|
|
|
|
|
| Mol Cell Biol, 1999 Nov, 19(11), 7519 - 28 Requirement for Ras/Rac1-mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein; Turkson J et al.; Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors . However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis . In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation . Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3 . We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis . Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK) . Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts . Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation . In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine . Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation . Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein . These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis. J Cell Sci, 1999 Nov, 112 ( Pt 21), 3769 - 77 Protein transport and flagellum assembly dynamics revealed by analysis of the paralysed trypanosome mutant snl-1; Bastin P et al.; The paraflagellar rod (PFR) of Trypanosoma brucei is a large, complex, intraflagellar structure that represents an excellent system in which to study flagellum assembly . Molecular ablation of one of its major constituents, the PFRA protein, in the snl-1 mutant causes considerable alteration of the PFR structure, leading to cell paralysis . Mutant trypanosomes sedimented to the bottom of the flask rather than staying in suspension but divided at a rate close to that of wild-type cells . This phenotype was complemented by transformation of snl-1 with a plasmid overexpressing an epitope-tagged copy of the PFRA gene . In the snl-1 mutant, other PFR proteins such as the second major constituent, PFRC, accumulated at the distal tip of the growing flagellum, forming a large dilation or 'blob' . This was not assembled as filaments and was removed by detergent-extraction . Axonemal growth and structure was unmodified in the snl-1 mutant and the blob was present only at the tip of the new flagellum . Strikingly, the blob of unassembled material was shifted towards the base of the flagellum after cell division and was not detectable when the daughter cell started to produce a new flagellum in the next cell cycle . The dynamics of blob formation and regression are likely indicators of anterograde and retrograde transport systems operating in the flagellum . In this respect, the accumulation of unassembled PFR precursors in the flagellum shows interesting similarities with axonemal mutants in other systems, illustrating transport of components of a flagellar structure during both flagellum assembly and maintenance . Observation of PFR components indicate that these are likely to be regulated and modulated throughout the cell cycle. J Cell Sci, 1999 Nov, 112 ( Pt 21), 3619 - 26 Ca(2+)/calmodulin and p85 cooperatively regulate an initiation of cytokinesis in Tetrahymena; Gonda K et al.; Tetrahymena p85 differs in mobility in two-dimensional SDS-polyacrylamide gel electrophoresis between wild-type and temperature-sensitive cell-division-arrest mutant cdaA1 cell extracts, and is localized to the presumptive division plane before the formation of the division furrow . The p85 contained three identical sequences which show homology to the calmodulin binding site of Ca(2+)/calmodulin dependent protein kinase Type II in Saccharomyces cerevisiae . We found the p85 directly interacts with Tetrahymena calmodulin in a Ca(2+)-dependent manner, using a co-sedimentation assay . We next examined the localization of p85 and calmodulin during cytokinesis using indirect immunofluorescence . The results showed that both proteins colocalize in the division furrow . This is the first observation that calmodulin is localized in the division furrow . Moreover, the direct interaction between p85 and Ca(2+)/calmodulin was inhibited by Ca(2+)/calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl . When the cells were treated with the drug just before the beginning of cytokinesis, the drug also inhibited the localization of p85 and calmodulin to the division plane, and the formation of the contractile ring and division furrow . Therefore, we propose that the Ca(2+)/calmodulin signal and its target protein p85 cooperatively regulate an initiation of cytokinesis and may be also concerned with the progression of cytokinesis in Tetrahymena. J Cell Sci, 1999 Nov, 112 ( Pt 21), 3591 - 601 Aurora/Ipl1p-related kinases, a new oncogenic family of mitotic serine-threonine kinases; Giet R et al.; During the past five years, a growing number of serine-threonine kinases highly homologous to the Saccharomyces cerevisiae Ipl1p kinase have been isolated in various organisms . A Drosophila melanogaster homologue, aurora, was the first to be isolated from a multicellular organism . Since then, several related kinases have been found in mammalian cells . They localise to the mitotic apparatus: in the centrosome, at the poles of the bipolar spindle or in the midbody . The kinases are necessary for completion of mitotic events such as centrosome separation, bipolar spindle assembly and chromosome segregation . Extensive research is now focusing on these proteins because the three human homologues are overexpressed in various primary cancers . Furthermore, overexpression of one of these kinases transforms cells . Because of the myriad of kinases identified, we suggest a generic name: Aurora/Ipl1p-related kinase (AIRK) . We denote AIRKs with a species prefix and a number, e.g . HsAIRK1. Nature, 1999 Sep 30, 401(6752), 485 - 9 Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase; van Wilpe S et al.; Mitochondrial preproteins are imported by a multisubunit translocase of the outer membrane (TOM), including receptor proteins and a general import pore . The central receptor Tom22 binds preproteins through both its cytosolic domain and its intermembrane space domain and is stably associated with the channel protein Tom40 (refs 11-13) . Here we report the unexpected observation that a yeast strain can survive without Tom22, although it is strongly reduced in growth and the import of mitochondrial proteins . Tom22 is a multifunctional protein that is required for the higher-level organization of the TOM machinery . In the absence of Tom22, the translocase dissociates into core complexes, representing the basic import units, but lacks a tight control of channel gating . The single membrane anchor of Tom22 is required for a stable interaction between the core complexes, whereas its cytosolic domain serves as docking point for the peripheral receptors Tom20 and Tom70 . Thus a preprotein translocase can combine receptor functions with distinct organizing roles in a multidomain protein. EMBO J, 1999 Oct 15, 18(20), 5778 - 88 Rat8p/Dbp5p is a shuttling transport factor that interacts with Rat7p/Nup159p and Gle1p and suppresses the mRNA export defect of xpo1-1 cells; Hodge CA et al.; In a screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective for mRNA export, we previously identified the essential DEAD-box protein Dbp5p/Rat8p and the nucleoporin Rat7p/Nup159p . Both are essential for mRNA export . Here we report that Dbp5p and Rat7p interact through their Nterminal domains . Deletion of this portion of Rat7p (Rat7pDeltaN) results in strong defects in mRNA export and eliminates association of Dbp5p with nuclear pores . Overexpression of Dbp5p completely suppressed the growth and mRNA export defects of rat7DeltaN cells and resulted in weaker suppression in cells carrying rat7-1 or the rss1-37 allele of GLE1 . Dbp5p interacts with Gle1p independently of the N-terminus of Dbp5p . Dbp5p shuttles between nucleus and cytoplasm in an Xpo1p-dependent manner . It accumulates in nuclei of xpo1-1 cells and in cells with mutations affecting Mex67p (mex67-5), Gsp1p (Ran) or Ran effectors . Overexpression of Dbp5p prevents nuclear accumulation of mRNA in xpo1-1 cells, but does not restore growth, suggesting that the RNA export defect of xpo1-1 cells may be indirect . In a screen for high-copy suppressors of the rat8-2 allele of DBP5, we identified YMR255w, now called GFD1 . Gfd1p is not essential, interacts with Gle1p and Rip1p/Nup42p, and is found in the cytoplasm and at the nuclear rim. EMBO J, 1999 Oct 15, 18(20), 5703 - 13 Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication; Jiang W et al.; The Cdc7-Dbf4 kinase is essential for regulating initiation of DNA replication in Saccharomyces cerevisiae . Previously, we identified a human Cdc7 homolog, HsCdc7 . In this study, we report the identification of a human Dbf4 homolog, HsDbf4 . We show that HsDbf4 binds to HsCdc7 and activates HsCdc7 kinase activity when HsDbf4 and HsCdc7 are coexpressed in insect and mammalian cells . HsDbf4 protein levels are regulated during the cell cycle with a pattern that matches that of HsCdc7 protein kinase activity . They are low in G(1), increase during G(1)-S, and remain high during S and G(2)-M . Purified baculovirus-expressed HsCdc7-HsDbf4 selectively phosphorylates the MCM2 subunit of the minichromosome maintenance (MCM) protein complex isolated by immunoprecipitation with MCM7 antibodies in vitro . Two-dimensional tryptic phosphopeptide-mapping analysis of in vivo (32)P-labeled MCM2 from HeLa cells reveals that several major tryptic phosphopeptides of MCM2 comigrate with those of MCM2 phosphorylated by HsCdc7-HsDbf4 in vitro, suggesting that MCM2 is a physiological HsCdc7-HsDbf4 substrate . Immunoneutralization of HsCdc7-HsDbf4 activity by microinjection of anti-HsCdc7 antibodies into HeLa cells blocks initiation of DNA replication . These results indicate that the HsCdc7-HsDbf4 kinase is directly involved in regulating the initiation of DNA replication by targeting MCM2 protein in mammalian cells. EMBO J, 1999 Oct 15, 18(20), 5622 - 33 Functional interaction between GCN5 and polyamines: a new role for core histone acetylation; Pollard KJ et al.; Polyamines are organic polycations essential for a wide variety of cellular functions, including nuclear integrity and chromosome condensation . Here we present genetic evidence that depletion of cellular polyamines partially alleviates the defects in HO and SUC2 expression caused by inactivation of the GCN5 histone acetyltransferase . In addition, the combination of polyamine depletion and a sin(-) allele of the histone H4 gene leads to almost complete bypass of the transcriptional requirement for GCN5 . In contrast, polyamine depletion does not alter the transcriptional requirements for the SWI/SNF chromatin remodeling complex nor does depletion lead to global defects in transcriptional regulation . In addition to these genetic studies, we show that polyamines facilitate oligomerization of nucleosomal arrays in vitro, and that polyamine-mediated condensation requires intact core histone N-terminal domains and is inhibited by histone hyperacetylation . Our studies suggest that polyamines are repressors of transcription in vivo, and that one role of histone hyperacetylation is to antagonize the ability of polyamines to stabilize highly condensed states of chromosomal fibers. Plant Cell, 1999 Oct, 11(10), 2045 - 58 Independent signaling pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection by Magnaporthe grisea; Dixon KP et al.; The phytopathogenic fungus Magnaporthe grisea elaborates a specialized infection cell called an appressorium with which it mechanically ruptures the plant cuticle . To generate mechanical force, appressoria produce enormous hydrostatic turgor by accumulating molar concentrations of glycerol . To investigate the genetic control of cellular turgor, we analyzed the response of M . grisea to hyperosmotic stress . During acute and chronic hyperosmotic stress adaptation, M . grisea accumulates arabitol as its major compatible solute in addition to smaller quantities of glycerol . A mitogen-activated protein kinase-encoding gene OSM1 was isolated from M . grisea and shown to encode a functional homolog of HIGH-OSMOLARITY GLYCEROL1 (HOG1), which encodes a mitogen-activated protein kinase that regulates cellular turgor in yeast . A null mutation of OSM1 was generated in M . grisea by targeted gene replacement, and the resulting mutants were sensitive to osmotic stress and showed morphological defects when grown under hyperosmotic conditions . M . grisea deltaosm1 mutants showed a dramatically reduced ability to accumulate arabitol in the mycelium . Surprisingly, glycerol accumulation and turgor generation in appressoria were unaltered by the Deltaosm1 null mutation, and the mutants were fully pathogenic . This result indicates that independent signal transduction pathways regulate cellular turgor during hyperosmotic stress and appressorium-mediated plant infection . Consistent with this, exposure of M . grisea appressoria to external hyperosmotic stress induced OSM1-dependent production of arabitol. J Biol Chem, 1999 Oct 22, 274(43), 30963 - 8 The ubiquitin-conjugating enzymes UbcH7 and UbcH8 interact with RING finger/IBR motif-containing domains of HHARI and H7-AP1; Moynihan TP et al.; Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) . However, cognate E3s and/or substrate proteins have been identified for only a few E2s . To identify proteins that can interact with the human E2 UbcH7, a yeast two-hybrid screen was performed . Two proteins were identified and termed human homologue of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1) . Both proteins, which are widely expressed, are characterized by the presence of RING finger and in between RING fingers (IBR) domains . No other overt structural similarity was observed between the two proteins . In vitro binding studies revealed that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in the interaction of these proteins with UbcH7 . Furthermore, binding of these two proteins to UbcH7 is specific insofar that both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1 . Although it is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3 activity, our data suggests that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome pathway. J Biol Chem, 1999 Oct 22, 274(43), 30896 - 905 Identification of Tek/Tie2 binding partners . Binding to a multifunctional docking site mediates cell survival and migration; Jones N et al.; The Tek/Tie2 receptor tyrosine kinase plays a pivotal role in vascular and hematopoietic development . To study the signal transduction pathways that are mediated by this receptor, we have used the yeast two-hybrid system to identify signaling molecules that associate with the phosphorylated Tek receptor . Using this approach, we demonstrate that five molecules, Grb2, Grb7, Grb14, Shp2, and the p85 subunit of phosphatidylinositol 3-kinase can interact with Tek in a phosphotyrosine-dependent manner through their SH2 domains . Mapping of the binding sites of these molecules on Tek reveals the presence of a multisubstrate docking site in the carboxyl tail of Tek (Tyr(1100)) . Mutation of this site abrogates binding of Grb2 and Grb7 to Tek in vivo, and this site is required for tyrosine phosphorylation of Grb7 and p85 in vivo . Furthermore, stimulation of Tek-expressing cells with Angiopoietin-1 results in phosphorylation of both Tek and p85 and in activation of endothelial cell migration and survival pathways that are dependent in part on phosphatidylinositol 3-kinase . Taken together, these results demonstrate that Angiopoietin-1-induced signaling from the Tek receptor is mediated by a multifunctional docking site that is responsible for activation of both cell migration and cell survival pathways. J Biol Chem, 1999 Oct 22, 274(43), 30887 - 95 Leucine zipper-mediated homodimerization of the adaptor protein c-Cbl . A role in c-Cbl's tyrosine phosphorylation and its association with epidermal growth factor receptor; Bartkiewicz M et al.; The 120-kDa proto-oncogenic protein c-Cbl is a multidomain adaptor protein that is phosphorylated in response to the stimulation of a broad range of cell surface receptors and participates in the assembly of signaling complexes that are formed as a result of the activation of various signal transduction pathways . Several structural features of c-Cbl, including the phosphotyrosine-binding domain, proline-rich domain, and motifs containing phosphotyrosine and phosphoserine residues, mediate the association of c-Cbl with other components of these complexes . In addition to those domains that have been demonstrated to play a role in the binding of c-Cbl to other signaling molecules, c-Cbl also contains a RING finger motif and a putative leucine zipper . In this study, we demonstrate that the previously identified putative leucine zipper mediates the formation of Cbl homodimers . Using the yeast two-hybrid system, we show that deletion of the leucine zipper domain is sufficient to abolish Cbl homodimerization, while Cbl mutants carrying extensive N-terminal truncations retain the ability to dimerize with the full-length Cbl . The requirement of the leucine zipper for the homodimerization of Cbl was confirmed by in vitro binding assays, using deletion variants of the C-terminal half of Cbl with and without the leucine zipper domain, and in cells using Myc and green fluorescent protein (GFP) N-terminal-tagged Cbl variants . In cells, the deletion of the leucine zipper caused a decrease in both the tyrosine phosphorylation of Cbl and its association with the epidermal growth factor receptor following stimulation with epidermal growth factor, thus demonstrating a role for the leucine zipper in c-Cbl's signaling functions . Thus, the leucine zipper domain enables c-Cbl to homodimerize, and homodimerization influences Cbl's signaling function, modulating the activity of Cbl itself and/or affecting Cbl's associations with other signaling proteins in the cell. J Biol Chem, 1999 Oct 22, 274(43), 30527 - 33 The human PICD gene encodes a cytoplasmic and peroxisomal NADP(+)-dependent isocitrate dehydrogenase; Geisbrecht BV et al.; Human PICD was identified by homology probing the data base of expressed sequence tags with the protein sequence of Saccharomyces cerevisiae Idp3p, a peroxisomal NADP(+)-dependent isocitrate dehydrogenase . The human PICD cDNA contains a 1242-base pair open reading frame, and its deduced protein sequence is 59% identical to yeast Idp3p . Expression of PICD partially rescued the fatty acid growth defect of the yeast idp3 deletion mutant suggesting that PICD is functionally homologous to Idp3p . Kinetic studies on bacterially expressed PICD demonstrated that this enzyme catalyzed the oxidative decarboxylation of isocitrate to 2-oxoglutarate with a specific activity of 22.5 units/mg and that PICD displayed K(M) values of 76 microM for isocitrate and 112 microM for NADP(+) . In subcellular fractionation experiments, we found PICD in both peroxisomes and cytoplasm of human and rat liver cells, with approximately 27% of total PICD protein associated with peroxisomes . The presence of PICD in mammalian peroxisomes suggests roles in the regeneration of NADPH for intraperoxisomal reductions, such as the conversion of 2, 4-dienoyl-CoAs to 3-enoyl-CoAs, as well as in peroxisomal reactions that consume 2-oxoglutarate, namely the alpha-hydroxylation of phytanic acid . As for cytoplasmic PICD, the phenotypes of patients with glucose-6-phosphate dehydrogenase deficiency (Luzzatto, L., and Mehta, A . (1995) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C . R., Beaudet, A . L., Sly, W . S., and Valle, D., eds) Vol . 3, 7th Ed., pp . 