Here we describe the isolation of a novel member of the SNF2 superfamily, characterized with sequence motifs similar to those in DNA and RNA helicases . The gene, designated RAD54B, is significantly homologous to the RAD54 recombination gene . The expression of RAD54B was high in testis and spleen, which are active in meiotic and mitotic recombination . These findings suggest that RAD54B may play an active role in recombination processes in concert with other members of the RAD52 epistasis group . RAD54B maps to human chromosome 8q21.3-q22 in a region associated with cancer-related chromosomal abnormalities . Homozygous mutations at highly conserved positions of RAD54B were observed in human primary lymphoma and colon cancer . These findings suggest that some cancers arise through alterations of the RAD54B function. DNA Cell Biol, 1999 May, 18(5), 381 - 96 Comparative analysis of KRAB zinc finger proteins in rodents and man: evidence for several evolutionarily distinct subfamilies of KRAB zinc finger genes; Mark C et al.; Although the KRAB zinc finger proteins probably constitute the single largest class of transcription factors within the human genome, almost nothing is known about their biological function . To increase our knowledge about this interesting and relatively unexplored family of potent transcriptional repressors, we here present the cloning, structural analysis, and expression study of three novel mouse KRAB zinc finger proteins . In addition, we present an extensive comparative analysis of various members of this gene family based on the structure of the common KRAB A motif . At least three larger subfamilies of KRAB zinc finger proteins are identified: one carrying the classical KRAB A motif only, another holding both a classical KRAB A and a classical KRAB B motif, and a third holding a classical KRAB A and a highly divergent KRAB B domain, named b . A large variation both in size and in primary amino acid sequence was observed in the linker region between the KRAB domain and the C-terminally located zinc finger repeats . This variability indicates that this region is of minor importance for the biological function of KRAB-containing zinc finger proteins . The fact that in many zinc finger genes, the entire or almost the entire linker region is composed of degenerate finger motifs substantiates this conclusion . The absence of identifiable KRAB A and B motifs in the genome of yeast, Saccharomyces cerevisiae, indicates a relatively late appearance of the KRAB domain in evolution and may suggest that the biological functions are restricted to multicellular organisms . In addition, we show that the expression of individual members of one subfamily of KRAB zinc finger genes is restricted to specific hematopoietic cell lineages . This finding suggests that KRAB zinc finger proteins may play a role in lineage commitment, possibly silencing leakage transcription from nonlineage-expressed genes. Mol Cell, 1999 May, 3(5), 673 - 8 GAL4 is regulated by the RNA polymerase II holoenzyme-associated cyclin-dependent protein kinase SRB10/CDK8; Hirst M et al.; Phosphorylation of the yeast transcription factor GAL4 at S699 is required for efficient galactose-inducible transcription . We demonstrate that this site is a substrate for the RNA polymerase holoenzyme-associated CDK SRB10 . S699 phosphorylation requires SRB10 in vivo, and this site is phosphorylated by purified SRB10/ SRB11 CDK/cyclin in vitro . RNA Pol II holoenzymes purified from WT yeast phosphorylate GAL4 at sites observed in vivo whereas holoenzymes from srb10 yeast are incapable of phosphorylating GAL4 at S699 . Mutations at GAL4 S699 and srb10 are epistatic for GAL induction, demonstrating that SRB10 regulates GAL4 activity through this phosphorylation in vivo . These results demonstrate a function for the SRB10/ CDK8 holoenzyme-associated CDK that involves regulation of transactivators by phosphorylation during transcriptional activation. J Allergy Clin Immunol, 1999 Jun, 103(6), 1158 - 65 Purification of the antigenic components of pigeon dropping extract, the responsible agent for cellular immunity in pigeon breeder's disease; Hisauchi-Kojima K et al.; BACKGROUND: Pigeon breeder's disease (PBD) is a lung disease caused by inhalation of antigens derived from pigeons . OBJECTIVE: This study was undertaken to characterize the responsible component of pigeon dropping extract (PDE) for PBD . METHODS: First, crude PDE was applied to SDS-PAGE followed by immunoblotting by using antibodies in bronchoalveolar lavage (BAL) fluid . Second, 9 bands of PDE were separated by SDS-PAGE and used for antigen-induced PBMCs . Finally, amino-terminal sequencing was conducted on an isolated 21-kd protein by 2-dimensional electrophoresis . RESULTS: Immunoblots with BAL fluid from patients with PBD identified 9 bands . Similar patterns were observed by using BAL fluid from 10 control patients (9 with summer-type hypersensitivity pneumonitis or idiopathic pulmonary fibrosis and 1 asymptomatic breeder), except for the 21-kd protein, which was detected in 10 patients with PBD and 1 