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 sel