3367-3398, McGraw-Hill Inc., New York) suggest that PICD serves a significant role in cytoplasmic NADPH production, particularly under conditions that do not favor the use of the hexose monophosphate shunt (Luzzatto et al.). J Biol Chem, 1999 Oct 22, 274(43), 30393 - 401 An RNA binding motif in the Cbp2 protein required for protein-stimulated RNA catalysis; Tirupati HK et al.; The fifth and terminal intron of yeast cytochrome b pre-mRNA (a group I intron) requires a protein encoded by the nuclear gene CBP2 for splicing . Because catalysis is intrinsic to the RNA, the protein is believed to promote formation of secondary and tertiary structure of the RNA, resulting in a catalytically competent intron . In vitro, this mitochondrial intron can be made to self-splice or undergo protein-facilitated splicing by varying the Mg(2+) and monovalent salt concentrations . This two-component system, therefore, provides a good model for understanding the role of proteins in RNA folding . A UV cross-linking experiment was initiated to identify RNA binding sites on Cbp2 and gain insights into Cbp2-intron interactions . A 12-amino acid region containing a presumptive contact site near the amino terminus was targeted for mutagenesis, and mutant proteins were characterized for RNA binding and stimulation of splicing . Mutations in this region resulted in partial or complete loss of function, demonstrating the importance of this determinant for stimulation of RNA splicing . Several of the mutations that severely reduced splicing did not significantly shift the overall binding isotherm of Cbp2 for the precursor RNA, suggesting that contacts critical for activity are not necessarily reflected in the dissociation constant . This analysis has identified a unique RNA binding motif of alternating basic and aromatic residues that is essential for protein facilitated splicing. Biochemistry, 1999 Oct 12, 38(41), 13670 - 83 Structure-activity relationships in the oxidation of para-substituted benzylamine analogues by recombinant human liver monoamine oxidase A; Miller JR et al.; Monoamine oxidase A (MAO A) plays a central role in the oxidation of amine neurotransmitters . To investigate the structure and mechanism of this enzyme, recombinant human liver MAO A was expressed and purified from Saccharomyces cerevisiae . Anaerobic titrations of the enzyme require only 1 mol of substrate per mole of enzyme-bound flavin for complete reduction . This demonstrates that only one redox-active group (i.e., the covalent FAD cofactor) is involved in catalysis . The reaction rates and binding affinities of 17 para-substituted benzylamine analogues with purified MAO A were determined by steady state and stopped flow kinetic experiments . For each substrate analogue that was tested, the rates of steady state turnover (k(cat)) and anaerobic flavin reduction (k(red)) are similar in value . Deuterium kinetic isotope effects on k(cat), k(red), k(cat)/K(m), and k(red)/K(s) with alpha, alpha-{(2)H}benzylamines are similar for each substrate analogue that was tested and range in value from 6 to 13, indicating that alpha-C-H bond cleavage is rate-limiting in catalysis . Substrate analogue dissociation constants determined from reductive half-reaction experiments as well as from steady state kinetic isotope effect data {Klinman, J . P., and Matthews, R . G . (1985) J . Am . Chem . Soc . 107, 1058-1060} are in excellent agreement . Quantitative structure-activity relationship (QSAR) analysis of dissociation constants shows that the binding of para-substituted benzylamine analogues to MAO A is best correlated with the van der Waals volume of the substituent, with larger substituents binding most tightly . The rate of para-substituted benzylamine analogue oxidation and/or substrate analogue-dependent flavin reduction is best correlated with substituent electronic effects (sigma) . Separation of the electronic substituent parameter (sigma) into field-inductive and resonance effects provides a more comprehensive treatment of the electronic correlations . The positive correlation of rate with sigma (rho approximately 2.0) suggests negative charge development at the benzyl carbon position occurs and supports proton abstraction as the mode of alpha-C-H bond cleavage . These results are discussed in terms of several mechanisms proposed for MAO catalysis and with previous structure-activity studies published with bovine liver MAO B {Walker, M . C., and Edmondson, D . E . (1994) Biochemistry 33, 7088-7098}. Cell Res, 1999 Sep, 9(3), 179 - 88 The Notch-Hes pathway in mammalian neural development; Kageyama R et al.; A wide variety of neurons and glial cells differentiate from common precursor cells in the developing nervous system . During this process, Notch-mediated cell-cell interaction is essential for maintenance of dividing cells and subsequent generation of cell type diversity . Activation of Notch inhibits cellular differentiation, and abnormality of the Notch pathway leads to premature neuronal differentiation, the lack of some cell types, and severe defects of tissue morphogenesis . Recent data demonstrate that Notch fails to inhibit cellular differentiation in the absence of the bHLH genes Hes1 and Hes5, which functionally antagonize the neuronal bHLH genes such as Mash1 . These results indicate that the two Hes genes are essential effectors for the Notch pathway and that neuronal differentiation is controlled by the pathway "Notch-->Hes1/Hes5- inverted question markMash1". Cell Res, 1999 Sep, 9(3), 163 - 70 Regulation of eukaryotic DNA replication and nuclear structure; Rui WJ; In eukaryote, nuclear structure is a key component for the functions of eukaryotic cells . More and more evidences show that the nuclear structure plays important role in regulating DNA replication . The nuclear structure provides a physical barrier for the replication licensing, participates in the decision where DNA replication initiates, and organizes replication proteins as replication factory for DNA replication . Through these works, new concepts on the regulation of DNA replication have emerged, which will be discussed in this minireview. Cancer Res, 1999 Oct 1, 59(19), 4864 - 9 Adrenal androgens stimulate the proliferation of breast cancer cells as direct activators of estrogen receptor alpha; Maggiolini M et al.; Estrogens stimulate the proliferation of many breast tumors and cell lines derived from them . Antiestrogens have therefore become a powerful therapeutic agent to treat breast tumors that express estrogen receptor (ER) alpha . In addition, aromatase inhibitors are now used in postmenopausal women to block the in situ conversion of adrenal androgens to estrogens . This approach can only be successful if ER-alpha in a particular tumor is not directly stimulated by adrenal androgens . We have examined this possibility using several different cell lines as model systems: (a) wild-type MCF7 cells, an ER-alpha-dependent human breast cancer cell line; (b) MCF7SH cells, an estrogen-independent MCF7 variant; (c) Ishikawa cells, an ER-alpha-containing human uterine cell line; (d) ER-negative HeLa cells; and (e) budding yeast . Transactivation assays with transfected ER-alpha reporter genes reveal a direct activation of ER-alpha by dehydroepiandrosterone (DHEA), 5alpha-androstene-3beta,17beta-diol, testosterone, and the two nonaromatizable androgens, dihydrotestosterone and 5alpha-androstane-3beta,17beta-diol . The involvement of other steroid receptors could be ruled out with specific antihormones . Moreover, the same set of ligands stimulates the proliferation of the two breast cancer cell lines . At subsaturating and physiologically relevant concentrations of DHEA, DHEA stimulates the proliferation of MCF7SH cells, which correlates with a substantial, albeit submaximal, transcriptional response . Thus, adrenal androgens must also be considered as risk factors in postmenopausal women. Cancer Res, 1999 Oct 1, 59(19), 4765 - 9 Dominant-negative mutations of the tumor suppressor p53 relating to early onset of glioblastoma multiforme; Marutani M et al.; Previous experiments have suggested that some mutant forms of p53 are able to inactivate the endogenous wild-type p53 protein in a dominant-negative fashion . However, it remains unknown whether tumors with such dominant-negative (transdominant) p53 mutants have a biological significance that is different from that of recessive p53 mutants . In this study, we examined the dominant-negative potential of various p53 mutants using a yeast-based assay in which both wild-type and mutant p53 were efficiently expressed . We tested a total of 106 p53 mutants, which were identified in brain tumors, glioblastoma multiforme-derived cell lines, breast cancers, or premalignant lesions and squamous cell carcinomas of oral epithelium or were otherwise created by mutagenesis . In agreement with the previous studies, our results demonstrated that transdominant mutations affected amino acid residues that are essential for the stabilization of the DNA-binding surface in the p53 core domain and for the direct interaction of p53 with its DNA-binding sequence . Among 40 patients with sporadic glioblastomas, the average age at diagnosis was significantly younger in the patients with tumors harboring dominant-negative mutations (30.4 +/- 14.7 years, n = 7) than it was in those with recessive mutations (55.2 +/- 18.6 years, n = 9, P < 0.012) and in those without mutations (54.7 +/- 17.1 years, n = 24, P < 0.003) . Our data suggest that dominant-negative p53 mutants accelerate development and/or growth of glioblastoma anlagen. Biol Cell, 1999 Jul, 91(6), 461 - 70 The Xenopus laevis centrosome aurora/Ipl1-related kinase; Giet R et al.; The cDNA encoding the protein kinase pEg2 was originally cloned through a differential screening performed during the early development of Xenopus laevis . pEg2 orthologues were found in various organisms and were classified in a new family of oncogenic mitotic protein kinases named 'aurora/Ipl1-related kinases' after the Drosophila melanogaster gene aurora and the Saccharomyces cerevisiae gene Ipl1 . The catalytic activity of pEg2 is necessary for the mitotic microtubule spindle formation in Xenopus laevis egg extracts . The addition of a dominant negative form of pEg2 to in vitro spindle assembly assays leads to monopolar spindles generated by a defect of centrosome separation . In Xenopus cultured cells, pEg2 was confined around the pericentriolar material once centrosomes were duplicated . The centrosome localization does not depend on the presence of microtubules . However, in vitro, the protein binds to taxol-stabilized microtubules independently of its kinase activity . During mitosis the location of the protein changes, in metaphase the kinase localizes on the microtubules at the poles of the mitotic spindle whereas it is not present on astral microtubules . This localization persists until the segregation of the chromosomes is completed . The presence of the kinase on the spindle may reveal another yet unknown function. FEBS Lett, 1999 Sep 10, 458(1), 11 - 6 Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors; Tanaka AS et al.; The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage . A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid . The mutations were restricted to the P1-P4' positions of the reactive site . Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing . The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system . The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase . The variant LDTI-10T binds to thrombin but does not inhibit it . The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V) . The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions. Proc Natl Acad Sci U S A, 1999 Oct 12, 96(21), 11752 - 7 An internal targeting signal directing proteins into the mitochondrial intermembrane space; Diekert K et al.; Import of most nucleus-encoded preproteins into mitochondria is mediated by N-terminal presequences and requires a membrane potential and ATP hydrolysis . Little is known about the chemical nature and localization of other mitochondrial targeting signals or of the mechanisms by which they facilitate membrane passage . Mitochondrial heme lyases lack N-terminal targeting information . These proteins are localized in the intermembrane space and are essential for the covalent attachment of heme to c type cytochromes . For import of heme lyases, the translocase of the mitochondrial outer membrane complex is both necessary and sufficient . Here, we report the identification of the targeting signal of mitochondrial heme lyases in the third quarter of these proteins . The targeting sequence is highly conserved among all known heme lyases . Its chemical character is hydrophilic because of a large fraction of both positively and negatively charged amino acid residues . These features clearly distinguish this signal from classical presequences . When inserted into a cytosolic protein, the targeting sequence directs the fusion protein into the intermembrane space, even in the absence of a membrane potential or ATP hydrolysis . The heme lyase targeting sequence represents the first topogenic signal for energy-independent transport into the intermembrane space and harbors two types of information . It assures accurate recognition and translocation by the translocase of the mitochondrial outer membrane complex, and it is responsible for driving the import reaction by undergoing high-affinity interactions with components of the intermembrane space. Mol Cell, 1999 Sep, 4(3), 445 - 50 The centromeric sister chromatid cohesion site directs Mcd1p binding to adjacent sequences; Megee PC et al.; Cohesion between sister chromatids occurs along the length of chromosomes, where it plays essential roles in chromosome segregation . We show here that the centromere, a cis-acting cohesion factor, directs the binding of Mcd1p, a cohesin subunit, to at least 2 kb regions flanking centromeres in a sequence-independent manner . The centromere is essential for the maintenance as well as the establishment of this cohesin domain . The efficiency of Mcd1p binding within the cohesin domain is independent of the primary nucleotide sequence of the centromere-flanking DNA but correlates with high A + T DNA content . Thus, the function of centromeres in the cohesion of centromere-proximal regions may be analogous to that of enhancers, nucleating cohesin complex binding over an extended chromosomal domain of A + T-rich DNA. Mol Cell, 1999 Sep, 4(3), 353 - 63 Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase; Levine K et al.; To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding . Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes . By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer . Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation. FEBS Lett, 1999 Oct 8, 459(2), 263 - 6 Pore-forming properties of elicitors of plant defense reactions and cellulolytic enzymes; Klusener B et al.; Using the planar lipid bilayer technique, it is shown that a yeast elicitor as well as several cellulolytic enzymes used in protoplasting plant cells contain components which strongly interact with the bilayers . This results in the appearance of transmembrane ion fluxes which may pass through membrane defect structures and even large conductance pores with unitary conductances above 400 pS . Since membrane depolarization is an immediate response in the process of defense elicitation in plant cells, elicitors may act directly with the lipid phase of cell membranes, causing depolarizations and thus initiating the process of elicitation . When using enzymatically prepared protoplasts in electrophysiological work, contributions to electrical activity by membrane active constituents originating from the enzymes used must be expected. Plant Physiol, 1999 Oct, 121(2), 391 - 7 The enzymatic activity of fungal xylanase is not necessary for its elicitor activity; Enkerli J et al.; Fungal xylanases from Trichoderma spp . are potent elicitors of defense responses in various plants . To determine whether enzymatic activity is necessary for elicitor activity, we used site-directed mutagenesis to reduce the catalytic activity of xylanase II from Trichoderma reesei . For this, the glutamic acid residue at position 210, which is part of the active center in this family of enzymes, was changed to either aspartic acid (E210D) or serine (E210S) . Wild-type and mutated forms of xylanase II were expressed in yeast cells and purified to homogeneity . Compared with the wild-type form of xylanase II, E210D had >100-fold and E210S 1,000-fold lower enzymatic activity . In contrast, these mutated forms showed no comparable drop in elicitor activity . They fully stimulated medium alkalinization and ethylene biosynthesis in suspension-cultured tomato (Lycopersicon esculentum) cells, as well as hypersensitive necrosis in leaves of tomato and tobacco (Nicotiana tabacum) plants . These results provide direct evidence that enzyme activity is not necessary for elicitor activity of fungal xylanase. Plant Physiol, 1999 Oct, 121(2), 353 - 61 Overexpression of a gene that encodes the first enzyme in the biosynthesis of asparagine-linked glycans makes plants resistant to tunicamycin and obviates the tunicamycin-induced unfolded protein response; Koizumi N et al.; The cytotoxic drug tunicamycin kills cells because it is a specific inhibitor of UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase (GPT), an enzyme that catalyzes the initial step of the biosynthesis of dolichol-linked oligosaccharides . In the presence of tunicamycin, asparagine-linked glycoproteins made in the endoplasmic reticulum are not glycosylated with N-linked glycans, and therefore may not fold correctly . Such proteins may be targeted for breakdown . Cells that are treated with tunicamycin normally experience an unfolded protein response and induce genes that encode endoplasmic reticulum chaperones such as the binding protein (BiP) . We isolated a cDNA clone for Arabidopsis GPT and overexpressed it in Arabidopsis . The transgenic plants have a 10-fold higher level of GPT activity and are resistant to 1 microg/mL tunicamycin, a concentration that kills control plants . Transgenic plants grown in the presence of tunicamycin have N-glycosylated proteins and the drug does not induce BiP mRNA levels as it does in control plants . BiP mRNA levels are highly induced in both control and GPT-expressing plants by azetidine-2-carboxylate . These observations suggest that excess GPT activity obviates the normal unfolded protein response that cells experience when exposed to tunicamycin. Mol Gen Genet, 1999 Sep, 262(2), 230 - 8 Molecular characterization of mitotic cyclins in rice plants; Umeda M et al.; Cyclins are known to activate cyclin-dependent protein kinases, which are essential for cell cycle progression in eukaryotes . We isolated full-length cDNAs encoding rice mitotic cyclins named CycA1; os; 1 and CycB2;os;1, which are related to A- and B-type cyclins, respectively, from animals . To characterize the function of these mitotic cyclins, as well as that of another B-type cyclin, CycB2;os;2, each cDNA was introduced into yeast cells . When cDNAs encoding CycA1;os;1, CycB2;os; or CycB2;os;2 were overexpressed in the yeast mutant DLI, which is deficient in G1 cyclins, the mutant phenotype was rescued, indicating that these mitotic cyclins are functional in yeast cells . When the cDNA encoding CycB2;os;1 was expressed in the wild-type yeast strain, the cells lost the ability to grow, whereas the expression of either cycA1;os: 1 or cycB2;os;2 did not inhibit growth . In situ hybridization of these mitotic cyclin genes with rice root apices and counterstaining of chromosomes with a DNA-specific dye revealed that cycA1;os;1 is expressed from the G2 phase to the early M phase, while transcripts of cycB2:os;1 and cycB2;os;2 accumulated until the end of mitosis . Our results indicate that these B2-type cyclins may be involved in the control of mitosis, in combination with a G2/M-phase CDK. Mol Gen Genet, 1999 Sep, 262(2), 220 - 9 Identification, characterization and chromosomal localization of the cognate human and murine DBF4 genes; Lepke M et al.; The kinase Dbf4p/Cdc7p is required for the G1/S phase transition during the cell cycle and plays a direct role in the activation of individual origins of replication in Saccharomyces cerevisiae . Here, we report the identification and characterization of mouse and human cDNAs whose products are related in sequence to Saccharomyces cerevisiae DBF4 cDNA . Both mammalian Dbf4 proteins contain a putative site for phosphorylation by CDK, PEST protease cleavage sites, nuclear localization signals and a short-looped zinc finger-like domain . Transcription of MmDBF4 is suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation . Upon replenishment of the medium, transcript levels increase during progression through G1, peaking as cells enter S phase . MmDbf4p interacts physically with Cdc7p and Mcm2p in vivo . Using fluorescence in situ hybridization (FISH), the human DBF4 gene was localized to chromosome 7 (q21.3), whereas FISH mapped the murine counterpart to band A2 on chromosome 5 . The results of chromosome mapping indicate that in both mouse and human the gene is present as a single copy . The structural conservation between Dbf4-related proteins suggests that these proteins play a key role in the regulation of DNA replication during the cell cycle in all eukaryotes. Philos Trans R Soc Lond B Biol Sci, 1999 Aug 29, 354(1388), 1471 - 8 The Croonian Lecture 1999 . Intracellular membrane traffic: getting proteins sorted; Pelham HR; The secretory and endocytic pathways within higher cells consist of multiple membrane-bound compartments, each with a characteristic composition, through which proteins move on their way to or from the cell surface . Sorting of proteins within this system is achieved by their selective incorporation into budding vesicles and the specific fusion of these with an appropriate target membrane . Cytosolic coat proteins help to select vesicle