asymptomatic breeder . The stimulation indices of PBMCs determined by using proteins electroeluted from the 9 bands were higher in patients with PBD than in the 10 control patients . The 21-kd protein was separated into 5 spots by 2-dimensional electrophoresis; these spots were all reactive with BAL fluid from patients with PBD as determined by immunoblotting . The sequence of the 21-kd protein had 57% identity to a Saccharomyces cerevisiae chromosome X reading frame . A synthetic peptide, derived from the amino acid sequence of the N-terminal of the native protein, induced significant proliferation of PBMCs obtained from 5 patients with PBD, but not with PBMCs obtained from control patients . CONCLUSION: The 21-kd protein is the only protein that identified individuals exposed to pigeons by immunoblotting . Only PBMCs from patients with PBD showed significant proliferation to the 21-kd protein and to the synthetic peptide on the basis of the N-terminal sequence of the native peptide . The 21-kd protein will be an important antigen for studies on the epidemiology, diagnosis, and pathogenesis of PBD. Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 7110 - 5 AtPCS1, a phytochelatin synthase from Arabidopsis: isolation and in vitro reconstitution; Vatamaniuk OK et al.; Phytochelatins, a class of posttranslationally synthesized peptides, play a pivotal role in heavy metal, primarily Cd2+, tolerance in plants and fungi by chelating these substances and decreasing their free concentrations . Derived from glutathione and related thiols by the action of gamma-glutamylcysteine dipeptidyl transpeptidases (phytochelatin synthases; EC 2.3.2.15), phytochelatins consist of repeating units of gamma-glutamylcysteine followed by a C-terminal Gly, Ser, or beta-Ala residue {poly-(gamma-Glu-Cys)n-Xaa} . Here we report the suppression cloning of a cDNA (AtPCS1) from Arabidopsis thaliana encoding a 55-kDa soluble protein that enhances heavy-metal tolerance and elicits Cd2+-activated phytochelatin accumulation when expressed in Saccharomyces cerevisiae . On the basis of these properties and the sufficiency of immunoaffinity-purified epitope-tagged AtPCS1 polypeptide for high rates of Cd2+-activated phytochelatin synthesis from glutathione in vitro, AtPCS1 is concluded to encode the enzyme phytochelatin synthase. Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6739 - 44 A Crm1p-independent nuclear export path for the mRNA-associated protein, Npl3p/Mtr13p; Liu Y et al.; mRNA export involves association of mRNAs with nucleoplasmic proteins, delivery to the nuclear pore complex, translocation to the cytoplasm, and reimport of recycling components . Many yeast mutants inhibit mRNA export, but there is little information concerning the RNA carriers and steps of transport that they affect . The hnRNP/serine-arginine-rich-like protein, Npl3p/Mtr13p, binds poly(A)+ RNA and shuttles between the nucleus and cytoplasm . Its export accelerates on inhibition of RNA synthesis . In vivo tests show that its export requires two proteins with putative leucine-rich nuclear export signals: Gle1p, Mex67p, and several additional nuclear and nuclear pore complex-associated proteins . Surprisingly, a nonnuclear pool of an import factor (the importin alpha homologue, Srp1p) is also required . Changes in the methylation status of Npl3p do not correlate with its nucleocytoplasmic distribution . A crm1 mutant that inhibits export of proteins with leucine-rich nuclear export signals and mRNAs does not inhibit Npl3p export . Moreover, several proteins needed for Npl3p export are not needed for export of a typical Crm1p cargo . Thus, Npl3p export requires only a subset of proteins implicated in mRNA export, suggesting that more than one mRNA export path exists . A distinct group of mutants, including a mutation of a member of the importin beta superfamily, inhibits Npl3p reimport from the cytoplasm. Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6609 - 14 A small nucleolar RNA:ribozyme hybrid cleaves a nucleolar RNA target in vivo with near-perfect efficiency; Samarsky DA et al.; A hammerhead ribozyme has been localized to the yeast nucleolus by using the U3 small nucleolar RNA as a carrier . The hybrid small nucleolar RNA:ribozyme, designated a "snorbozyme," is metabolically stable and cleaves a target U3 RNA with nearly 100% efficiency in vivo . This is the most efficient in vivo cleavage reported for a trans-acting ribozyme . A key advantage of the model substrate featured is that a stable, trimmed cleavage product accumulates . This property allows accurate kinetic measurements of authentic cleavage in vivo . The system offers new avenues for developing effective ribozymes for research and therapeutic applications. Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6591 - 6 Accurate quantitation of protein expression and site-specific phosphorylation; Oda Y et al.; A mass spectrometry-based method is described for simultaneous identification and quantitation of individual proteins and for determining changes in the levels of modifications at specific sites on individual proteins . Accurate quantitation is achieved through the use of whole-cell stable isotope labeling . This approach was applied to the detection of abundance differences of proteins present in wild-type versus mutant cell populations and to the identification of in vivo phosphorylation sites in the PAK-related yeast Ste20 protein kinase that depend specifically on the G1 cyclin Cln2 . The present method is general and affords a quantitative description of cellular differences at the level of protein expression and modification, thus providing information that is critical to the understanding of complex biological phenomena. Biochem J, 1999 Jun 15, 340 ( Pt 3), 855 - 62 Transcription factor GCN4 for control of amino acid biosynthesis also regulates the expression of the gene for lipoamide dehydrogenase; Zaman Z et al.; The yeast LPD1 gene encoding lipoamide dehydrogenase is subject to the general control of amino acid biosynthesis mediated by the GCN4 transcription factor . This is striking in that it demonstrates that GCN4-mediated regulation extends much farther upstream than simply to the direct pathways for amino acid and purine biosynthesis . In yeast, lipoamide dehydrogenase functions in at least three multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase (which function in the entry of pyruvate into, and metabolism via, the citric acid cycle) and glycine decarboxylase . When wild-type cells were shifted from growth on amino acid-rich to amino acid-deficient medium, the expression of lipoamide dehydrogenase was induced approx . 2-fold . In a similar experiment no such induction was observed in isogenic gcn4 mutant cells . Northern analysis indicated that amino acid starvation affected levels of the LPD1 transcript . In the upstream region of LPD1 are three matches to the consensus for control mediated by GCN4 . Directed mutagenesis of each site, and of all combinations of sites, suggests that only one site might be important for the general control response under the conditions tested . Gel-retardation analysis with GCN4 protein synthesized in vitro has indicated that GCN4 can bind in vitro to at least two of the consensus motifs. Mol Biol Cell, 1999 Jun, 10(6), 1939 - 55 Localization and recycling of gp27 (hp24gamma3): complex formation with other p24 family members; Fullekrug J et al.; We report here the characterization of gp27 (hp24gamma3), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secretory pathway . Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to the cis side of the Golgi apparatus . In addition, some gp27 was detected in COPI- and COPII-coated structures throughout the cytoplasm . This indicated cycling that was confirmed in three ways . First, 15 degrees C temperature treatment resulted in accumulation of gp27 in pre-Golgi structures colocalizing with anterograde cargo . Second, treatment with brefeldin A caused gp27 to relocate into peripheral structures positive for both KDEL receptor and COPII . Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticulum (ER) by blocking ER export . Together, this shows that gp27 cycles extensively in the early secretory pathway . Immunoprecipitation and coexpression studies further revealed that a significant fraction of gp27 existed in a hetero-oligomeric complex . Three members of the p24 family, GMP25 (hp24alpha2), p24 (hp24beta1), and p23 (hp24delta1), coprecipitated in what appeared to be stochiometric amounts . This heterocomplex was specific . Immunoprecipitation of p26 (hp24gamma4) failed to coprecipitate GMP25, p24, or p23 . Also, very little p26 was found coprecipitating with gp27 . A functional requirement for complex formation was suggested at the level of ER export . Transiently expressed gp27 failed to leave the ER unless other p24 family proteins were coexpressed . Comparison of attached oligosaccharides showed that gp27 and GMP25 recycled differentially . Only a very minor portion of GMP25 displayed complex oligosaccharides . In contrast, all of gp27 showed modifications by medial and trans enzymes at steady state . We conclude from these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibrium with individual p24 proteins to allow for differential recycling and distributions. Mol Biol Cell, 1999 Jun, 10(6), 1873 - 89 The phosphatidylinositol 3-phosphate binding protein Vac1p interacts with a Rab GTPase and a Sec1p homologue to facilitate vesicle-mediated vacuolar protein sorting; Tall GG et al.; Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events . Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport . Little is known about the functional relationship between Rabs and SNARE protein complexes . We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p . Vac1p was found to bind the Sec1p homologue Vps45p . Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed . Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate {PtdIns(3)P} . In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association . Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p . Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p . Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34 . These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein-protein interactions . We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex. J Exp Med, 1999 Jun 7, 189(11), 1707 - 14 Identification of Grb2 as a novel binding partner of tumor necrosis factor (TNF) receptor I; Hildt E et al.; Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine . Its pleiotropic biological properties are signaled through two distinct cell surface receptors: the TNF receptor type I (TNFR-I) and the TNF receptor type II . Neither of the two receptors possesses tyrosine kinase activity . A large majority of TNF-alpha-dependent activities can be mediated by TNFR-I . Recently, c-Raf-1 kinase was identified as an intracellular target of a signal transduction cascade initiated by binding of TNF-alpha to TNFR-I . However, the mechanism engaged in TNF-alpha-dependent activation of c-Raf-1 kinase is still enigmatic . Here we report that the cytosolic adapter protein Grb2 is a novel binding partner of TNFR-I . Grb2 binds with its COOH-terminal SH3 domain to a PLAP motif within TNFR-I and with its NH2-terminal SH3 domain to SOS (son of sevenless) . A PLAP deletion mutant of TNFR-I fails to bind Grb2 . The TNFR-I/Grb2 interaction is essential for the TNF-alpha-dependent activation of c-Raf-1 kinase; activation of c-Raf-1 kinase by TNF-alpha can be blocked by coexpression of Grb2 mutants harboring inactivating point mutations in the NH2- or COOH-terminal SH3 domain, cell-permeable peptides that disrupt the Grb2/TNFR-I interaction or transdominant negative Ras . Functionality of the TNFR-I/Grb2/SOS/Ras interaction is a prerequisite but not sufficient for TNF-alpha-dependent activation of c-Raf-1 kinase . Inhibition of the TNFR-I/FAN (factor associated with neutral sphingomyelinase) interaction, which is essential for TNF-alpha-dependent activation of the neutral sphingomyelinase, either by cell-permeable peptides or by deletion of the FAN binding domain, prevents activation of c-Raf-1 kinase . In conclusion, binding of the Grb2 adapter protein via its COOH-terminal SH3 domain to the nontyrosine kinase receptor TNFR-I results in activation of a signaling cascade known so far to be initiated, in the case of the tyrosine kinase receptors, by binding of the SH2 domain of Grb2 to phosphotyrosine. Chem Phys Lipids, 1999 Apr, 98(1-2), 69 - 77 Pathways for phosphoinositide synthesis; Tolias KF et al.; In eukaryotic cells, phosphatidylinositol can be phosphorylated on the inositol ring by a series of kinases to produce at least seven distinct phosphoinositides . These lipids have been implicated in a variety of cellular processes, including calcium regulation, actin rearrangement, vesicle trafficking, cell survival and mitogenesis . The phosphorylated lipids can act as precursors of second messengers or act directly to recruit specific signaling proteins to the membrane . A number of the kinases responsible for producing these lipids have been purified and their cDNA clones have been isolated . The most well characterized of these enzymes are the phosphoinositide 3-kinases . However, progress has also been made in the characterization of phosphatidylinositol 4-kinases and phosphatidylinositol-4-phosphate 5-kinases . In addition, new pathways involving phosphatidylinositol-5-phosphate 4-kinases, phosphatidylinositol-3-phosphate 5-kinases and phosphatidylinositol-3-phosphate 4-kinases have recently been described . The various enzymes and pathways involved in the synthesis of cellular phosphoinositides will be discussed. Chem Phys Lipids, 1999 Apr, 98(1-2), 23 - 33 Mammalian phosphatidylinositol transfer proteins: emerging roles in signal transduction and vesicular traffic; Cockcroft S; Phosphatidylinositol transfer proteins (PITP) are abundant cytosolic proteins found in all mammalian cells . Two cytosolic isoforms of 35 and 36 kDa (PITP alpha and PITP beta) have been identified which share 77% identity . These proteins are characterized by having a single phospholipid binding site which exhibits dual headgroup specificity . The preferred lipid that can occupy the site can be either phosphatidylinositol (PI) or phosphatidylcholine (PC) . In addition, PITP beta can also bind sphingomyelin . A second characteristic of these proteins is the ability to transfer PI and PC (or SM) from one membrane compartment to another in vitro . The function of PITP in mammalian cells has been examined mainly using reconstitution studies utilizing semi-intact cells or cell-free systems . From such analyses, a requirement for PITP has been identified in phospholipase C-mediated phosphatidylinositol bisphosphate (PI(4,5)P2) hydrolysis, in phosphoinositide 3-kinase catalyzed PIP3 generation, in regulated exocytosis, in the biogenesis of secretory granules and vesicles and in intra-golgi transport . Studies aimed at elucidating the mechanism of action of PITP in each of these seemingly disparate processes have yielded a singular theme: the activity of PITP stems from its ability to transfer PI from its site of synthesis to sites of cellular activity . This function was predicted from its in vitro characteristics . The second feature of PITP that was not predicted is the ability to stimulate the local synthesis of several phosphorylated forms of PI including PI(4)P, PI(4,5)P2, PI(3)P, PI(3,4,5)P3 by presenting PI to the lipid kinases involved in phosphoinositide synthesis . We conclude that PITP contributes in multiple aspects of cell biology ranging from signal transduction to membrane trafficking events where a central role for phosphoinositides is recognized either as a substrate or as an intact lipid signalling molecule. J Biol Chem, 1999 Jun 11, 274(24), 17219 - 25 Modulation of human heat shock factor trimerization by the linker domain; Liu PC et al.; Heat shock transcription factors (HSFs) are stress-responsive proteins that activate the expression of heat shock genes and are highly conserved from bakers' yeast to humans . Under basal conditions, the human HSF1 protein is maintained as an inactive monomer through intramolecular interactions between two coiled-coil domains and interactions with heat shock proteins; upon environmental, pharmacological, or physiological stress, HSF1 is converted to a homotrimer that binds to its cognate DNA binding site with high affinity . To dissect regions of HSF1 that make important contributions to the stability of the monomer under unstressed conditions, we have used functional complementation in bakers' yeast as a facile assay system . Whereas wild-type human HSF1 is restrained as an inactive monomer in yeast that is unable to substitute for the essential yeast HSF protein, mutations in the linker region between the DNA binding domain and the first coiled-coil allow HSF1 to homotrimerize and rescue the viability defect of a hsfDelta strain . Fine mapping by functional analysis of HSF1-HSF2 chimeras and point mutagenesis revealed that a small region in the amino-terminal portion of the HSF1 linker is required for maintenance of HSF1 in the monomeric state in both yeast and in transfected human 293 cells . Although linker regions in transcription factors are known to modulate DNA binding specificity, our studies suggest that the human HSF1 linker plays no role in determining HSF1 binding preferences in vivo but is a critical determinant in regulating the HSF1 monomer-trimer equilibrium. J Biol Chem, 1999 Jun 11, 274(24), 17058 - 62 Cloning and characterization of ATRAP, a novel protein that interacts with the angiotensin II type 1 receptor; Daviet L et al.; The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 (AT1) receptor has recently been shown to interact with several classes of cytoplasmic proteins that regulate different aspects of AT1 receptor physiology . Employing yeast two-hybrid screening of a mouse kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the murine AT1a receptor as a bait, we have isolated a novel protein with a predicted molecular mass of 18 kDa, which we have named ATRAP (for AT1 receptor-associated protein) . ATRAP interacts specifically with the carboxyl-terminal domain of the AT1a receptor but not with those of angiotensin II type 2 (AT2), m3 muscarinic acetylcholine, bradykinin B2, endothelin B, and beta2-adrenergic receptors . The mRNA of ATRAP was abundantly expressed in kidney, heart, and testis but was poorly expressed in lung, liver, spleen, and brain . The ATRAP-AT1a receptor association was confirmed by affinity chromatography, by specific co-immunoprecipitation of the two proteins, and by fluorescence microscopy, showing co-localization of these proteins in intact cells . Overexpression of ATRAP in COS-7 cells caused a marked inhibition of AT1a receptor-mediated activation of phospholipase C without affecting m3 receptor-mediated activation . In conclusion, we have isolated a novel protein that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT1a receptor and affects AT1a receptor signaling. J Biol Chem, 1999 Jun 11, 274(24), 16747 - 53 Homo- and heterodimerization of synapsins; Hosaka M et al.; In vertebrates, synapsins constitute a family of synaptic vesicle proteins encoded by three genes . Synapsins contain a central ATP-binding domain, the C-domain, that is highly homologous between synapsins and evolutionarily conserved in invertebrates . The crystal structure of the C-domain from synapsin I revealed that it constitutes a large (>300 amino acids), independently folded domain that forms a tight dimer with or without bound ATP . We now show that the C-domains of all synapsins form homodimers, and that in addition, C-domains from different synapsins associate into heterodimers . This conclusion is based on four findings: 1) in yeast two-hybrid screens with full-length synapsin IIa as a bait, the most frequently isolated prey cDNAs encoded the C-domain of synapsins; 2) quantitative yeast two-hybrid protein-protein binding assays demonstrated pairwise strong interactions between all synapsins; 3) immunoprecipitations from transfected COS cells confirmed that synapsin II heteromultimerizes with synapsins I and III in intact cells, and similar results were obtained with bacterial expression