contents, while fusion is mediated by membrane proteins termed SNAREs present in both vesicles and target membranes . SNAREs are not the sole determinants of target specificity, but they lie at the heart of the fusion process . The complete set of SNAREs is known in yeast, and analysis of their locations, interactions and functions in vivo gives a comprehensive picture of the traffic routes and the ways in which organelles such as the Golgi apparatus are formed . The principles of protein and lipid sorting revealed by this analysis are likely to apply to a wide variety of eukaryotic cells. Yeast, 1999 Oct, 15(14), 1529 - 39 Disruption and functional analysis of six ORFs on chromosome XV: YOL117w, YOL115w ( TRF4), YOL114c, YOL112w ( MSB4), YOL111c and YOL072w; Iwanejko L et al.; We have carried out the systematic disruption of six ORFs on chromosome XV, of Saccharomyces cerevisiae using the long flanking homology technique to replace each with the KanMX cassette; we have also constructed plasmids containing replacement cassettes and cognate clones for each ORF . Disruption of three of the ORFs-YOL117w, YOL114c, and YOL112w (also known as MSB4)-does not result in any noteworthy phenotype with respect to temperature or nutritional requirements, but yol112w mutants with an additional disruption of YNL293w, which encodes a protein similar to Yol112w, exhibit a slow growth phenotype . The protein specified by YOL114c shares similarity with the human DS-1 protein . Disruption of YOL115w confers slow growth, cold sensitivity and poor sporulation; this ORF has been described elsewhere as TRF4, which encodes a topoisomerase I-related protein . Cells with disruptions of YOL111c, whose product is weakly similar to the human ubiquitin-like protein GdX, are slightly impaired in mating . Mutants disrupted for YOL072w, the predicted product of which is unrelated to any protein of known function, grow slowly, are cold-sensitive and sporulate with reduced efficiency . J Biol Chem, 1999 Oct 15, 274(42), 30297 - 302 Mutations in a GTP-binding motif of eukaryotic elongation factor 1A reduce both translational fidelity and the requirement for nucleotide exchange; Carr-Schmid A et al.; A series of mutations in the highly conserved N(153)KMD(156)GTP-binding motif of the Saccharomyces cerevisiae translation elongation factor 1A (eEF1A) affect the GTP-dependent functions of the protein and increase misincorporation of amino acids in vitro . Two critical regulatory processes of translation elongation, guanine nucleotide exchange and translational fidelity, were analyzed in strains with the N153T, D156N, and N153T/D156E mutations . These strains are omnipotent suppressors of nonsense mutations, indicating reduced A site fidelity, which correlates with changes either in total translation rates in vivo or in GTPase activity in vitro . All three mutant proteins also show an increase in the K(m) for GTP . An in vivo system lacking the guanine nucleotide exchange factor eukaryotic elongation factor 1Balpha (eEF1Balpha) and supported for growth by excess eEF1A was used to show the two mutations with the highest K(m) for GTP restore most but not all growth defects found in these eEF1Balpha deficient-strains to near wild type . An increase in K(m) alone, however, is not sufficient for suppression and may indicate eEF1Balpha performs additional functions . Additionally, eEF1A mutations that suppress the requirement for guanine nucleotide exchange may not effectively perform all the functions of eEF1A in vivo. J Biol Chem, 1999 Oct 15, 274(42), 29897 - 904 Characterization of human and murine PMP20 peroxisomal proteins that exhibit antioxidant activity in vitro; Yamashita H et al.; We have isolated the cDNAs encoding human and mouse homologues of a yeast protein, termed peroxisomal membrane protein 20 (PMP20) . Comparison of the amino acid sequences of human (HsPMP20) and mouse (MmPMP20) PMP20 proteins revealed a high degree of identity (93%), whereas resemblance to the yeast Candida boidinii PMP20A and PMP20B (CbPMP20A and CbPMP20B) was less (30% identity) . Both HsPMP20 and MmPMP20 lack transmembrane regions, as do CbPMP20A and CbPMP20B . HsPMP20 mRNA expression was low in human fetal tissues, especially in the brain . In adult tissues, HsPMP20 mRNA was expressed in the majority of tissues tested . HsPMP20 and MmPMP20 contained the C-terminal tripeptide sequence Ser-Gln-Leu (SQL), which is similar to the peroxisomal targeting signal 1 utilized for protein import into peroxisomes . HsPMP20 bound directly to the human peroxisomal targeting signal 1 receptor, HsPEX5 . Mutagenesis analysis showed that the C-terminal tripeptide sequence, SQL, of HsPMP20 is necessary for its binding to HsPEX5 . Subcellular fractionation of HeLa cells, expressing epitope-tagged PMP20, revealed that HsPMP20 is localized in the cytoplasm and in a particulate fraction containing peroxisomes . Double-staining immunofluorescence studies showed colocalization of HsPMP20 and thiolase, a bona fide peroxisomal protein . The amino acid sequence alignment of HsPMP20, MmPMP20, CbPMP20A, and CbPMP20B displayed high similarity to thiol-specific antioxidant proteins . HsPMP20 exerted an inhibitory effect on the inactivation of glutamine synthetase in the thiol metal-catalyzed oxidation system but not in the nonthiol metal-catalyzed oxidation system, suggesting that HsPMP20 possesses thiol-specific antioxidant activity . In addition, HsPMP20 removed hydrogen peroxide by its thiol-peroxidase activity . These results indicate that HsPMP20 is imported into the peroxisomal matrix via PEX5p and may work to protect peroxisomal proteins against oxidative stress . Because some portion of PMP20 might also be present in the cytosol, HsPMP20 may also have a protective effect in the cytoplasm. Immunity, 1999 Sep, 11(3), 339 - 48 Ku in the cytoplasm associates with CD40 in human B cells and translocates into the nucleus following incubation with IL-4 and anti-CD40 mAb; Morio T et al.; CD40 plays a critical role in survival, growth, differentiation, and class switching of B lymphocytes . Although Ku is required for immunoglobulin class switching, how CD40 signal transduction is coupled to Ku is still unknown . Here, we show that CD40 directly interacts with Ku through the membrane-proximal region of cytoplasmic CD40 . Ku was confined to the cytoplasm in human primary B cells, and the engagement of CD40 on the B cells cultured in the presence of IL-4 resulted in the dissociation of Ku from CD40, translocation of Ku into the nucleus, and increase in the activity of DNA-dependent protein kinase . These findings indicate that Ku is involved in the CD40 signal transduction pathway and may play an important role in the CD40-mediated events. J Neurobiol, 1999 Nov 5, 41(2), 189 - 207 Genetic analysis of the Drosophila ellipsoid body neuropil: organization and development of the central complex; Renn SC et al.; The central complex is an important center for higher-order brain function in insects . It is an intricate neuropil composed of four substructures . Each substructure contains repeated neuronal elements which are connected by processes such that topography is maintained . Although the neuronal architecture has been described in several insects and the behavioral role investigated in various experiments, the exact function of this neuropil has proven elusive . To describe the architecture of the central complex, we study 15 enhancer-trap lines that label various ellipsoid body neuron types . We find evidence for restriction of gene expression that is correlated with specific neuronal types: such correlations suggest functional classifications as well . We show that some enhancer-trap patterns reveal a single ellipsoid body neuron type, while others label multiple types . We describe the development of the ellipsoid body neuropil in wild-type animals and propose developmental mechanisms based on animals displaying structural mutations of this neuropil . The experiments performed here demonstrate the degree of resolution possible from the analysis of enhancer-trap lines and form a useful library of tools for future structure/function studies of the ellipsoid body . Mol Biol Cell, 1999 Oct, 10(10), 3317 - 29 High-copy suppressor analysis reveals a physical interaction between Sec34p and Sec35p, a protein implicated in vesicle docking; Kim DW et al.; A temperature-sensitive mutant, sec34-2, is defective in the late stages of endoplasmic reticulum (ER)-to-Golgi transport . A high-copy suppressor screen that uses the sec34-2 mutant has resulted in the identification of the SEC34 structural gene and a novel gene called GRP1 . GRP1 encodes a previously unidentified hydrophilic yeast protein related to the mammalian Golgi protein golgin-160 . Although GRP1 is not essential for growth, the grp1Delta mutation displays synthetic lethal interactions with several mutations that result in ER accumulation and a block in the late stages of ER-to-Golgi transport, but not with those that block the budding of vesicles from the ER . Our findings suggest that Grp1p may facilitate membrane traffic indirectly, possibly by maintaining Golgi function . In an effort to identify genes whose products physically interact with Sec34p, we also tested the ability of overexpressed SEC34 to suppress known secretory mutations that block vesicular traffic between the ER and the Golgi . This screen revealed that SEC34 specifically suppresses sec35-1 . SEC34 encodes a hydrophilic protein of approximately 100 kDa . Like Sec35p, which has been implicated in the tethering of ER-derived vesicles to the Golgi, Sec34p is predominantly soluble . Sec34p and Sec35p stably associate with each other to form a multiprotein complex of approximately 480 kDa . These data indicate that Sec34p acts in conjunction with Sec35p to mediate a common step in vesicular traffic. Mol Biol Cell, 1999 Oct, 10(10), 3263 - 77 Phosphorylation controls timing of Cdc6p destruction: A biochemical analysis; Elsasser S et al.; The replication initiation protein Cdc6p forms a tight complex with Cdc28p, specifically with forms of the kinase that are competent to promote replication initiation . We now show that potential sites of Cdc28 phosphorylation in Cdc6p are required for the regulated destruction of Cdc6p that has been shown to occur during the Saccharomyces cerevisiae cell cycle . Analysis of Cdc6p phosphorylation site mutants and of the requirement for Cdc28p in an in vitro ubiquitination system suggests that targeting of Cdc6p for degradation is more complex than previously proposed . First, phosphorylation of N-terminal sites targets Cdc6p for polyubiquitination probably, as expected, through promoting interaction with Cdc4p, an F box protein involved in substrate recognition by the Skp1-Cdc53-F-box protein (SCF) ubiquitin ligase . However, in addition, mutation of a single, C-terminal site stabilizes Cdc6p in G2 phase cells without affecting substrate recognition by SCF in vitro, demonstrating a second and novel requirement for specific phosphorylation in degradation of Cdc6p . SCF-Cdc4p- and N-terminal phosphorylation site-dependent ubiquitination appears to be mediated preferentially by Clbp/Cdc28p complexes rather than by Clnp/Cdc28ps, suggesting a way in which phosphorylation of Cdc6p might control the timing of its degradation at then end of G1 phase of the cell cycle . The stable cdc6 mutants show no apparent replication defects in wild-type strains . However, stabilization through mutation of three N-terminal phosphorylation sites or of the single C-terminal phosphorylation site leads to dominant lethality when combined with certain mutations in the anaphase-promoting complex. Biochem Biophys Res Commun, 1999 Oct 5, 263(3), 743 - 8 Protein kinase CKII interacts with and phosphorylates the SAG protein containing ring-H2 finger motif; Son MY et al.; To investigate the biological function of CKII, we have identified proteins that interact with the subunits of CKII using the yeast two-hybrid system . Here we report that SAG, an antioxidant protein containing Ring-H2 finger motif, is a cellular partner associating with the beta subunit of CKII . SAG does not interact with the alpha subunit of CKII . Analysis of SAG deletion mutants indicates that the Ring-H2 motif of SAG is necessary and sufficient for its binding to the beta subunit of CKII . Recombinant SAG can be phosphorylated by CKII in vitro, providing evidence that the beta subunit mediates the interaction of CKII enzyme with substrate proteins . Overlay experiment shows that SAG and the beta subunit of CKII associate directly in vitro and that CKII-mediated phosphorylation of SAG does not affect the interaction between SAG and the beta subunit of CKII . Northern blot analysis indicates that both SAG and the beta subunit of CKII were relatively rich in human heart, liver, skeletal muscle, and pancreas, but were detected in only trace amounts in brain, placenta, and lung . Our present results suggest that CKII may play a role in the regulation of SAG function . J Mol Biol, 1999 Oct 8, 292(5), 987 - 1001 The maturase encoded by a group I intron from Aspergillus nidulans stabilizes RNA tertiary structure and promotes rapid splicing; Ho Y et al.; The AnCOB group I intron from Aspergillus nidulans self-splices, providing the Mg2+ concentration is >/= 15 mM . The splicing reaction is greatly stimulated by a maturase protein encoded within the intron itself . An initial structural and biochemical analysis of the splicing reaction has now been performed . The maturase bound rapidly to the precursor RNA (kon approximately 3 x 10(9) M(-1) min(-1)) and remained tightly bound (koff </= 0.04 min(-1)) . The catalytic step of 5' splice-site cleavage occurred at a rate of up to 11 min(-1) under single turnover conditions . The maturase-assisted reaction of heat-denatured RNA proceeded at a rate of about 1 min(-1), arguing that there are early steps of folding that cannot be readily facilitated by the protein . pH analysis revealed a biphasic profile with a pKa of 7.0 . The rate of the maturase-assisted reaction was independent of the Mg2+ concentration down to 3 mM . Self-splicing in optimal Mg2+ (>/= 150 mM) was tenfold slower, in part because of the existence of an equilibrium between folded and partially folded RNA . In contrast, the maturase very effectively stabilized tertiary structure in 5 mM Mg2+, a noticeable example being an interaction between the P8 helix and a GNRA sequence that constitutes the L2 terminal loop of the P2 helix . Formation of the 5' splice-site recognition helix was assisted by either the maturase or high concentrations of Mg2+ . The maturase was required during splicing so it is not a true chaperone . However, RNase protection assays and kinetic studies suggest that the maturase recognizes and facilitates folding of an intron with limited tertiary structure and even incomplete secondary structure . Biochem J, 1999 Oct 15, 343 Pt 2, 281 - 99 The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation; Halestrap AP et al.; Monocarboxylates such as lactate and pyruvate play a central role in cellular metabolism and metabolic communication between tissues . Essential to these roles is their rapid transport across the plasma membrane, which is catalysed by a recently identified family of proton-linked monocarboxylate transporters (MCTs) . Nine MCT-related sequences have so far been identified in mammals, each having a different tissue distribution, whereas six related proteins can be recognized in Caenorhabditis elegans and 4 in Saccharomyces cerevisiae . Direct demonstration of proton-linked lactate and pyruvate transport has been demonstrated for mammalian MCT1-MCT4, but only for MCT1 and MCT2 have detailed analyses of substrate and inhibitor kinetics been described following heterologous expression in Xenopus oocytes . MCT1 is ubiquitously expressed, but is especially prominent in heart and red muscle, where it is up-regulated in response to increased work, suggesting a special role in lactic acid oxidation . By contrast, MCT4 is most evident in white muscle and other cells with a high glycolytic rate, such as tumour cells and white blood cells, suggesting it is expressed where lactic acid efflux predominates . MCT2 has a ten-fold higher affinity for substrates than MCT1 and MCT4 and is found in cells where rapid uptake at low substrate concentrations may be required, including the proximal kidney tubules, neurons and sperm tails . MCT3 is uniquely expressed in the retinal pigment epithelium . The mechanisms involved in regulating the expression of different MCT isoforms remain to be established . However, there is evidence for alternative splicing of the 5'- and 3'-untranslated regions and the use of alternative promoters for some isoforms . In addition, MCT1 and MCT4 have been shown to interact specifically with OX-47 (CD147), a member of the immunoglobulin superfamily with a single transmembrane helix . This interaction appears to assist MCT expression at the cell surface . There is still much work to be done to characterize the properties of the different isoforms and their regulation, which may have wide-ranging implications for health and disease . In the future it will be interesting to explore the linkage of genetic diseases to particular MCTs through their chromosomal location. Curr Biol, 1999 Sep 23, 9(18), R708 - 10 The missing (L) UNC? Raff JW. In many cells, centrosomes are required to position nuclei at specific locations in the cytoplasm . The nature of the link between centrosomes and nuclei is mysterious, but the recently characterised UNC84 protein appears to be involved. Yeast, 1999 Sep 30, 15(13), 1347 - 55 ade9 is an allele of SER1 and plays an indirect role in purine biosynthesis; Buc PS et al.; In this study we demonstrate that ade9 plays an indirect role in purine biosynthesis as a non-functional allele of SER1 in Saccharomyces cerevisiae . The SER1 locus, encoding 3-phosphoserine aminotransferase required for serine biosynthesis, is located on chromosome XV and resides approximately where ade9 had previously been mapped genetically . A minimal functional construct of SER1 is necessary and sufficient to complement both the adenine- and serine-requiring phenotypes of ade9 strains . In addition, adequate exogenous serine levels mask the adenine phenotype of ade9 . A disruption of SER1 behaves in the same manner phenotypically as the original ade9 strain . Therefore, ade9 can be more accurately described as an allele of SER1 . J Cell Biol, 1999 Oct 4, 147(1), 163 - 74 Cell wall stress depolarizes cell growth via hyperactivation of RHO1; Delley PA et al.; Cells sense and physiologically respond to environmental stress via signaling pathways . Saccharomyces cerevisiae cells respond to cell wall stress by transiently depolarizing the actin cytoskeleton . We report that cell wall stress also induces a transient depolarized distribution of the cell wall biosynthetic enzyme glucan synthase FKS1 and its regulatory subunit RHO1, possibly as a mechanism to repair general cell wall damage . The redistribution of FKS1 is dependent on the actin cytoskeleton . Depolarization of the actin cytoskeleton and FKS1 is mediated by the plasma membrane protein WSC1, the RHO1 GTPase switch, PKC1, and a yet-to-be defined PKC1 effector branch . WSC1 behaves like a signal transducer or a stress-specific actin landmark that both controls and responds to the actin cytoskeleton, similar to the bidirectional signaling between integrin receptors and the actin cytoskeleton in mammalian cells . The PKC1-activated mitogen-activated protein kinase cascade is not required for depolarization, but rather for repolarization of the actin cytoskeleton and FKS1 . Thus, activated RHO1 can mediate both polarized and depolarized cell growth via the same effector, PKC1, suggesting that RHO1 may function as a rheostat rather than as a simple on-off switch. Curr Biol, 1999 Sep 9, 9(17), 963 - 6 DNA damage triggers disruption of telomeric silencing and Mec1p-dependent relocation of Sir3p; McAinsh AD et al.; In eukaryotic cells, surveillance mechanisms detect and respond to DNA damage by triggering cell-cycle arrest and inducing the expression of DNA-repair genes {1} . In budding yeast, a single DNA double-strand break (DSB) is sufficient to trigger cell-cycle arrest {2} . One highly conserved pathway for repairing DNA DSBs is DNA non-homologous end-joining (NHEJ), which depends on the DNA end-binding protein Ku {3} . NHEJ also requires the SIR2, SIR3 and SIR4 gene products {4} {5}, which are responsible for silencing at telomeres and the mating-type loci {6} . Because of the link between NHEJ and the Sir proteins, we investigated whether DNA damage influences telomeric silencing . We found that DNA damage triggers the reversible loss of telomeric silencing and relocation of Sir3p from telomeres . Complete Sir3p relocation was triggered by a single DNA DSB, suggesting that the singal is amplified . Consistent with this idea, Sir3p relocation depended on the DNA damage-signalling components Ddc1p and Mec1p . Thus, signalling of DNA damage may release Sir3p from telomeres and permit its subsequent association with other nuclear subdomains to regulate transcription, participate in DNA repair and/or enhance genomic stability by other mechanisms. Nat Genet, 1999 Oct, 23(2), 245 - 8 Loss of Cul1 results in early embryonic lethality and dysregulation of cyclin E; Dealy MJ et al.; The sequential timing of cell-cycle transitions is primarily governed by the availability and activity of key cell-cycle proteins . Recent studies in yeast have identified a class of ubiquitin ligases (E3 enzymes) called SCF complexes, which regulate the abundance of proteins that promote and inhibit cell-cycle progression at the G1-S phase transition . SCF complexes consist of three invariable components, Skp1, Cul-1 (Cdc53 in yeast) and Rbx1, and a variable F-box protein that recruits a specific cellular protein to the ubquitin pathway for degradation . To study the role of Cul-1 in mammalian development and cell-cycle regulation, we generated mice deficient for Cul1 and analysed null embryos and heterozygous cell lines . We show that Cul1 is required for early mouse development and that Cul1 mutants fail to regulate the abundance of the G1 cyclin, cyclin E (encoded by Ccne), during embryogenesis. Nat Biotechnol, 1999 Oct, 17(10), 994 - 9 Quantitative analysis of complex protein mixtures using isotope-coded affinity tags; Gygi SP et al.; We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures . The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry . Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source . The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions . The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques . The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues. Biochemistry, 1999 Sep 14, 38(37), 12150 - 8 DNA bending induced by high mobility group proteins studied by fluorescence resonance energy transfer; Lorenz M et al.; The HMG domains of the chromosomal high mobility group proteins homologous to the vertebrate HMG1 and HMG2 proteins preferentially recognize distorted DNA structures . DNA binding also induces a substantial bend . Using fluorescence resonance energy transfer (FRET), we have determined the changes in the end-to-end distance consequent on the binding of selected insect counterparts of HMG1 to two DNA fragments, one of 18 bp containing a single dA(2) bulge and a second of 27 bp with two dA(2) bulges . The observed changes are consistent with overall bend angles for the complex of the single HMG domain with one bulge and of two domains with two bulges of approximately 90-100 degrees and approximately 180-200 degrees, respectively . The former value contrasts with an inferred value of 150 degrees reported by Heyduk et al . (1) for the bend induced by a single domain . We also observe that the induced bend angle is unaffected by the presence of the C-terminal acidic region . The DNA bend of approximately 95 degrees observed in the HMG domain complexes is similar in magnitude to that induced by the TATA-binding protein (80 degrees), each monomeric unit of the integration host factor (80 degrees), and the LEF-1 HMG domain (107 degrees) . We suggest this value may represent a steric limitation on the extent of DNA bending induced by a single DNA-binding motif. Eur J Immunol, 1999 Sep, 29(9), 2988 - 98 Reversal of Blimp-1-mediated apoptosis by A1, a member of the Bcl-2 family; Knodel M et al.; Blimp-1 (B lymphocyte-induced maturation protein 1) is strongly expressed during the late stages of B cell differentiation to immunoglobulin-secreting plasma cells . Overexpression of Blimp-1 in B lymphoma cells has been reported to induce either growth arrest and cell death or Ig secretion and terminal differentiation, depending on the developmental stage of the recipient lymphomas . By using a retroviral expression system we show that Blimp-1-transduced immature WEHI 231 murine B lymphoma cells produce J chain, increased levels of the secretory form of micro heavy chain mRNA and secrete IgM for a short period of time . Concomitantly, they exhibit altered ratios of c-myc/mad4 mRNA levels, a reduction in the expression of the anti-apoptotic bcl-2 family member A1 and a distinct growth disadvantage, followed by cell death . Reintroduction of A1 by retroviral transduction greatly extends the life span of Blimp-1-expressing WEHI 231 cells which continue to secrete IgM . These data suggest that levels of A1 may determine the checkpoint between death and survival of Blimp-1-expressing B cells at different stages of differentiation. EMBO J, 1999 Oct 1, 18(19), 5399 - 410 Functions of the exosome in rRNA, snoRNA and snRNA synthesis; Allmang C et al.; The yeast nuclear exosome contains multiple 3'-->5' exoribonucleases, raising the question of why so many activities are present in the complex . All components are required during the 3' processing of the 5.8S rRNA, together with the putative RNA helicase Dob1p/Mtr4p . During this processing three distinct steps can be resolved, and hand-over between different exonucleases appears to occur at least twice . 3' processing of snoRNAs (small nucleolar RNAs) that are excised from polycistronic precursors or from mRNA introns is also a multi-step process that involves the exosome, with final trimming specifically dependent on the Rrp6p component . The spliceosomal U4 snRNA (small nuclear RNA) is synthesized from a 3' extended precursor that is cleaved by Rnt1p at sites 135 and 169 nt downstream of the mature 3' end . This cleavage is followed by 3'-->5' processing of the pre-snRNA involving the exosome complex and Dob1p . The exosome, together with Rnt1p, also participates in the 3' processing of the U1 and U5 snRNAs . We conclude that the exosome is involved in the processing of many RNA substrates and that different components can have distinct functions. EMBO J, 1999 Oct 1, 18(19), 5370 - 9 Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus; Egea-Cortines M et al.; In Antirrhinum, floral meristems are established by meristem identity genes . Floral meristems give rise to floral organs in whorls, with their identity established by combinatorial activities of organ identity genes . Double mutants of the floral meristem identity gene SQUAMOSA and organ identity genes DEFICIENS or GLOBOSA produce flowers in which whorled patterning is partially lost . In yeast, SQUA, DEF and GLO proteins form ternary complexes via their C-termini, which in gel-shift assays show increased DNA binding to CArG motifs compared with DEF/GLO heterodimers or SQUA/SQUA homodimers . Formation of ternary complexes by plant MADS-box factors increases the complexity of their regulatory functions and might be the molecular basis for establishment of whorled phyllotaxis and combinatorial interactions of floral organ identity genes. EMBO J, 1999 Oct 1, 18(19), 5334 - 46 Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway; Weinreich M et al.; Eukaryotic cells coordinate chromosome duplication by assembly of protein complexes at origins of DNA replication and by activation of cyclin-dependent kinase and Cdc7p-Dbf4p kinase . We show in Saccharomyces cerevisiae that although Cdc7p levels are constant during the cell division cycle, Dbf4p and Cdc7p-Dbf4p kinase activity fluctuate . Dbf4p binds to chromatin near the G(1)/S-phase boundary well after binding of the minichromosome maintenance (Mcm) proteins, and it is stabilized at the non-permissive temperature in mutants of the anaphase-promoting complex, suggesting that Dbf4p is targeted for destruction by ubiquitin-mediated proteolysis . Arresting cells with hydroxyurea (HU) or with mutations in genes encoding DNA replication proteins results in a more stable, hyper-phosphorylated form of Dbf4p and an attenuated kinase activity . The Dbf4p phosphorylation in response to HU is RAD53 dependent . This suggests that an S-phase checkpoint function regulates Cdc7p-Dbf4p kinase activity . Cdc7p may also play a role in adapting from the checkpoint response since deletion of CDC7 results in HU hypersensitivity . Recombinant Cdc7p-Dbf4p kinase was purified and both subunits were autophosphorylated . Cdc7p-Dbf4p efficiently phosphorylates several proteins that are required for the initiation of DNA replication, including five of the six Mcm proteins and the p180 subunit of DNA polymerase alpha-primase. EMBO J, 1999 Oct 1, 18(19), 5226 - 33 Bcs1p, an AAA-family member, is a chaperone for the assembly of the cytochrome bc(1) complex; Cruciat CM et al.; Bcs1p, a mitochondrial protein and member of the conserved AAA protein family, is involved in the biogenesis of the cytochrome bc(1) complex . We demonstrate here that Bcs1p is directly required for the assembly of the Rieske FeS and Qcr10p proteins into the cytochrome bc(1) complex . Bcs1p binds to a precomplex in the assembly pathway of the cytochrome bc(1) complex . Binding of Bcs1p to and release from this assembly intermediate is driven by ATP hydrolysis . We propose that Bcs1p acts as an ATP-dependent chaperone, maintaining the precomplex in a competent state for the subsequent assembly of the Rieske FeS and Qcr10p proteins. Br J Cancer, 1999 Oct, 81(3), 503 - 9 Detection of loss of heterozygosity at RAD51, RAD52, RAD54 and BRCA1 and BRCA2 loci in breast cancer: pathological correlations; Gonzalez R et al.; Loss of heterozygosity (LOH) in loci of the 15q15.1, 12p13, 1p32, 17q21 and 13q12-13 regions may collaborate in the inactivation of RAD51, RAD52, RAD54, BRCA1, BRCA2 and possibly other genes implicated in the repair of double-stranded DNA and in DNA recombination . We investigate allelic losses in microsatellites of the RAD51, RAD52, RAD54, BRCA1 and BRCA2 regions, and their correlations with nine pathologic parameters in 127 breast carcinomas . The LOH analysis was performed by amplifying DNA by PCR, using 15 markers of the 15q15.1, 12p13.3, 1p32, 17q21 and 13q12-13 regions . LOH was found in the RAD51 region in 32% of tumours, in the RAD52 region in 16%, in RAD54 in 20% and in the BRCA1 and BRCA2 regions in 49% and 44% respectively . Significant correlations between one or more regions with concomitant LOH and pathologic parameters were observed with respect to age (P = 0.008), oestrogen receptor content (P = 0.03), progesterone receptors (P = 0.003), higher grade (P = 0.001), more advanced stage (P = 0.004) and peritumoural vessel involvement (P < 0.0001) . The number of cases in which LOH was observed simultaneously in two or more regions was always higher than expected on the basis of their statistical probability, and curiously, the three patients with LOH at five regions concomitantly were under the age of 30 years . These results suggest that LOH at these regions could be related to breast cancer, and probably to a poor tumour prognosis. Science, 1999 Oct 1, 286(5437), 117 - 20 Est1 and Cdc13 as comediators of telomerase access; Evans SK et al.; Cdc13 and Est1 are single-strand telomeric DNA binding proteins that contribute to telomere replication in the yeast Saccharomyces cerevisiae . Here it is shown that fusion of Cdc13 to the telomerase-associated Est1 protein results in greatly elongated telomeres . Fusion proteins consisting of mutant versions of Cdc13 or Est1 confer similar telomere elongation, indicating that close physical proximity can bypass telomerase-defective mutations in either protein . Fusing Cdc13 directly to the catalytic core of telomerase allows stable telomere maintenance in the absence of Est1, consistent with a role for Est1 in mediating telomerase access . Telomere length homeostasis therefore is maintained in part by restricting access of telomerase to chromosome termini, but this limiting situation can be overcome by directly tethering telomerase to the telomere. J Biol Chem, 1999 Oct 8, 274(41), 29500 - 4 Three conserved transcriptional repressor domains are a defining feature of the TIEG subfamily of Sp1-like zinc finger proteins; Cook T et al.; Sp1-like transcription factors are characterized by three highly homologous C-terminal zinc finger motifs that bind GC-rich sequences . These proteins behave as either activators or repressors and have begun to be classified into different subfamilies based upon the presence of conserved motifs outside the zinc finger domain . This classification predicts that different Sp1-like subfamilies share certain functional properties . TIEG1 and TIEG2 constitute a new subfamily of transforming growth factor-beta-inducible Sp1-like proteins whose zinc finger motifs also bind GC-rich sequences . However, regions outside of the DNA-binding domain that differ in structure from other Sp1-like family members remain poorly characterized . Here, we have used extensive mutagenesis and GAL4-based transcriptional assays to identify three repression domains within TIEG1 and TIEG2 that we call R1, R2, and R3 . R1 is 10 amino acids, R2 is 12 amino acids, and R3 is approximately 80 amino acids long . None of these domains share homology with previously described transcriptional regulatory motifs, but they share strong sequence homology and are functionally conserved between TIEG1 and TIEG2 . Together, these data demonstrate that TIEG proteins are capable of repressing transcription, define domains critical for this function, and further support the idea that different subfamilies of Sp1-like proteins have evolved to mediate distinct transcriptional functions. J Biol Chem, 1999 Oct 8, 274(41), 29102 - 7 Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C; Zhu T et al.; Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are rapidly deactivated by a membrane-bound phosphatase activity . The efficiency of this regulation suggests CFTR and protein phosphatases may be associated within a regulatory complex . In this paper we test that possibility using co-immunoprecipitation and cross-linking experiments . A monoclonal anti-CFTR antibody co-precipitated type 2C protein phosphatase (PP2C) from baby hamster kidney cells stably expressing CFTR but did not co-precipitate PP1, PP2A, or PP2B . Conversely, a polyclonal anti-PP2C antibody co-precipitated CFTR from baby hamster kidney membrane extracts . Exposing baby hamster kidney cell lysates to dithiobis (sulfosuccinimidyl propionate) caused the cross-linking of histidine-tagged CFTR (CFTR(His10)) and PP2C into high molecular weight complexes that were isolated by chromatography on Ni(2+)-nitrilotriacetic acid-agarose . Chemical cross-linking was specific for PP2C, because PP1, PP2A, and PP2B did not co-purify with CFTR(His10) after dithiobis (sulfosuccinimidyl propionate) exposure . These results suggest CFTR and PP2C exist in a stable complex that facilitates regulation of the channel. J Biol Chem, 1999 Oct 8, 274(41), 29031 - 7 Kap104p-mediated nuclear import . Nuclear localization signals in mRNA-binding proteins and the role of Ran and Rna; Lee DC et al.; Kap104p is a Saccharomyces cerevisiae nuclear import receptor for two essential mRNA-binding proteins, Nab2p and Nab4p/Hrp1p . We demonstrate direct binding of Kap104p to each of these substrates . We have defined the nuclear localization signals in both Nab2p and Nab4p/Hrp1p by Kap104p binding in vitro and KAP104-dependent nuclear import in vivo . The nuclear localization signals map to similar arginine/glycine-rich RNA-binding domains in both proteins and are thus termed rg-nuclear localization signals to distinguish them from classical nuclear localization signals . We also demonstrate that Kap104p, like other known beta-karyopherins (or importins), interacts directly with the small GTPase Ran/Gsp1 . However, unlike other known import factors, Ran binding is not sufficient to mediate release of substrates from Kap104p; efficient Ran-GTP-mediated substrate release requires RNA . Also, addition of Kap104p to Nab2p and Nab4p/Hrp1p prebound to single-stranded DNA-cellulose stimulated release of both proteins from the resin . We suggest a simple cycle in which Nab2p and Nab4p/Hrp1p, upon import, are released in the nucleus at sites of transcription by the concerted action of Ran-GTP and binding to newly synthesized mRNA . The resulting ribonucleoprotein complexes are exported to the cytoplasm, where Kap104p rebinds to Nab2p and Nab4p/Hrp1p, contributing to their release from mRNA. Plant J, 1999 Sep, 19(5), 599 - 603 The pre-vacuolar t-SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP; Bassham DC et al.; Many proteins are transported to the plant vacuole through the secretory pathway in small transport vesicles by a series of vesicle budding and fusion reactions . Vesicles carrying vacuolar cargo bud from the trans-Golgi network are thought to fuse with a pre-vacuolar compartment before being finally transported to the vacuole . In mammals and yeast, the fusion of a vesicle with its target organelle is mediated by a 20S protein complex containing membrane and soluble proteins that appear to be conserved between different species . A number of membrane proteins have been identified in plants that show sequence similarity with a family of integral membrane proteins (t-SNAREs) on target organelles that are required for the fusion of transport vesicles with that organelle . However, the biochemical function of these proteins has remained elusive . Here, we demonstrate for the first time the formation of a 20S complex in plants that has characteristics of complexes involved in vesicle fusion . This complex contains AtPEP12p, an Arabidopsis protein thought to be involved in protein transport to the prevacuolar compartment . In addition, we have shown that AtPEP12p can bind to alpha-SNAP, indicating that AtPEP12p does indeed function as a SNAP receptor or SNARE . These preliminary data suggest that AtPEP12p may function jointly with alpha-SNAP and NSF in the fusion of transport vesicles containing vacuolar cargo proteins with the pre-vacuolar compartment. Eur J Biochem, 1999 Oct, 265(2), 809 - 14 Identification of Delta5-fatty acid desaturase from the cellular slime mold dictyostelium discoideum; Saito T et al.; cDNA fragments putatively encoding amino acid sequences characteristic of the fatty acid desaturase were obtained using expressed sequence tag (EST) information of the Dictyostelium cDNA project . Using this sequence, we have determined the cDNA sequence and genomic sequence of a desaturase . The cloned cDNA is 1489 nucleotides long and the deduced amino acid sequence comprised 464 amino acid residues containing an N-terminal cytochrome b5 domain . The whole sequence was 38.6% identical to the initially identified Delta5-desaturase of Mortierella alpina . We have confirmed its function as Delta5-desaturase by over expression mutation in D . discoideum and also the gain of function mutation in the yeast Saccharomyces cerevisiae . Analysis of the lipids from transformed D . discoideum and yeast demonstrated the accumulation of Delta5-desaturated products . This is the first report concering fatty acid desaturase in cellular slime molds. Eur J Biochem, 1999 Oct, 265(2), 754 - 62 Characterization of a mammalian homolog of the GCN2 eukaryotic initiation factor 2alpha kinase; Berlanga JJ et al.; In eukaryotic cells, protein synthesis is regulated in response to various environmental stresses by phosphorylating the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) . Three different eIF2alpha kinases have been identified in mammalian cells, the heme-regulated inhibitor (HRI), the interferon-inducible RNA-dependent kinase (PKR) and the endoplasmic reticulum-resident kinase (PERK) . A fourth eIF2alpha kinase, termed GCN2, was previously characterized from Saccharomyces cerevisiae, Drosophila melanogaster and Neurospora crassa . Here we describe the cloning of a mouse GCN2 cDNA (MGCN2), which represents the first mammalian GCN2 homolog . MGCN2 has a conserved motif, N-terminal to the kinase subdomain V, and a large insert of 139 amino acids located between subdomains IV and V that are characteristic of the known eIF2alpha kinases . Furthermore, MGCN2 contains a class II aminoacyl-tRNA synthetase domain and a degenerate kinase segment, downstream and upstream of the eIF2alpha kinase domain, respectively, and both are singular features of GCN2 protein kinases . MGCN2 mRNA is expressed as a single message of approximately 5.5 kb in a wide range of different tissues, with the highest levels in the liver and the brain . Specific polyclonal anti-(MGCN2) immunoprecipitated an eIF2alpha kinase activity and recognized a 190 kDa phosphoprotein in Western blots from either mouse liver or MGCN2-transfected 293 cell extracts . Interestingly, serum starvation