systems; and 4) quantification of the synapsin III level in synapsin I/II double knockout mice showed that the level of synapsin III is decreased by 50%, indicating that heteromultimerization of synapsin III with synapsins I or II occurs in vivo and is required for protein stabilization . These data suggest that synapsins coat the surface of synaptic vesicles as homo- and heterodimers in which the C-domains of the various subunits have distinct regulatory properties and are flanked by variable C-terminal sequences . The data also imply that synapsin III does not compensate for the loss of synapsins I and II in the double knockout mice. J Biol Chem, 1999 Jun 11, 274(24), 16677 - 80 The eukaryotic polypeptide chain releasing factor (eRF3/GSPT) carrying the translation termination signal to the 3'-Poly(A) tail of mRNA . Direct association of erf3/GSPT with polyadenylate-binding protein; Hoshino S et al.; The mammalian GTP-binding protein GSPT, whose carboxyl-terminal sequence is homologous to the eukaryotic elongation factor EF1alpha, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in the translation termination . The amino-terminal domain of GSPT was, however, not required for the binding . Search for other GSPT-binding proteins in yeast two-hybrid screening system resulted in the identification of a cDNA encoding polyadenylate-binding protein (PABP), whose amino terminus is associating with the poly(A) tail of mRNAs presumably for their stabilization . The interaction appeared to be mediated through the carboxyl-terminal domain of PABP and the amino-terminal region of GSPT . Interestingly, multimerization of PABP with poly(A), which is ascribed to the action of its carboxyl-terminal domain, was completely inhibited by the interaction with the amino-terminal domain of GSPT . These results indicate that GSPT/eRF3 may play important roles not only in the termination of protein synthesis but also in the regulation of mRNA stability . Thus, the present study is the first report showing that GSPT/eRF3 carries the translation termination signal to 3'-poly(A) tail ubiquitously present in eukaryotic mRNAs. EMBO J, 1999 Jun 1, 18(11), 3119 - 32 Mechanism of non-spliceosomal mRNA splicing in the unfolded protein response pathway; Gonzalez TN et al.; The unfolded protein response is an intracellular signaling pathway that, in response to accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER), upregulates transcription of ER resident chaperones . A key step in this pathway is the non-conventional, regulated splicing of the mRNA encoding the positive transcriptional regulator Hac1p . In the yeast Saccharomyces cerevisiae, the bifunctional transmembrane kinase/endoribonuclease Ire1p cleaves HAC1 mRNA at both splice junctions and tRNA ligase joins the two exons together . We have reconstituted HAC1 mRNA splicing in an efficient in vitro reaction and show that, in many ways, the mechanism of HAC1 mRNA splicing resembles that of pre-tRNA splicing . In particular, Ire1p endonucleolytic cleavage leaves 2', 3'-cyclic phosphates, the excised exons remain associated by base pairing, and exon ligation by tRNA ligase follows the same chemical steps as for pre-tRNA splicing . To date, this mechanism of RNA processing is unprecedented for a messenger RNA . In contrast to the striking similarities to tRNA splicing, the structural features of the splice junctions recognized by Ire1p differ from those recognized by tRNA endonuclease . We show that small stem-loop structures predicted to form at both splice junctions of HAC1 mRNA are required and sufficient for Ire1p cleavage. EMBO J, 1999 Jun 1, 18(11), 3101 - 6 The nucleosome remodeling complex, Snf/Swi, is required for the maintenance of transcription in vivo and is partially redundant with the histone acetyltransferase, Gcn5; Sudarsanam P et al.; Snf/Swi, a nucleosome remodeling complex, is important for overcoming nucleosome-mediated repression of transcription in Saccharomyces cerevisiae . We have addressed the mechanism by which Snf/Swi controls transcription in vivo of an Snf/Swi-dependent promoter, that of the SUC2 gene . By single-cell analysis, our results show that Snf/Swi is required for activated levels of SUC2 expression in every cell of a population . In addition, Snf/Swi is required for maintenance of SUC2 transcription, suggesting that continuous chromatin remodeling is necessary to maintain an active transcriptional state . Finally, Snf/Swi and Gcn5, a histone acetyltransferase, have partially redundant roles in the control of SUC2 transcription, suggesting a functional overlap between two different mechanisms believed to overcome repression by nucleosomes, nucleosome remodeling and histone acetylation. EMBO J, 1999 Jun 1, 18(11), 3090 - 100 Repression by Ikaros and Aiolos is mediated through histone deacetylase complexes; Koipally J et al.; Here we show that the lymphoid lineage-determining factors Ikaros and Aiolos can function as strong transcriptional repressors . This function is mediated through two repression domains and is dependent upon the promoter context and cell type . Repression by Ikaros proteins correlates with hypo-acetylation of core histones at promoter sites and is relieved by histone deacetylase inhibitors . Consistent with these findings, Ikaros and its repression domains can interact in vivo and in vitro with the mSin3 family of co-repressors which bind to histone deacetylases . Based on these and our recent findings of associations between Ikaros and Mi-2-HDAC, we propose that Ikaros family members modulate gene expression during lymphocyte development by recruiting distinct histone deacetylase complexes to specific promoters. J Mol Biol, 1999 Jun 11, 289(3), 565 - 77 Engineered mutants in the switch II loop of Ran define the contribution made by key residues to the interaction with nuclear transport factor 2 (NTF2) and the role of this interaction in nuclear protein import; Kent HM et al.; Nuclear protein import requires a precisely choreographed series of interactions between nuclear pore components and soluble factors such as importin-beta, Ran, and nuclear transport factor 2 (NTF2) . We used the crystal structure of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction . X-ray crystallography showed that the F72Y, F72W and R76E mutations did not introduce major structural changes into the mutant Ran . The GDP-bound form of the switch II mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Phe72 are all crucial for the interaction between Ran and NTF2 . Nuclear protein accumulation in digitonin-permeabilzed cells was impaired with Ran mutants deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport . We used mutants of the yeast Ran homologue Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo . Although neither mutant was viable when integrated into the genome as a single copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72Y on a centromeric plasmid were viable, confirming that this mutant retained the essential properties of wild-type Ran . However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, although strains overexpressing the mutant to higher levels using an episomal 2micrometers plasmid were viable, indicating that the R76E mutation may also have interfered with other interactions made by Gsp1p . Biotechnol Prog, 1999 May-Jun, 15(3), 304 - 11 Functional genomic technologies: creating new paradigms for fundamental and applied biology; Kao CM; New technologies that analyze the behavior of thousands of genes in parallel are creating, for the first time, a foundation of data for building integrated models of cellular processes . This review discusses the general issues of utilizing genomic methods in fundamental and applied research settings, using the study of stress responses and improvement of secondary metabolite production as examples . A fusion of concepts from biological and nonbiological disciplines, including mathematics, computer science, physics, chemistry, and engineering, is required to address the theoretical and experimental challenges facing the field of genomics, and together promise great breakthroughs in our understanding and engineering of cellular systems. Nucleic Acids Res, 1999 Jun 15, 27(12), 2538 - 44 Interaction between human topoisomerase I and a novel RING finger/arginine-serine protein; Haluska P Jr et al.; The N-terminus of human topoisomerase I participates in the binding of this enzyme to helicases and other proteins . Using the N-terminal 250 amino acids of human topoisomerase I and a yeast two-hybrid/ in vitro binding screen, a novel arginine-serine-rich peptide was identified as a human topoisomerase I-binding protein . The corresponding full-length protein, named topors, contains a consensus RING zinc finger domain and nuclear localization signals in addition to the arginine-serine-rich region . The RING finger domain of topors is homologous to a similar domain in a family of viral proteins that are involved in the regulation of viral transcription . When expressed in HeLa cells as a green fluorescent protein fusion, topors localizes in the nucleus in a punctate pattern and co-immunoprecipitates with topoisomerase I . These data suggest that topors is involved in trans-cription, possibly recruiting topoisomerase I to RNA polymerase II transcriptional complexes. Scand J Immunol, 1999 Jun, 49(6), 620 - 8 Protein-protein interactions between native Ro52 and immunoglobulin G heavy chain; Yang Y et al.; Using a yeast two-hybrid system to search for proteins interacting with Ro52 autoantigen, we identified a novel protein-protein interaction . Two different cDNA clones, which interacted with Ro52 in the yeast two-hybrid system, were identified and isolated from a human B-cell library . Surprisingly, both clones encoded the heavy chain of human IgG1 . The expression of both HIS3 and beta-galactosidase reporter genes in yeast suggested that the interaction between Ro52 and IgG occurred in vivo . In vitro studies utilizing recombinant Ro52 and purified immunoglobulins indicated that the interaction was immunoglobulin class and subclass specific . Ro52 interacted with IgG1 and IgG4, but not with IgG2, IgG3, IgA or IgM . Ro52 