increased eIF2alpha phosphorylation in MGCN2-transfected human 293T cells . This finding provides evidence that GCN2 is the unique eIF2alpha kinase present in all eukaryotes from yeast to mammals and underscores the role of MGCN2 kinase in translational control and its potential physiological significance. J Cell Sci, 1999 Oct, 112 ( Pt 20), 3529 - 35 Evidence for the presence of an NF-kappaB signal transduction system in Dictyostelium discoideum; Traincard F et al.; The Rel/NF-kappaB family of transcription factors and regulators has so far only been described in vertebrates and arthropods, where they mediate responses to many extracellular signals . No counterparts of genes coding for such proteins have been identified in the Caenorhabditis elegans genome and no NF-kappaB activity was found in Saccharomyces cerevisiae . We describe here the presence of an NF-kappaB transduction pathway in the lower eukaryote Dictyostelium discoideum . Using antibodies raised against components of the mammalian NF-kappaB pathway, we demonstrate in Dictyostelium cells extracts the presence of proteins homologous to Rel/NF-kappaB, IkappaB and IKK components . Using gel-shift experiments in nuclear extracts of developing Dictyostelium cells, we demonstrate the presence of proteins binding to kappaB consensus oligonucleotides and to a GC-rich kappaB-like sequence, lying in the promoter of cbpA, a developmentally regulated Dictyostelium gene encoding the Ca(2+)-binding protein CBP1 . Using immunofluorescence, we show specific nuclear translocation of the p65 and p50 homologues of the NF-kappaB transcription factors as vegetatively growing cells develop to the slug stage . Taken together, our results strongly indicate the presence of a complete NF-kappaB signal transduction system in Dictyostelium discoideum that could be involved in the developmental process. Biochemistry, 1999 Sep 28, 38(39), 12727 - 34 FTIR-Spectroscopy of multistranded coiled coil proteins; Heimburg T et al.; Coiled coils of different order were investigated using infrared (IR) spectroscopy . Recently, we demonstrated that dimeric coiled coils display unique vibrational spectra with at least three separable bands instead of only one band of a classical alpha-helix in the amide I region.This was attributed to a distortion of the helical structure by the supercoil bending, giving rise to bands that are not observed in the undistorted helix . Here, we investigated coiled coils forming trimers, tetramers, and pentamers . These higher order coiled coils, in general, possess larger superhelical pitches, resulting in a smaller helical distortion . We found that all coiled coils studied, including the native dimeric GCN4 leucine zipper and its variants leading to parallel trimers and tetramers as well as the rod portions of fibritin (parallel trimer), alpha-actinin (antiparallel spectrin type trimer), and COMP (parallel pentamer), displayed the typical three band pattern of the coiled coil amide I spectra . However, the separation of these three bands and their positional deviation from the classical alpha-helical band position was correlated to the extent of the helical distortion as reflected by the pitch values of the supercoils . The most pronounced spectral anomaly was found for the tropomyosin dimer with a reported helical pitch of 137 A, whereas the smallest spectral distortion was found for the pentameric COMP complex and the tetrameric leucine zipper mutant, both with a pitch of about 205 A. Biochemistry, 1999 Sep 28, 38(39), 12621 - 8 Mapping of a DNA binding region of the PI-sceI homing endonuclease by affinity cleavage and alanine-scanning mutagenesis; Hu D et al.; The PI-SceI protein is a member of the LAGLIDADG family of homing endonucleases that is generated by a protein splicing reaction . PI-SceI has a bipartite domain structure, and the protein splicing and endonucleolytic reactions are catalyzed by residues in domains I and II, respectively . Structural and mutational evidence indicates that both domains mediate DNA binding . Treatment of the protein with trypsin breaks a peptide bond within a disordered region of the endonuclease domain situated between residues Val-270 and Leu-280 and interferes with the ability of this domain to bind DNA . To identify specific residues in this region that are involved in DNA binding and/or catalysis, alanine-scanning mutagenesis was used to create a set of PI-SceI mutant proteins that were assayed for activity . One of these mutants, N281A, was >300-fold less active than wild-type PI-SceI, and two other proteins, R277A and N284A, were completely inactive . These decreases in cleavage activity parallel similar decreases in substrate binding by the endonuclease domains of these mutant proteins . We mapped the approximate position of the disordered region to one of the ends of the 31 base pair PI-SceI recognition sequence using mutant proteins that were substituted with cysteine at residues Asn-274 and Glu-283 and tethered to the chemical nuclease FeBABE . These mutational and affinity cleavage data strongly support a model of PI-SceI docked to its DNA substrate that suggests that one or more residues identified here are responsible for contacting base pair A/T(-)(9), which is essential for substrate binding. Peptides, 1999, 20(8), 899 - 905 A cDNA, from Agrotis ipsilon, that encodes the pheromone biosynthesis activating neuropeptide (PBAN) and other FXPRL peptides; Duportets L et al.; A cDNA encoding the prohormone of the pheromone biosynthesis activating neuropeptide (PBAN) in the moth Agrotis ipsilon was isolated . The cDNA contains 834 nucleotides, coding for a 193-amino acid protein that exhibits 89% identity with PBAN prohormones of other moths . The prohormone contains five potential peptides belonging to the FXPRL family . The peptide corresponding to the Bombyx mori diapause hormone exhibits an extra residue, and the C-terminal leucine is replaced by an isoleucine, introducing a new type of variability in this family of peptides . Northern blot analysis revealed expression in suboesophagal ganglion complexes . Constitutive heterologous expression of Agi-PBAN cDNA in yeast, using three different antibodies, did not produce PBAN-immunoreactive material. J Gen Virol, 1999 Sep, 80 ( Pt 9), 2319 - 27 Lower concentration of La protein required for internal ribosome entry on hepatitis C virus RNA than on poliovirus RNA; Isoyama T et al.; Translation initiation of poliovirus and hepatitis C virus (HCV) RNA occurs by entry of ribosomes to the internal RNA sequence, called the internal ribosomal entry site (IRES) . Both IRES bind to the La protein and are thought to require the protein for their translation initiation activity, although they are greatly different in both the primary and predicted secondary structures . To compare the La protein requirement for these IRES, we took advantage of I-RNA from the yeast Saccharomyces cerevisiae, which has been reported to bind to La protein and block poliovirus IRES-mediated translation initiation . In a cell-free translation system prepared from HeLa cells, yeast I-RNA inhibited translation initiation on poliovirus RNA as expected, but did not significantly inhibit translation initiation on HCV RNA . However, the translation initiation directed by either IRES was apparently inhibited by I-RNA in rabbit reticulocyte lysates, in which La protein is limiting . I-RNA-mediated inhibition of HCV IRES-dependent translation in rabbit reticulocyte lysates was reversed by exogenous addition of purified recombinant La protein of smaller amounts than necessary to reverse poliovirus IRES-dependent translation . These results suggest that HCV IRES requires lower concentrations of La protein for its function than does poliovirus IRES . Immunofluorescence studies showed that HCV infection appeared not to affect the subcellular localization of La protein, which exists mainly in the nucleus, although La protein redistributed to the cytoplasm after poliovirus infection . The data are compatible with the low requirement of La protein for HCV IRES activity. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11440 - 5 CDC45 and DPB11 are required for processive DNA replication and resistance to DNA topoisomerase I-mediated DNA damage; Reid RJ et al.; The antitumor agent camptothecin targets DNA topoisomerase I by reversibly stabilizing a covalent enzyme-DNA intermediate . The subsequent collision of DNA replication forks with these drug-enzyme-DNA complexes produces the cytotoxic DNA lesions that signal cell cycle arrest and ultimately lead to cell death . Despite intense investigation, the character of the lesions produced and the repair processes that resolve the damage remain poorly defined . A yeast genetic screen was implemented to isolate conditional mutants with enhanced sensitivity to DNA topoisomerase I-mediated DNA damage . Cells exhibiting temperature-sensitive growth in the presence of the DNA topoisomerase I mutant, Top1T722Ap, were selected . Substitution of Ala for Thr722 increases the stability of the covalent Top1T722Ap-DNA intermediate, mimicking the cytotoxic action of camptothecin . Two mutants isolated, cdc45-10 and dpb11-10, exhibited specific defects in DNA replication and a synthetic lethal phenotype in the absence of DNA damaging agents . The accumulation of Okazaki fragments under nonpermissive conditions suggests a common function in promoting processive DNA replication through polymerase switching . These results provide a mechanistic basis for understanding the cellular processes involved in the resolution of DNA damage induced by camptothecin and DNA topoisomerase I. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11317 - 22 Expression of the CDH1-associated form of the anaphase-promoting complex in postmitotic neurons; Gieffers C et al.; The anaphase-promoting complex/cyclosome (APC) is a tightly cell cycle-regulated ubiquitin-protein ligase that targets cyclin B and other destruction box-containing proteins for proteolysis at the end of mitosis and in G1 . Recent work has shown that activation of the APC in mitosis depends on CDC20, whereas APC is maintained active in G1 via association with the CDC20-related protein CDH1 . Here we show that the mitotic activator CDC20 is the only component of the APC ubiquitination pathway whose expression is restricted to proliferating cells, whereas the APC and CDH1 are also expressed in several mammalian tissues that predominantly contain differentiated cells, such as adult brain . Immunocytochemical analyses of cultured rat hippocampal neurons and of mouse and human brain sections indicate that the APC and CDH1 are ubiquitously expressed in the nuclei of postmitotic terminally differentiated neurons . The APC purified from brain contains all core subunits known from proliferating cells and is tightly associated with CDH1 . Purified brain APC(CDH1) has a high cyclin B ubiquitination activity that depends less on the destruction box than on the activity of mitotic APC(CDC20) . On the basis of these results, we propose that the functions of APC(CDH1) are not restricted to controlling cell-cycle progression but may include the ubiquitination of yet unidentified substrates in differentiated cells. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11206 - 10 Chitin synthase III: synthetic lethal mutants and "stress related" chitin synthesis that bypasses the CSD3/CHS6 localization pathway; Osmond BC et al.; We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3 . In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Delta, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion . In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant . This finding suggests that "stress response" chitin synthesis proceeds through an alternate Chs3p targeting pathway. Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 10976 - 83 The catalytic sites of 20S proteasomes and their role in subunit maturation: a mutational and crystallographic study; Groll M et al.; We present a biochemical and crystallographic characterization of active site mutants of the yeast 20S proteasome with the aim to characterize substrate cleavage specificity, subunit intermediate processing, and maturation . beta1(Pre3), beta2(Pup1), and beta5(Pre2) are responsible for the postacidic, tryptic, and chymotryptic activity, respectively . The maturation of active subunits is independent of the presence of other active subunits and occurs by intrasubunit autolysis . The propeptides of beta6(Pre7) and beta7(Pre4) are intermediately processed to their final forms by beta2(Pup1) in the wild-type enzyme and by beta5(Pre2) and beta1(Pre3) in the beta2(Pup1) inactive mutants . A role of the propeptide of beta1(Pre3) is to prevent acetylation and thereby inactivation . A gallery of proteasome mutants that contain active site residues in the context of the inactive subunits beta3(Pup3), beta6(Pre7), and beta7(Pre4) show that the presence of Gly-1, Thr1, Asp17, Lys33, Ser129, Asp166, and Ser169 is not sufficient to generate activity. Genes Dev, 1999 Sep 15, 13(18), 2375 - 87 Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells; Singer JD et al.; Cyclin E is an unstable protein that is degraded in a ubiquitin- and proteasome- dependent pathway . Two factors stimulate cyclin E ubiquitination in vivo: when it is free of its CDK partner, and when it is phosphorylated on threonine 380 . We pursued the first of these pathways by using a two-hybrid screen to identify proteins that could bind only to free cyclin E . This resulted in the isolation of human Cul-3, a member of the cullin family of E3 ubiquitin-protein ligases . We found that Cul-3 was bound to cyclin E but not to cyclin E-Cdk2 complexes in mammalian cells, and that overexpression of Cul-3 increased ubiquitination of cyclin E but not other cyclins . Conversely, deletion of the Cul-3 gene in mice caused increased accumulation of cyclin E protein, and had cell-type-specific effects on S-phase regulation . In the extraembryonic ectoderm, in which cells undergo a standard mitotic cycle, there was a greatly increased number of cells in S phase . In the trophectoderm, in which cells go through endocycles, there was a block to entry into S phase . The SCF pathway, which targets cyclins for ubiquitination on the basis of their phosphorylation state, and the Cul-3 pathway, which selects cyclin E for ubiquitination on the basis of its assembly into CDK complexes, may be complementary ways to control cyclin abundance. Genes Dev, 1999 Sep 15, 13(18), 2369 - 74 Recruitment of the SWI/SNF chromatin remodeling complex by transcriptional activators; Yudkovsky N et al.; SWI/SNF is a chromatin remodeling complex that facilitates expression of a number of yeast genes . Here we demonstrate that SWI/SNF can be recruited from yeast nuclear extracts by a transcriptional activator . Recruitment is dependent on an activation domain but not on promoter sequences, TBP, or RNA polymerase II holoenzyme . We also show that acidic activation domains can target SWI/SNF remodeling activity . These results demonstrate that SWI/SNF activity can be targeted by gene-specific activators and that this recruitment can occur independently of Pol II holoenzyme. Cell, 1999 Sep 17, 98(6), 791 - 8 Electron crystal structure of an RNA polymerase II transcription elongation complex; Poglitsch CL et al.; The structure of an actively transcribing complex, containing yeast RNA polymerase II with associated template DNA and product RNA, was determined by electron crystallography . Nucleic acid, in all likelihood the "transcription bubble" at the active center of the enzyme, occupies a previously noted 25 A channel in the protein structure . Details are indicative of a roughly 90 degrees bend of the DNA between upstream and downstream regions . The DNA apparently lies entirely on one face of the polymerase, rather than passing through a hole to the opposite side, as previously suggested. J Biol Inorg Chem, 1999 Feb, 4(1), 21 - 31 Solution structure of reduced horse heart cytochrome c; Banci L et al.; In the frame of a broad study on the structural differences between the two redox forms of cytochromes to be related to the electron transfer process, the NMR solution structure of horse heart cytochrome c in the reduced form has been determined . The structural data obtained in the present work are compared to those already available in the literature on the same protein and the presence of conformational differences is discussed in the light of the experimental method employed for the structure determination . Redox-state dependent changes are analyzed and in particular they are related to the role of propionate-7 of the heme . Also some hydrogen bonds are changed upon reduction of the heme iron . A substantial similarity is observed for the backbone fold, independently of the oxidation state . At variance, some meaningful differences are observed in the orientation of a few side chains . These changes are related to those found in the case of the highly homologous cytochrome c from Saccharomyces cerevisiae . The exchangeability of the NH protons has been investigated and found to be smaller than in the case of the oxidized protein . We think that this is a characteristic of reduced cytochromes and that mobility is a medium for molecular recognition in vivo. J Biol Inorg Chem, 1999 Apr, 4(2), 209 - 19 X-ray crystal structures of active site mutants of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis; Macedo-Ribeiro S et al.; The X-ray structures of the chloroperoxidase from Curvularia inaequalis, heterologously expressed in Saccharomyces cerevisiae, have been determined both in its apo and in its holo forms at 1.66 and 2.11 A resolution, respectively . The crystal structures reveal that the overall structure of this enzyme remains nearly unaltered, particularly at the metal binding site . At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interactions and hydrogen bonds with positively charged residues involved in the interactions with the vanadate in the native protein . The vanadate binding pocket seems to form a very rigid frame stabilising oxyanion binding . The rigidity of this active site matrix is the result of a large number of hydrogen bonding interactions involving side chains and the main chain of residues lining the active site . The structures of single site mutants to alanine of the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed . Additionally we determined the structural effects of mutations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involved in forming a salt bridge with Arg490 which also interacts with the vanadate . The enzymatic chlorinating activity is drastically reduced to approximately 1% in mutants D292A, H404A and H496A . The structures of the mutants confirm the view of the active site of this chloroperoxidase as a rigid matrix providing an oxyanion binding site . No large changes are observed at the active site for any of the analysed mutants . The empty space left by replacement of large side chains by alanines is usually occupied by a new solvent molecule which partially replaces the hydrogen bonding interactions to the vanadate . The new solvent molecules additionally replace part of the interactions the mutated side chains were making to other residues lining the active site frame . When this is not possible, another side chain in the proximity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame. Oncogene, 1999 Sep 16, 18(37), 5177 - 86 NF-kappaB subunit p65 binds to 53BP2 and inhibits cell death induced by 53BP2; Yang JP et al.; Nuclear factor kappaB (NF-kappaB) is a transcription factor that controls the expression of many cellular and viral genes . The p65 (RelA) subunit plays a critical role as a transcriptional activator and recent observations have highlighted its role in the control of apoptosis . Here we report that 53BP2, a protein previously identified by interaction with wild type p53 and Bcl-2, also binds to p65 in a yeast two-hybrid system . This specific interaction was confirmed by pull-down assay in vitro and by a mammalian two-hybrid assay in vivo . We observed that full-length 53BP2 fused to GFP had a punctate distribution in cytoplasm, predominantly in perinuclear region whereas the N-terminal 53BP2 localized in cytoplasm and C-terminal 53BP2 localized in the nucleus . Furthermore, we found that overexpression of GFP-53BP2 induced apoptosis in transiently transfected cells . Neither the N-terminal nor the C-terminal of 53BP2 fused to GFP induced cell death . Interestingly, co-transfection with a p65 expression plasmid significantly inhibited 53BP2-induced cell death . The previous findings that 53BP2 bound to p53 and Bcl-2 together with our present observations suggest that 53BP2 may play a central role in the regulation of apoptosis and cell growth. Oncogene, 1999 Sep 16, 18(37), 5131 - 7 Identification of human APC10/Doc1 as a subunit of anaphase promoting complex; Kurasawa Y et al.; Anaphase-promoting complex or cyclosome (APC) is a ubiquitin ligase which specifically targets mitotic regulatory factors such as Pds1/Cut2 and cyclin B . Identification of the subunits of multiprotein complex APC in several species revealed the highly conserved composition of APC from yeast to human . It has been reported, however, that vertebrate APC is composed of at least eight subunits, APC1 to APC8, while budding yeast APC is constituted of at least 12 components, Apc1 to Apc13 . It has not yet been clearly understood whether additional components found in budding yeast, Apc9 to Apc13, are actually composed of mammalian APC . Here we isolated and characterized human APC10/Doc1, and found that APC10/Doc1 binds to APC core subunits throughout the cell cycle . Further, it was found that APC10/Doc1 is localized in centrosomes and mitotic spindles throughout mitosis, while it is also localized in kinetochores from prophase to anaphase and in midbody in telophase and cytokinesis . These results strongly support the notion that human APC10/Doc1 may be one of the APC core subunits rather than the transiently associated regulatory factor. J Mol Biol, 1999 Sep 24, 292(3), 697 - 705 Crystal structure of the conserved subdomain of human protein SRP54M at 2.1 A resolution: evidence for the mechanism of signal peptide binding; Clemons WM Jr et al.; Protein SRP54 is an integral part of the mammalian signal recognition particle (SRP), a cytosolic ribonucleoprotein complex which associates with ribosomes and serves to recognize, bind, and transport proteins destined for the membrane or secretion . The methionine-rich M-domain of protein SRP54 (SRP54M) binds the SRP RNA and the signal peptide as the nascent protein emerges from the ribosome . A focal point of this critical cellular function is the detailed understanding of how different hydrophobic signal peptides are recognized efficiently and transported specifically, despite considerable variation in sequence . We have solved the crystal structure of a conserved functional subdomain of the human SRP54 protein (hSRP54m) at 2.1 A resolution showing a predominantly alpha helical protein with a large fraction of the structure available for binding . RNA binding is predicted to occur in the vicinity of helices 4 to 6 . The N-terminal helix extends significantly from the core of the structure into a large but constricted hydrophobic groove of an adjacent molecule, thus revealing molecular details of possible interactions between alpha helical signal peptides and human SRP54 . Bioessays, 1999 Oct, 21(10), 824 - 32 Chromosomal elements conferring epigenetic inheritance; Lyko F et al.; Epigenetic regulation of transcription can lead to a stable differential expression of identical genetic information in the same cell or cell population . There is increasing evidence that higher order chromatin structures, involving specific multiprotein complexes, constitute one device to establish and maintain epigenetic marks . In addition, defined chromosomal elements conferring epigenetic inheritance of transcriptional expression states have recently been identified . During the period where the difference in expression of identical genes is established, these sequences appear to be used as switch elements by both negative and positive regulators . Once the epigenetic mark is "set", the elements maintain either the silenced or the activated expression state over many cell generations . Here we review recent data obtained from analyzing epigenetic gene regulation in different organisms and show that similarities in the underlying mechanisms appear to exist . Nucleic Acids Res, 1999 Oct 15, 27(20), 4059 - 70 Assignment of the L30-mRNA complex using selective isotopic labeling and RNA mutants; Mao H et al.; The helix-loop-helix structure formed in the pre-mRNA and the mRNA of L30, a ribosomal protein from the yeast Saccharomyces cerevisiae, serves as an auto-regulatory binding site for the protein to suppress the L30 synthesis upon overproduction . Using a 33-nucleotide model RNA, the structures of the L30 binding site RNA in the presence and absence of the protein were investigated using nuclear magnetic resonance (NMR) spectroscopy . Homonuclear and(13)C/(15)N-based resonance assignments and spectral comparisons indicated that the purine-rich internal loop is dynamic in the free RNA but becomes ordered in the presence of L30 protein . Although the resonances in the loop region are sharper and more disperse in the bound RNA, their assignment was extremely challenging, due to spectral complexity and broadened resonances caused by local dynamics . Two strategies, namely selective(13)C/(15)N-labeling and NMR analyses of five complexes with RNA mutants, were used to overcome these difficulties . Only using these approaches could assignment of the internal loop resonances and identification of the unusual NOEs and nucleotide conformations within the internal loop be made . In the case of structural determination of the L30-mRNA complex, it was critical to be able to take advantage of the available biochemical information in order to complete the structure determination. J Biol Chem, 1999 Oct 1, 274(40), 28803 - 7 Isolation of the protein kinase TAO2 and identification of its mitogen-activated protein kinase/extracellular signal-regulated kinase kinase binding domain; Chen Z et al.; We previously reported the cloning of the thousand and one-amino acid protein kinase 1 (TAO1), a rat homolog of the Saccharomyces cerevisiae protein kinase sterile 20 protein . Here we report the complete sequence and properties of a related rat protein kinase TAO2 . Like TAO1, recombinant TAO2 selectively activated mitogen-activated protein/extracellular signal-regulated kinase kinases (MEKs) 3, 4, and 6 of the stress-responsive mitogen-activated protein kinase pathways in vitro and copurified with MEK3 endogenous to Sf9 cells . To examine TAO2 interactions with MEKs, the MEK binding domain of TAO2 was localized to an approximately 135-residue sequence just C-terminal to the TAO2 catalytic domain . In vitro this MEK binding domain associated with MEKs 3 and 6 but not MEKs 1, 2, or 4 . Using chimeric MEK proteins, we found that the MEK N terminus was sufficient for binding to TAO2 . Catalytic activity of full-length TAO2 enhanced its binding to MEKs . However, neither the autophosphorylation of the MEK binding domain of TAO2 nor the activity of MEK itself was required for MEK binding . These results suggest that TAO proteins lie in stress-sensitive kinase cascades and define a mechanism by which these kinases may organize downstream targets. J Biol Chem, 1999 Oct 1, 274(40), 28736 - 44 Alignment of the B" subunit of RNA polymerase III transcription factor IIIB in its promoter complex; Shah SM et al.; TFIIIB, the central transcription initiation factor of the eukaryotic nuclear RNA polymerase (pol) III is composed of three subunits: the TATA-binding protein; Brf, the TFIIB-related subunit; and B", the Saccharomyces cerevisiae, TFC5 gene product . The orientation of the B" subunit within the TFIIIB-DNA complex has been analyzed at two promoters by two approaches that involve site-specific photochemical protein-DNA cross-linking: a collection of B" internal and external deletion proteins has been surveyed for those deletions that alter the interaction of B" with DNA or change the orientation of B" relative to DNA; a method for regionally mapping cross-links between specific DNA sites and (32)P-end-labeled protein has also been applied . The results map an N-proximal segment of B" to the upstream end of the TFIIIB-DNA complex and amino acids 299-315 to the principal DNA-contact site, approximately 8 base pairs upstream of the TATA box . The analysis also indicates that a segment comprising amino acids 316-434 loops away from DNA, and locates the C-proximal 170 amino acids of B" downstream of the TATA box . Examination of two-cross-link products formed by DNA with adjacent and nearby photoactive nucleotides supports the conclusion that Brf and B" share an extended interface along the length of the TFIIIB-DNA complex. J Biol Chem, 1999 Oct 1, 274(40), 28716 - 23 E1A inhibits transforming growth factor-beta signaling through binding to Smad proteins; Nishihara A et al.; Smads form a recently identified family of proteins that mediate intracellular signaling of the transforming growth factor (TGF)-beta superfamily . Smads bind to DNA and act as transcriptional regulators . Smads interact with a variety of transcription factors, and the interaction is likely to determine the target specificity of gene induction . Smads also associate with transcriptional coactivators such as p300 and CBP . E1A, an adenoviral oncoprotein, inhibits TGF-beta-induced transactivation, and the ability of E1A to bind p300/CBP is required for the inhibition . Here we determined the Smad interaction domain (SID) in p300 and found that two adjacent regions are required for the interaction . One of the regions is the C/H3 domain conserved between p300 and CBP, and the other is a nonconserved region . p300 mutants containing SID inhibit transactivation by TGF-beta in a dose-dependent manner . E1A inhibits the interaction of Smad3 with a p300 mutant that contains SID but lacks the E1A binding domain . We found that E1A interacts specifically with receptor-regulated Smads, suggesting a novel mechanism whereby E1A antagonizes TGF-beta signaling. J Biol Chem, 1999 Oct 1, 274(40), 28584 - 9 Hypermethylation of metallothionein-I promoter and suppression of its induction in cell lines overexpressing the large subunit of Ku protein; Majumder S et al.; We have shown previously that the heavy metal-induced metallothionein-I (MT-I) gene expression is specifically repressed in a rat fibroblast cell line (Ku-80) overexpressing the 80-kDa subunit of Ku autoantigen but not in cell lines overexpressing the 70-kDa subunit or Ku heterodimer . Here, we explored the molecular mechanism of silencing of MT-I gene in Ku-80 cells . Genomic footprinting analysis revealed both basal and heavy metal-inducible binding at specific cis elements in the parental cell line (Rat-1) . By contrast, MT-I promoter in Ku-80 cells was refractory to any transactivating factors, implying alteration of chromatin structure . Treatment of two clonal lines of Ku-80 cells with 5-azacytidine, a potent DNA demethylating agent, rendered MT-I gene inducible by heavy metals, suggesting that the gene is methylated in these cells . Bisulfite genomic sequencing revealed that all 21 CpG dinucleotides in MT-I immediate promoter were methylated in Ku-80 cells, whereas only four CpG dinucleotides were methylated in Rat-1 cells . Almost all methylated CpG dinucleotides were demethylated in Ku-80 cells after 5-azacytidine treatment . To our knowledge, this is the first report that describes hypermethylation of a specific gene promoter and its resultant silencing in response to overexpression of a cellular protein. J Biol Chem, 1999 Oct 1, 274(40), 28528 - 36 Identification of a human histone acetyltransferase related to monocytic leukemia zinc finger protein; Champagne N et al.; We describe here the identification and functional characterization of a novel human histone acetyltransferase, termed MORF (monocytic leukemia zinc finger protein-related factor) . MORF is a 1781-residue protein displaying significant sequence similarity to MOZ (monocytic leukemia zinc finger protein) . MORF is ubiquitously expressed in adult human tissues, and its gene is located at human chromosome band 10q22 . MORF has intrinsic histone acetyltransferase activity . In addition to its histone acetyltransferase domain, MORF possesses a strong transcriptional repression domain at its N terminus and a highly potent activation domain at its C terminus . Therefore, MORF is a novel histone acetyltransferase that contains multiple functional domains and may be involved in both positive and negative regulation of transcription. J Biol Chem, 1999 Oct 1, 274(40), 28385 - 94 Chicken ovalbumin upstream promoter-transcription factor II, a new partner of the glucose response element of the L-type pyruvate kinase gene, acts as an inhibitor of the glucose response; Lou DQ et al.; Transcription of the L-type pyruvate kinase (L-PK) gene is induced by glucose in the presence of insulin and repressed by glucagon via cyclic AMP . The DNA regulatory sequence responsible for mediating glucose and cyclic AMP responses, called glucose response element (GlRE), consists of two degenerated E boxes spaced by 5 base pairs and is able to bind basic helix-loop-helix/leucine zipper proteins, in particular the upstream stimulatory factors (USFs) . From ex vivo and in vivo experiments, it appears that USFs are required for correct response of the L-PK gene to glucose, but their expression and binding activity are not known to be regulated by glucose . A genetic screen in yeast has allowed us to identify a novel transcriptional factor binding to the GlRE, i.e . the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) . Binding of COUP-TFII to the GlRE was confirmed by electrophoretic mobility shift assays, and COUP-TFII-containing complexes were detectable in liver nuclear extracts . Neither abundance nor binding activity of COUP-TFII appeared to be significantly regulated by diets . In footprinting experiments, two COUP-TFII-binding sites overlapping the E boxes were detected . Overexpression of COUP-TFII abrogated the USF-dependent transactivation of an artificial GlRE-dependent promoter in COS cells and the glucose responsiveness of the L-PK promoter in hepatocytes in primary culture . In addition, a mutated GlRE with increased affinity for USF and very low affinity for COUP-TFII conferred a dramatically decreased glucose responsiveness on the L-PK promoter in hepatocytes in primary culture by increasing activity of the reporter gene in low glucose condition . We propose that COUP-TFII could be a negative regulatory component of the glucose sensor complex assembled on the GlRE of the L-PK gene and most likely of other glucose-responsive genes as well. Drug Metab Dispos, 1999 Oct, 27(10), 1117 - 22 Heterologous expression and kinetic characterization of human cytochromes P-450: validation of a pharmaceutical tool for drug metabolism research; Masimirembwa CM et al.; Drug metabolism studies in the early phases of drug discovery and development will improve the selection of new chemical entities that will be successful in clinical trials . To meet the expanding demands for these studies on the numerous chemicals generated through combinatorial chemistry, we have heterologously expressed nine human drug-metabolizing cytochromes P-450 (CYPs) in Saccharomyces cerevisiae . The enzymes were characterized using known marker substrates CYP1A1/1A2 (ethoxyresorufin), 2C8 (paclitaxel), 2C9 (diclofenac), 2C19 (S-mephenytoin), 2D6 (bufuralol), 2E1 (chlorzoxazone), and 3A4/3A5 (testosterone) . All of the CYPs showed the expected substrate specificity except for chlorzoxazone hydroxylation, which, in addition to CYP2E1 and 1A2, was also catalyzed by CYP1A1 with a high turnover . The apparent Michaelis-Menten parameters obtained for each CYP were within the ranges of those reported in the literature using human liver microsomes and/or recombinant CYPs . The K(m) for CYP2E1-catalyzed chlorzoxazone hydroxylation was, however, much higher (177 microM) than that obtained using liver microsomes (40 microM) . CYP-selective inhibitors, alpha-naphthoflavone (CYP1A1/1A2), quercetin (2C8), sulfaphenazole (2C9), quinidine (2D6), and ketoconazole (3A4/3A5) showed significant isoform-selective inhibitory effects . We have shown that ticlopidine is a potent inhibitor of CYP2C19 (IC(50) = 4 . 5 microM) and CYP2D6 (IC(50) = 3.5 microM) activities . We have therefore successfully set-up and validated an "in-house" heterologous system for the production of human recombinant CYPs for use in metabolism research. Arch Biochem Biophys, 1999 Oct 1, 370(1), 77 - 85 Complementation between mitochondrial processing peptidase (MPP) subunits from different species; Adamec J et al.; Mitochondrial processing peptidase (MPP), a dimer of nonidentical subunits, is the primary peptidase responsible for the removal of leader peptides from nuclearly encoded mitochondrial proteins . Alignments of the alpha and beta subunits of MPP (alpha- and beta-MPP) from different species show strong protein sequence similarity in certain regions, including a highly negatively charged region as well as a domain containing a putative metal ion binding site . In this report, we describe experiments in which we combine the subunits of MPP from yeast, rat, and Neurospora crassa, both in vivo and in vitro and mesure the resultant processing activity . For in vivo complementation, we used the temperature sensitive mif1 and mif2 yeast mutants, which lack MPP activity at the nonpermissive temperature (37 degrees C) . We found that the defective alpha-MPP of mif2 cannot be substituted for by the alpha-MPP from rat or Neurospora . On the other hand, the beta-MPP from rat and Neurospora can fully substitute for the defective beta-MPP in the mif1 mutant . These results were confirmed in in vitro experiments in which individually expressed subunits were combined . Only combinations of the alpha-MPP from yeast with the beta-MPP from rat or Neurospora produced active MPP . Toxicol Sci, 1999 Sep, 51(1), 54 - 70 Detection of thyroid toxicants in a tier I screening battery and alterations in thyroid endpoints over 28 days of exposure; O'Connor JC et al.; Phenobarbital (PB), a thyroid hormone excretion enhancer, and propylthiouracil (PTU), a thyroid hormone-synthesis inhibitor, have been examined in a Tier I screening battery for detecting endocrine-active compounds (EACs) . The Tier I battery incorporates two short-term in vivo tests (5-day ovariectomized female battery and 15-day intact male battery using Sprague-Dawley rats) and an in vitro yeast transactivation system (YTS) . In addition to the Tier I battery, thyroid endpoints (serum hormone concentrations, liver and thyroid weights, thyroid histology, and UDP-glucuronyltransferase {UDP-GT} and 5'-deiodinase activities) have been evaluated in a 15-day dietary restriction experiment . The purpose was to assess possible confounding of results due to treatment-related decreases in body weight . Finally, several thyroid-related endpoints (serum hormone concentrations, hepatic UDP-GT activity, thyroid weights, thyroid follicular cell proliferation, and histopathology of the thyroid gland) have been evaluated for their utility in detecting thyroid-modulating effects after 1, 2, or 4 weeks of treatment with PB or PTU . In the female battery, changes in thyroid endpoints following PB administration, were limited to decreased serum tri-iodothyronine (T3) and thyroxine (T4) concentrations . There were no changes in thyroid stimulating hormone (TSH) concentrations or in thyroid gland histology . In the male battery, PB administration increased serum TSH and decreased T3 and T4 concentrations . The most sensitive indicator of PB-induced thyroid effects in the male battery was thyroid histology (pale staining and/or depleted colloid) . In the female battery, PTU administration produced increases in TSH concentrations, decreases in T3 and T4 concentrations, and microscopic changes (hypertrophy/hyperplasia, colloid depletion) in the thyroid gland . In the male battery, PTU administration caused thyroid gland hypertrophy/hyperplasia and colloid depletion, and the expected thyroid hormonal alterations (increased TSH, and decreased serum T3 and T4 concentrations) . The dietary restriction study demonstrated that possible confounding of the data can occur with the thyroid endpoints when body weight decrements are 15% or greater . In the thyroid time course experiment, PB produced increased UDP-GT activity (at all time points), increased serum TSH (4-week time point), decreased serum T3 (1-and 2-week time points) and T4 (all time points), increased relative thyroid weight (2- and 4-week time points), and increased thyroid follicular cell proliferation (1- and 2-week time points) . Histological effects in PB-treated rats were limited to mild colloid depletion at the 2- and 4-week time points . At all three time points, PTU increased relative thyroid weight, increased serum TSH, decreased serum T3 and T4, increased thyroid follicular cell proliferation, and produced thyroid gland hyperplasia/hypertrophy . Thyroid gland histopathology, coupled with decreased serum T4 concentrations, has been proposed as the most useful criteria for identifying thyroid toxicants . These data suggest that thyroid gland weight, coupled with thyroid hormone analyses and thyroid histology, are the most reliable endpoints for identifying thyroid gland toxicants in a short-duration screening battery . The data further suggest that 2 weeks is the optimal time point for identifying thyroid toxicants based on the 9 endpoints examined . Hence, the 2-week male battery currently being validated as part of this report should be an effective screen for detecting both potent and weak thyroid toxicants. Toxicol Sci, 1999 Sep, 51(1), 44 - 53 Detection of the environmental antiandrogen p,p-DDE in CD and long-evans rats using a tier I screening battery and a Hershberger assay; O'Connor JC et al.; In this report, p,p'-DDE, a weak androgen receptor (AR) antagonist, has been examined in a Tier I screening battery designed to detect endocrine-active compounds (EACs) . The screening battery that was used to examine p,p'-DDE was an abbreviated version of a proposed Tier I screening battery (Cook et al., 1997, Regul . ToxicoL Pharmacol . 