could also precipitate IgG directly from serum . The identified cDNA clones did not include the variable region of IgG, which suggested a non-classical interaction independent of antibody specificity . We further mapped the domain of Ro52 responsible for this interaction to the C-terminus rfp-like region . In conclusion, our data support an unusual interaction between native Ro52 and IgG . The potential biological significance of this unusual protein-protein interaction is discussed. Biochemistry, 1999 Jun 1, 38(22), 7227 - 34 Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: mapping of residues that when altered give rise to an activated enzyme; Axelsen KB et al.; The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants . The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme . Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana . Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H+-ATPase . The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP . Residues that when altered lead to increased pump activity group together in two regions of the C-terminus . One region stretches from K863 to L885 and includes two residues (Q879 and R880) that are conserved between plant and fungal H+-ATPases . The other region, incorporating S904 to L919, is situated in an extension of the C-terminus unique to plant H+-ATPases . Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells . Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain exposes a latent binding site for activatory 14-3-3 proteins. Biochemistry, 1999 Jun 1, 38(22), 7111 - 7 Human bleomycin hydrolase binds ribosomal proteins; Koldamova RP et al.; Bleomycin hydrolase (BH) is a cysteine proteinase that inactivates the anticancer drug bleomycin . Yeast BH forms a homohexameric structure that resembles a 20S proteasome and binds to single-stranded RNA and DNA . We now demonstrate that human BH (hBH) interacts and colocalizes with ribosomal proteins . Using a yeast two-hybrid system, we found hBH bound to human homologues of rat ribosomal proteins L11 and L29 . The N-terminus of hBH (amino acids 14-175), which contains a catalytic Cys93, was critical for the binding to L11 in the two-hybrid environment . hBH precipitated 35S-labeled L11 and L29 in vitro, and hBH colocalized with L11 and L29 as determined by immunofluorescence . In addition to cytosolic bleomycin hydrolase, we found abundant bleomycin hydrolase activity associated with the ribosomal subcellular fraction by differential centrifugation . hBH was also detected by Western immunoblotting in a high-speed particulate fraction, where the majority of L11 and L29 were found . In vitro experiments showed recombinant hBH binds to Chinese hamster ovary cell microsomes . Thus, our data strongly suggest that hBH exists as both a free cytosolic and ribosome-associated protein. Biochem Cell Biol, 1998, 76(5), 735 - 41 Regulation and characterization of the Na+/H+ exchanger; Fliegel L et al.; The Na+/H+ exchanger is a ubiquitous protein present in all mammalian cell types that functions to remove one intracellular H+ for one extracellular Na+ . Several isoforms of the protein exist, which are referred to as NHE1 to NHE6 (for Na+/H+ exchanger one through six) . The NHE1 protein was the first isoform cloned and studied in a variety of systems . This review summarizes recent papers on this protein, particularly those that have examined regulation of the protein and its expression and activity. J Mol Med, 1999 Apr, 77(4), 386 - 92 The DNA-binding subunit p140 of replication factor C is upregulated in cycling cells and associates with G1 phase cell cycle regulatory proteins; van der Kuip H et al.; The DNA-binding subunit of replication factor C (RFCp140) plays an important role in both DNA replication and DNA repair . The mechanisms regulating activation of RFCp140 thereby controlling replication and cellular proliferation are largely unknown . We analyzed protein expression of RFCp140 during cell cycle progression and investigated the association of RFCp140 with cell cycle regulatory proteins in cell lines of various tissue origin and in primary hematopoietic cells . Western and Northern blot analyses of RFCp140 from synchronized cells showed downregulation of RFCp140 when cells enter a G0-like quiescent state and upregulation of RFCp140 in cycling cells . Translocation from the cytoplasmic compartment to the nucleus did not account for the significant increase in RFCp140 protein levels observed in cycling cells . To investigate a potential association of RFCp140 with cell cycle regulatory proteins coimmunoprecipitation assays were performed . These studies demonstrated specific binding of RFCp140 to cdk4-kinase in hematopoietic and fibroblast cell lines . Additional coimmunoprecipitation studies revealed specific association of RFCp140 with cyclin D1, p21, proliferating cell nuclear antigen, and retinoblastoma protein . These findings link DNA replication and repair factor RFCp140 to G1 phase cell cycle regulatory elements critically involved in cell cycle control.
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