26, 60-68) that consisted of a 15-day intact male in vivo battery and an in vitro yeast transactivation system (YTS) . In addition, strain sensitivity differences were evaluated using male Crl:CDIGS BR (CD) and Long-Evans (LE) rats . Finally, p,p'-DDE was examined in a Hershberger assay designed to detect AR agonists . In the in vivo male battery using CD rats, responses to p,p'-DDE included organ weight changes (increased relative liver weight and decreased absolute epididymis weight) and hormonal alterations (increased serum estradiol {E2} levels and decreased serum FSH and T4 levels) . Responses to p,p'-DDE in LE rats included organ weight changes (increased relative liver weight, absolute epididymis weight, relative accessory sex gland {ASG} unit weight, as well as the individual component weights of the ASG {prostate and seminal vesicles}), and hormonal alterations (increased serum testosterone {T}, E2, dihydrotestosterone {DHT}, thyroid-stimulating hormone {TSH}, and decreased T4 levels) . These data demonstrate that there are considerable strain-sensitivity differences to p,p'-DDE exposure . The described in vivo male battery using CD rats did not identify p,p'-DDE as an EAC . In contrast, the in vivo male battery using LE rats identified p,p'-DDE as a EAC . Evaluation of the data for the LE rats demonstrate that p,p'-DDE appears to be acting as an AR antagonist whose primary effects are more potent centrally than peripherally . In the YTS for the AR, p,p'-DDE had an EC50 value of 3.5 x 10(-4) M; however, in the AR YTS competition assay, p,p'-DDE did not inhibit DHT binding to the AR . p,p'-DDE was inactive in the YTS containing the estrogen receptor or progesterone receptor at the concentrations evaluated . In the Hershberger assay, p,p'-DDE administration caused antiandrogen-like effects characterized by attenuation of the testosterone propionate-induced increases in reproductive-organ weights . In summary, these data suggest that strain selection will affect the ability to detect certain weak EACs . However, a Tier I screening battery consisting of both in vivo and in vitro endpoints would reduce the chance that weak-acting compounds such as p,p'-DDE would not be identified as potential EACs. Int J Oncol, 1999 Oct, 15(4), 687 - 92 Prognostic significance of polo-like kinase expression in esophageal carcinoma; Tokumitsu Y et al.; PLK (polo-like kinase), which belongs to a family of serine/threonine kinases and represents the human counterpart of structurally related protein kinases, polo of Drosophila melanogaster and CDC5 of Saccharomyces cerevisiae, may be implicated in spindle formation and chromosome segregation during mitosis . There are, however, few reports on the significance of PLK gene expression in human carcinomas . In order to evaluate its clinical significance, we examined the expression of the PLK mRNA in 49 esophageal and 75 gastric carcinomas, using reverse transcription-polymerase chain reaction analysis . In esophageal carcinomas, PLK overexpression was detected in 47 carcinomas (97%) when compared to the corresponding normal tissues . It is noteworthy that the patients with high-grade PLK overexpression represented a significantly poorer prognosis group than those with low-grade PLK overexpression (3-year survival rate: 54.9% vs 24.8%, p<0.05) . A multivariate analysis demonstrated that the PLK mRNA expression status was an independent prognostic factor for patients with esophageal carcinoma . On the other hand, 55 gastric carcinomas (73%) were revealed to overexpress PLK mRNA, but the expression status showed no correlation with prognosis . This study demonstrated that the PLK overexpression was frequently observed in esophageal and gastric carcinomas, and appeared to be an independent prognostic factor for patients with esophageal carcinoma. Cancer Res, 1999 Sep 15, 59(18), 4519 - 24 Two organochlorine pesticides, toxaphene and chlordane, are antagonists for estrogen-related receptor alpha-1 orphan receptor; Yang C et al.; Estrogen-related receptor (ERR) alpha-1 shares a high amino acid sequence homology with estrogen receptor alpha . Although estrogens are not ligands of ERR alpha-1, our recent results suggest that toxaphene and chlordane, two organochlorine pesticides with estrogen-like activity, behave as antagonists for this orphan nuclear receptor . The two compounds increased ERR alpha-1-mediated expression of the reporter enzyme beta-galactosidase in a yeast-based assay . The screen was developed by expressing the hERR alpha-1-yeast Gal 4 activation domain fusion protein in yeast cells carrying the beta-galactosidase reporter plasmid, which contains an ERR alpha-1-binding element . In transfection experiments using mammalian cell lines, such as the SK-BR-3 breast cancer cell line, the compounds were found to have an antagonist activity against ERR alpha-1-mediated expression of the reporter chloramphenicol acetyltransferase . In contrast to the findings with ERR alpha-1, the two compounds were found to slightly induce the estrogen receptor a-mediated expression of chloramphenicol acetyltransferase in SK-BR-3 cells . In a ligand-independent manner, the ERR alpha-1 activity in SK-BR-3 cells was induced 3-fold by cotransfection with the GRIP1 coactivator expression plasmid . Toxaphene was found to be capable of suppressing the GRIP1 coactivator-induced ERR alpha-1 activity in SK-BR-3 cells . In addition, a stable ERR alpha-1 expressing HepG2 hepatoma cell line was generated, and the aromatase activity in the transfected cell line was found to be twice that in the untransfected cell line . The enzyme aromatase converts androgens to estrogens, and aromatase expression in HepG2 cells is regulated in part by an ERR alpha-1-modulating promoter . A 24-h incubation of an ERR alpha-1-transfected HepG2 cell line with 10 microM toxaphene reduced its aromatase activity to the level in the untransfected cell line . Because toxaphene is not an inhibitor of aromatase, it is thought that the decrease of the aromatase activity in ERR alpha-1 transfected HepG2 cells following toxaphene treatment resulted from a suppression of the aromatase expression by toxaphene acting as the antagonist of ERR alpha-1 . Toxaphene and chlordane are among the 12 persistent organic pollutants identified by the United Nations Environment Programme as requiring urgent attention . Their antagonistic effects on ERR alpha-1 should not be overlooked. Dev Biol, 1999 Oct 1, 214(1), 197 - 210 Study of the posterior spiracles of Drosophila as a model to understand the genetic and cellular mechanisms controlling morphogenesis; Hu N et al.; We have studied the posterior spiracles of Drosophila as a model to link patterning genes and morphogenesis . A genetic cascade of transcription factors downstream of the Hox gene Abdominal-B subdivides the primordia of the posterior spiracles into two cell populations that develop using two different morphogenetic mechanisms . The inner cells that give rise to the spiracular chamber invaginate by elongating into "bottle-shaped" cells . The surrounding cells give rise to a protruding stigmatophore by changing their relative positions in a process similar to convergent extension . The genetic cascades regulating spiracular chamber, stigmatophore, and trachea morphogenesis are different but coordinated to form a functional tracheal system . In the posterior spiracle, this coordination involves the control of the initiation of cell invagination that starts in the cells closer to the trachea primordium and spreads posteriorly . As a result, the opening of the tracheal system shifts back from the spiracular branch of the trachea into the posterior spiracle cells . We analyze the contribution of the ems gene to this coordination . In ems mutants, invagination of the spiracle cells adjacent to the trachea does not occur, but more posterior cells of the spiracle invaginate normally . This results in a spiracle without a lumen and with the tracheal opening located outside it . Eur J Biochem, 1999 Oct 1, 265(1), 210 - 20 Mutational analysis of Ser14 and Asp157 in the nucleotide-binding site of beta-actin; Schuler H et al.; This paper compares wild-type and two mutant beta-actins, one in which Ser14 was replaced by a cysteine, and a second in which both Ser14 and Asp157 were exchanged (Ser14-->Cys and Ser14-->Cys, Asp157-->Ala, respectively) . Both of these residues are part of invariant sequences in the loops, which bind the ATP phosphates, in the interdomain cleft of actin . The increased nucleotide exchange rate, and the decreased thermal stability and affinity for DNase I seen with the mutant actins indicated that the mutations disturbed the interdomain coupling . Despite this, the two mutant actins retained their ATPase activity . In fact, the mutated actins expressed a significant ATPase activity even in the presence of Ca2+ ions, conditions under which actin normally has a very low ATPase activity . In the presence of Mg2+ ions, the ATPase activity of actin was decreased slightly by the mutations . The mutant actins polymerized as the wild-type protein in the presence of Mg2+ ions, but slower than the wild-type in a K+/Ca2+ milieu . Profilin affected the lag phases and elongation rates during polymerization of the mutant and wild-type actins to the same extent, whereas at steady-state, the concentration of unpolymerized mutant actin appeared to be elevated . Decoration of mutant actin filaments with myosin subfragment 1 appeared to be normal, as did their movement in the low-load motility assay system . Our results show that Ser14 and Asp157 are key residues for interdomain communication, and that hydroxyl and carboxyl groups in positions 14 and 157, respectively, are not necessary for ATP hydrolysis in actin. Eur J Biochem, 1999 Sep, 264(2), 415 - 26 Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis; Findeisen M et al.; The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex . In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract . We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-dependent kinase activities . High cyclin A-dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins . Cyclin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei . In contrast, high cyclin E/cdk2 was not detected on chromatin, but was found soluble in the nucleoplasm . High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM directly . We conclude that chromatin-bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association. Mol Cell Biol, 1999 Oct, 19(10), 6729 - 41 Regulation of cell cycle transcription factor Swi4 through auto-inhibition of DNA binding; Baetz K et al.; In Saccharomyces cerevisiae, two transcription factors, SBF (SCB binding factor) and MBF (MCB binding factor), promote the induction of gene expression at the G(1)/S-phase transition of the mitotic cell cycle . Swi4 and Mbp1 are the DNA binding components of SBF and MBF, respectively . The Swi6 protein is a common subunit of both transcription factors and is presumed to play a regulatory role . SBF binding to its target sequences, the SCBs, is a highly regulated event and requires the association of Swi4 with Swi6 through their C-terminal domains . Swi4 binding to SCBs is restricted to the late M and G(1) phases, when Swi6 is localized to the nucleus . We show that in contrast to Swi6, Swi4 remains nuclear throughout the cell cycle . This finding suggests that the DNA binding domain of Swi4 is inaccessible in the full-length protein when not complexed with Swi6 . To explore this hypothesis, we expressed Swi4 and Swi6 in insect cells by using the baculovirus system . We determined that partially purified Swi4 cannot bind SCBs in the absence of Swi6 . However, Swi4 derivatives carrying point mutations or alterations in the extreme C terminus were able to bind DNA or activate transcription in the absence of Swi6, and the C terminus of Swi4 inhibited Swi4 derivatives from binding DNA in trans . Full-length Swi4 was determined to be monomeric in solution, suggesting an intramolecular mechanism for auto-inhibition of binding to DNA by Swi4 . We detected a direct in vitro interaction between a C-terminal fragment of Swi4 and the N-terminal 197 amino acids of Swi4, which contain the DNA binding domain . Together, our data suggest that intramolecular interactions involving the C-terminal region of Swi4 physically prevent the DNA binding domain from binding SCBs . The interaction of the carboxy-terminal region of Swi4 with Swi6 alleviates this inhibition, allowing Swi4 to bind DNA. Mol Cell Biol, 1999 Oct, 19(10), 6642 - 51 The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and functionally separated from NOT2, NOT4, and NOT5; Bai Y et al.; The CCR4-NOT complex (1 mDa in size), consisting of the proteins CCR4, CAF1, and NOT1 to NOT5, regulates gene expression both positively and negatively and is distinct from other large transcriptional complexes in Saccharomyces cerevisiae such as SNF/SWI, TFIID, SAGA, and RNA polymerase II holoenzyme . The physical and genetic interactions between the components of the CCR4-NOT complex were investigated in order to gain insight into how this complex affects the expression of diverse genes and processes . The CAF1 protein was found to be absolutely required for CCR4 association with the NOT proteins, and CCR4 and CAF1, in turn, physically interacted with NOT1 through its central amino acid region from positions 667 to 1152 . The NOT3, NOT4, and NOT5 proteins had no significant effect on the association of CCR4, CAF1, and NOT1 with each other . In contrast, the NOT2, NOT4, and NOT5 interacted with the C-terminal region (residues 1490 to 2108) of NOT1 in which NOT2 and NOT5 physically associated in the absence of CAF1, NOT3, and NOT4 . These and other data indicate that the physical ordering of these proteins in the complex is CCR4-CAF1-NOT1-(NOT2, NOT5), with NOT4 and NOT3 more peripheral to NOT2 and NOT5 . The physical separation of CCR4 and CAF1 from other components of the CCR4-NOT complex correlated with genetic analysis indicating partially separate functions for these two groups of proteins . ccr4 or caf1 deletion suppressed the increased 3-aminotriazole resistance phenotype conferred by not mutations, resulted in opposite effects on gene expression as compared to several not mutations, and resulted in a number of synthetic phenotypes in combination with not mutations . These results define the CCR4-NOT complex as consisting of at least two physically and functionally separated groups of proteins. Acta Crystallogr D Biol Crystallogr, 1999 Sep, 55 ( Pt 9), 1503 - 15 How RhoGDI binds Rho; Longenecker K et al.; Like all Rho (Ras homology) GTPases, RhoA functions as a molecular switch in cell signaling, alternating between GTP- and GDP-bound states, with its biologically inactive GDP-bound form maintained as a cytosolic complex with RhoGDI (guanine nucleotide-exchange inhibitor) . The crystal structures of RhoA-GDP and of the C-terminal immunoglobulin-like domain of RhoGDI (residues 67-203) are known, but the mechanism by which the two proteins interact is not known . The functional human RhoA-RhoGDI complex has been expressed in yeast and crystallized (P6(5)22, unit-cell parameters a = b = 139, c = 253 A, two complexes in the asymmetric unit) . Although diffraction from these crystals extends to 3.5 A and is highly anisotropic, the experimentally phased (MAD plus MIR) electron-density map was adequate to reveal the mutual disposition of the two molecules . The result was validated by molecular-replacement calculations when data were corrected for anisotropy . Furthermore, the N-terminus of RhoGDI (the region involved in inhibition of nucleotide exchange) can be identified in the electron-density map: it is bound to the switch I and switch II regions of RhoA, occluding an epitope which binds Dbl-like nucleotide-exchange factors . The entrance of the hydrophobic pocket of RhoGDI is 25 A from the last residue in the RhoA model, with its C-terminus oriented to accommodate the geranylgeranyl group without conformational change in RhoA. Cell, 1999 Sep 3, 98(5), 675 - 86 Regulation of hormone-induced histone hyperacetylation and gene activation via acetylation of an acetylase; Chen H et al.; Nuclear receptors have been postulated to regulate gene expression via their association with histone acetylase (HAT) or deacetylase complexes . We report that hormone induces dramatic hyperacetylation at endogenous target genes through the HAT activity of p300/CBP . Unexpectedly, this hyperacetylation is transient and coincides with attenuation of hormone-induced gene activation . In exploring the underlying mechanism, we found that the acetylase ACTR can be acetylated by p300/CBP . The acetylation neutralizes the positive charges of two lysine residues adjacent to the core LXXLL motif and disrupts the association of HAT coactivator complexes with promoter-bound estrogen receptors . These results provide strong in vivo evidence that histone acetylation plays a key role in hormone-induced gene activation and define cofactor acetylation as a novel regulatory mechanism in hormonal signaling. Folia Microbiol (Praha), 1999, 44(1), 19 - 24 Dimorphism in Yarrowia lipolytica: filament formation is suppressed by nitrogen starvation and inhibition of respiration; Szabo R; In contrast to Saccharomyces cerevisiae, nitrogen starvation inhibited formation of hyphae in liquid cultures of Y . lipolytica, while carbon source did not seem to be important for filament formation . Inhibitors of mitochondrial respiration strongly suppressed the development of hyphae, indicating that energy conversion processes, and thus carbon metabolism, may be involved . pH of the medium also strongly affected the morphology, but only in the presence of a complex nitrogen source, implying that the cells respond to altered nutrition in media with different pH rather than to pH itself . The results suggest that the XPR2 gene encoding Y . lipolytica alkaline extracellular proteinase is involved in the regulation of dimorphism in this species. Virology, 1999 Sep 15, 262(1), 200 - 9 Analysis of the RNA binding specificity of the human tap protein, a constitutive transport element-specific nuclear RNA export factor; Kang Y et al.; The human Tap protein has been proposed to mediate Mason Pfizer monkey virus constitutive transport element (CTE)-dependent nuclear RNA export and may also play a role in global mRNA export . Here, we have used in vivo assays, in both yeast and human cells, together with in vitro assays, to further characterize the RNA binding properties of Tap, which has been proposed to contain a novel leucine-rich RNA binding motif . Using the yeast three hybrid assay, we selected RNA molecules that retain Tap binding activity from a pool of randomized CTE sequences . The recovered RNA sequences differed only minimally from the wild-type CTE yet all displayed lower affinity for Tap both in vivo and in vitro . Analysis of the RNA export activity of the recovered CTE variants revealed that Tap affinity was highly predictive of CTE biological activity . Together, these observations provide additional evidence supporting the identification of Tap as the direct cofactor for CTE function and demonstrate that RNA binding by Tap is highly sequence specific . Mol Cell, 1999 Aug, 4(2), 153 - 66 Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding; Rittinger K et al.; We have solved the high-resolution X-ray structure of 14-3-3 bound to two different phosphoserine peptides, representing alternative substrate-binding motifs . These structures reveal an evolutionarily conserved network of peptide-protein interactions within all 14-3-3 isotypes, explain both binding motifs, and identify a novel intrachain phosphorylation-mediated loop structure in one of the peptides . A 14-3-3 mutation disrupting Raf signaling alters the ligand-binding cleft, selecting a different phosphopeptide-binding motif and different substrates than the wild-type protein . Many 14-3-3: peptide contacts involve a C-terminal amphipathic alpha helix containing a putative nuclear export signal, implicating this segment in both ligand and Crm1 binding . Structural homology between the 14-3-3 NES structure and those within I kappa B alpha and p53 reveals a conserved topology recognized by the Crm1 nuclear export machinery. Plant Cell, 1999 Sep, 11(9), 1743 - 54 A transmembrane hybrid-type histidine kinase in Arabidopsis functions as an osmosensor; Urao T et al.; Water deficit and the resulting osmotic stress affect plant growth . To understand how plant cells monitor and respond to osmotic change from water stress, we isolated a cDNA from dehydrated Arabidopsis plants . This cDNA encodes a novel hybrid-type histidine kinase, ATHK1 . Restriction fragment length polymorphism mapping showed that the ATHK1 gene is on chromosome 2 . The predicted ATHK1 protein has two putative transmembrane regions in the N-terminal half and has structural similarity to the yeast osmosensor synthetic lethal of N-end rule 1 (SLN1) . The ATHK1 transcript was more abundant in roots than other tissues under normal growth conditions and accumulated under conditions of high or low osmolarity . Histochemical analysis of beta-glucuronidase activities driven by the ATHK1 promoter further indicates that the ATHK1 gene is transcriptionally upregulated in response to changes in external osmolarity . Overexpression of the ATHK1 cDNA suppressed the lethality of the temperature-sensitive osmosensing-defective yeast mutant sln1-ts . By contrast, ATHK1 cDNAs in which conserved His or Asp residues had been substituted failed to complement the sln1-ts mutant, indicating that ATHK1 functions as a histidine kinase . Introduction of the ATHK1 cDNA into the yeast double mutant sln1Delta sho1Delta, which lacks two osmosensors, suppressed lethality in high-salinity media and activated the high-osmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK) . These results imply that ATHK1 functions as an osmosensor and transmits the stress signal to a downstream MAPK cascade. J Biol Chem, 1999 Sep 24, 274(39), 28019 - 25 Interaction of hHR23 with S5a . The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26 S proteasome; Hiyama H et al.; hHR23B is one of two human homologs of the Saccharomyces cerevisiae nucleotide excision repair (NER) gene product RAD23 and a component of a protein complex that specifically complements the NER defect of xeroderma pigmentosum group C (XP-C) cell extracts in vitro . Although a small proportion of hHR23B is tightly complexed with the XP-C responsible gene product, XPC protein, a vast majority exists as an XPC-free form, indicating that hHR23B has additional functions other than NER in vivo . Here we demonstrate that the human NER factor hHR23B as well as another human homolog of RAD23, hHR23A, interact specifically with S5a, a subunit of the human 26 S proteasome using the yeast two-hybrid system . Furthermore, hHR23 proteins were detected with S5a at the position where 26 S proteasome sediments in glycerol gradient centrifugation of HeLa S100 extracts . Intriguingly, hHR23B showed the inhibitory effect on the degradation of (125)I-lysozyme in the rabbit reticulocyte lysate . hHR23 proteins thus appear to associate with 26 S proteasome in vivo . From co-precipitation experiments using several series of deletion mutants, we defined the domains in hHR23B and S5a that mediate this interaction . From these results, we propose that part of hHR23 proteins are involved in the proteolytic pathway in cells. J Biol Chem, 1999 Sep 24, 274(39), 27845 - 56 The mammalian HSF4 gene generates both an activator and a repressor of heat shock genes by alternative splicing; Tanabe M et al.; The expression of heat shock genes is controlled at the level of transcription by members of the heat shock transcription factor family in vertebrates . HSF4 is a mammalian factor characterized by its lack of a suppression domain that modulates formation of DNA-binding homotrimer . Here, we have determined the exon structure of the human HSF4 gene and identified a major new isoform, HSF4b, derived by alternative RNA splicing events, in addition to a previously reported HSF4a isoform . In mouse tissues HSF4b mRNA was more abundant than HSF4a as examined by reverse transcription-polymerase chain reaction, and its protein was detected in the brain and lung . Although both mouse HSF4a and HSF4b form trimers in the absence of stress, these two isoforms exhibit different transcriptional activity; HSF4a acts as an inhibitor of the constitutive expression of heat shock genes, and hHSF4b acts as a transcriptional activator . Furthermore HSF4b but not HSF4a complements the viability defect of yeast cells lacking HSF . Moreover, heat shock and other stresses stimulate transcription of target genes by HSF4b in both yeast and mammalian cells . These results suggest that differential splicing of HSF4 mRNA gives rise to both an inhibitor and activator of tissue-specific heat shock gene expression. EMBO J, 1999 Sep 15, 18(18), 5108 - 19 NuA4, an essential transcription adaptor/histone H4 acetyltransferase complex containing Esa1p and the ATM-related cofactor Tra1p; Allard S et al.; Post-translational acetylation of histone H4 N-terminal tail in chromatin has been associated with several nuclear processes including transcription . We report the purification and characterization of a native multisubunit complex (NuA4) from yeast that acetylates nucleosomal histone H4 . NuA4 has an apparent molecular mass of 1.3 MDa . All four conserved lysines of histone H4 can be acetylated by NuA4 . We have identified the catalytic subunit of the complex as the product of ESA1, an essential gene required for cell cycle progression in yeast . Antibodies against Esa1p specifically immunoprecipitate NuA4 activity whereas the complex purified from a temperature-sensitive esa1 mutant loses its acetyltransferase activity at the restrictive temperature . Additionally, we have identified another subunit of the complex as the product of TRA1, an ATM-related essential gene homologous to human TRRAP, an essential cofactor for c-Myc- and E2F-mediated oncogenic transformation . Finally, the ability of NuA4 to stimulate GAL4-VP16-driven transcription from chromatin templates in vitro is also lost in the temperature-sensitive esa1 mutant . The function of the essential Esa1 protein as the HAT subunit of NuA4 and the presence of Tra1p, a putative transcription activator-interacting subunit, supports an essential link between nuclear H4 acetylation, transcriptional regulation and cell cycle control. EMBO J, 1999 Sep 15, 18(18), 5099 - 107 HDAC4 deacetylase associates with and represses the MEF2 transcription factor; Miska EA et al.; The acetylation state of histones can influence transcription . Acetylation, carried out by acetyltransferases such as CBP/p300 and P/CAF, is commonly associated with transcriptional stimulation, whereas deacetylation, mediated by the three known human deacetylases HDAC1, 2 and 3, causes transcriptional repression . The known human deacetylases represent a single family and are homologues of the yeast RPD3 deacetylase . Here we identify and characterize HDAC4, a representative of a new human histone deacetylase family, which is homologous to the yeast HDA1 deacetylase . We show that HDAC4, unlike other deacetylases, shuttles between the nucleus and the cytoplasm in a process involving active nuclear export . In the nucleus, HDAC4 associates with the myocyte enhancer factor MEF2A . Binding of HDAC4 to MEF2A results in the repression of MEF2A transcriptional activation, a function that requires the deacetylase domain of HDAC4 . These results identify MEF2A as a nuclear target for HDAC4-mediated repression and suggests that compartmentalization may be a novel mechanism for controlling the nuclear activity of this new family of deacetylases. Biochem Biophys Res Commun, 1999 Sep 16, 263(1), 213 - 8 Molecular cloning and expression of human carnitine octanoyltransferase: evidence for its role in the peroxisomal beta-oxidation of branched-chain fatty acids; Ferdinandusse S et al.; To study the putative role of human carnitine octanoyltransferase (COT) in the beta-oxidation of branched-chain fatty acids, we identified and cloned the cDNA encoding human COT and expressed it in the yeast Saccharomyces cerevisiae . Enzyme activity measurements showed that COT efficiently converts one of the end products of the peroxisomal beta-oxidation of pristanic acid, 4, 8-dimethylnonanoyl-CoA, to its corresponding carnitine ester . Production of the carnitine ester of this branched/medium-chain acyl-CoA within the peroxisome is required for its transport to the mitochondrion where further beta-oxidation occurs . In contrast, 4, 8-dimethylnonanoyl-CoA is not a substrate for carnitine acetyltransferase, another acyltransferase localized in peroxisomes, which catalyzes the formation of carnitine esters of the other products of pristanic acid beta-oxidation, namely acetyl-CoA and propionyl-CoA . Our results shed new light on the function of COT in fatty acid metabolism and point to a crucial role of COT in the beta-oxidation of branched-chain fatty acids . Biochem Biophys Res Commun, 1999 Sep 16, 263(1), 23 - 7 In vitro selective RNA synthesis with L-A virus nanoparticles; Ebihara T et al.; New in vitro RNA synthesis has been performed with an L-A virus nanoparticles, in which the gene and polymerase are integrated . The specific recognition sequence (packaging site) of L-A virus was inserted within a gene of interest . Based on the intrinsic replication cycle, the exogenous RNA with the packaging site was encapsulated by an empty L-A virus nanoparticle . The packaging site worked as a recognition site even for exogenous RNAs . The recognized RNA was replicated to dsRNA, and was then transcribed by empty L-A virus nanoparticles . These results indicate that empty L-A virus nanoparticles recognize an exogenous RNA with the packaging site and synthesize RNA in vitro . Arch Biochem Biophys, 1999 Sep 15, 369(2), 193 - 6 Purification and characterization of recombinant rat hepatic CYP4F1; Kikuta Y et al.; CYP4F1 was discovered by Chen and Hardwick (Arch . Biochem . Biophys . 300, 18-23, 1993) as a new CYP4 cytochrome P450 (P450) preferentially expressed in rat hepatomas . However, the catalytic function of this P450 remained poorly defined . We have purified recombinant CYP4F1 protein to a specific content of 12 nmol of P450/mg of protein from transfected yeast cells by chromatography of solubilized microsomes on an amino-n-hexyl Sepharose 4B column, followed by sequential HPLC on a DEAE column and two hydroxylapatite columns . The purified P450 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 53 kDa . The enzyme catalyzed the omega-hydroxylation of leukotriene B(4) with a K(m) of 134 microM and a V(max) of 6.5 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b(5) . In addition, 6-trans-LTB(4), lipoxin A(4), prostaglandin A(1), and several hydroxyeicosatetraenoic acids (HETEs) were also omega-hydroxylated . Of several eicosanoids examined, 8-HETE was the most efficient substrate, with a K(m) of 18.6 microM and a V(max) of 15.8 nmol/min/nmol of P450 . In contrast, no activity was detected toward lipoxin B(4), laurate, palmitate, arachidonate, and benzphetamine . The results suggest that CYP4F1 participates in the hepatic inactivation of several bioactive eicosanoids. Proc Natl Acad Sci U S A, 1999 Sep 14, 96(19), 10770 - 5 Deletion of Ku86 causes early onset of senescence in mice; Vogel H et al.; DNA double-strand breaks formed during the assembly of antigen receptors or after exposure to ionizing radiation are repaired by proteins important for nonhomologous end joining that include Ku86, Ku70, DNA-PK(CS), Xrcc4, and DNA ligase IV . Here we show that ku86-mutant mice, compared with control littermates, prematurely exhibited age-specific changes characteristic of senescence that include osteopenia, atrophic skin, hepatocellular degeneration, hepatocellular inclusions, hepatic hyperplastic foci, and age-specific mortality . Cancer and likely sepsis (indicated by reactive immune responses) partly contributed to age-specific mortality for both cohorts, and both conditions occurred earlier in ku86(-/-) mice . These data indicate that Ku86-dependent chromosomal metabolism is important for determining the onset of age-specific changes characteristic of senescence in mice. Proc Natl Acad Sci U S A, 1999 Sep 14, 96(19), 10699 - 704 Transition state heterogeneity in GCN4 coiled coil folding studied by using multisite mutations and crosslinking; Moran LB et al.; We have investigated the folding behavior of dimeric and covalently crosslinked versions of the 33-residue alpha-helical GCN4-p1 coiled coil derived from the leucine zipper region of the transcriptional activator GCN4 . The effects of multisite substitutions indicate that folding occurs along multiple routes with nucleation sites located throughout the protein . The similarity in activation energies of the different routes together with an analysis of intrinsic helical propensities indicate that minimal helix is present before a productive collision of the two chains . However, approximately one-third to one-half of the total helical structure is formed in the postcollision transition state ensemble . For the crosslinked, monomeric version, folding occurs along a single robust pathway . Here, the region nearest the crosslink, with the least helical propensity, is structured in the transition state whereas the region farthest from the tether, with the most propensity, is completely unstructured . Hence, the existence of transition state heterogeneity and the selection of folding routes critically depend on chain topology. Genes Dev, 1999 Sep 1, 13(17), 2284 - 300 Nuclear export of Far1p in response to pheromones requires the export receptor Msn5p/Ste21p; Blondel M et al.; Far1p is a bifunctional protein that is required to arrest the cell cycle and to establish cell polarity during yeast mating . Far1p is localized predominantly in the nucleus but accumulates in the cytoplasm in cells exposed to pheromones . Here we show that Far1p functions in both subcellular compartments: nuclear Far1p is required to arrest the cell cycle, whereas cytoplasmic Far1p is involved in the establishment of cell polarity . The subcellular localization of Far1p is regulated by two mechanisms: (1) Far1p contains a functional bipartite nuclear localization signal (NLS), and (2) Far1p is exported from the nucleus by Msn5p/Ste21p, a member of the exportin family . Cells deleted for Msn5p/Ste21p failed to export Far1p in response to pheromones, whereas overexpression of Msn5p/Ste21p was sufficient to accumulate Far1p in the cytoplasm in the absence of pheromones . Msn5p/Ste21p was localized in the nucleus and interacted with Far1p in a manner dependent on GTP-bound Gsp1p . Two-hybrid analysis identified a small fragment within Far1p that is necessary and sufficient for binding to Msn5p/Ste21p, and is also required to export Far1p in vivo . Finally, similar to Deltamsn5/ste21 strains, cells expressing a mutant Far1p, which can no longer be exported, exhibit a mating defect, but are able to arrest their cell cycle in response to pheromones . Taken together, our results suggest that nuclear export of Far1p by Msn5p/Ste21p coordinates the two separable functions of Far1p during mating. Genes Dev, 1999 Sep 1, 13(17), 2242 - 57 Components of an SCF ubiquitin ligase localize to the centrosome and regulate the centrosome duplication cycle; Freed E et al.; Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis . The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown . In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown . Using immunofluorescence microscopy, we show that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus . Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome . Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting . Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation . Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos . We identify candidate centrosomal F-box proteins, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle. Biophys Chem, 1999 Aug 30, 80(3), 217 - 26 A thermodynamic study on the formation and stability of DNA duplex at transcription site for DNA binding proteins GCN4; Cao W et al.; Using isothermal titration calorimetry (ITC), we studied the thermodynamic parameters of the 15-mer duplex dsDNA {d(GAGATGACTCATCTC)}.{d(GAGATGAGTCATCTC)} formation from its two complementary single strands (S1 and S2) over a range of temperatures . The two complementary single strands d(GAGATGACTCATCTC) (herein called S1) and d(GAGATGAGTCATCTC) (herein called S2) containing palindromic sequences may assume ordered structures at low temperatures, which made the duplex dsDNA formation rather complicated . The thermodynamic parameters for the duplex formation, such as the binding constants (Kb), the enthalpies (delta H0), the free energies (delta G0), the entropies (delta S0) are strongly temperature-dependent . The thermally-induced disruptions of the duplex and its two complementary single strands, S1 and S2, were measured using differential scanning calorimetry (DSC) and CD spectroscopy, the results demonstrate that the DNA duplex is very stable, and its component single strands have an ordered structure at low temperature . This 15-mer specific sequence DNA may act as recognition site for DNA binding proteins GCN4 and plays a key role in transcription regulation of gene expression . Our analyses of the thermodynamic data suggest that the duplex formation is a coupled process between conformational transitions in the two single strands and their binding to form duplex dsDNA. J Biotechnol, 1999 May 28, 71(1-3), 207 - 23 Analysis of the pathway structure of metabolic networks; Simpson TW et al.; Metabolic networks comprise a multitude of enzymatic reactions carrying out various functions related to cell growth and product formation . Although such reactions are occasionally organized into biochemical pathways, a formal procedure is desired to identify the independent pathways in a bioreaction network and the degree of engagement of each individual reaction in these pathways . We present a procedure for the identification of the independent pathways of bioreaction networks of any size and complexity . The method makes use of the steady-state internal metabolite stoichiometry matrix and defines the independent pathways through the reaction membership of its kernel matrix . Examples from the aromatic amino acid biosynthetic pathway and central carbon metabolism of cells in culture are provided to illustrate the method . Applications to the analysis of the control structure of bioreaction networks are also discussed. J Virol, 1999 Oct, 73(10), 8732 - 40 Functional analysis of the interaction between VPg-proteinase (NIa) and RNA polymerase (NIb) of tobacco etch potyvirus, using conditional and suppressor mutants; Daros JA et al.; The tobacco etch potyvirus (TEV) RNA-dependent RNA polymerase (NIb) has been shown to interact with the proteinase domain of the VPg-proteinase (NIa) . To investigate the significance of this interaction, a Saccharomyces cerevisiae two-hybrid assay was used to isolate conditional NIa mutant proteins with temperature-sensitive (ts) defects in interacting with NIb . Thirty-six unique tsNIa mutants with substitutions affecting the proteinase domain were recovered . Most of the mutants coded for proteins with little or no proteolytic activity at permissive and nonpermissive temperatures . However, three mutant proteins retained proteolytic activity at both temperatures and, in two cases (tsNIa-Q384P and tsNIa-N393D), the mutations responsible for the ts interaction phenotype could be mapped to single positions . One of the mutations (N393D) conferred a ts-genome-amplification phenotype when it was placed in a recombinant TEV strain . Suppressor NIb mutants that restored interaction with the tsNIa-N393D protein at the restrictive temperature were recovered by a two-hybrid selection system . Although most of the suppressor mutants failed to stimulate amplification of genomes encoding the tsNIa-N393D protein, two suppressors (NIb-I94T and NIb-C380R) stimulated amplification of virus containing the N393D substitution by approximately sevenfold . These results support the hypothesis that interaction between NIa and NIb is important during TEV genome replication. Ann Neurol, 1999 Sep, 46(3), 409 - 12 Role of very-long-chain acyl-coenzyme A synthetase in X-linked adrenoleukodystrophy; Steinberg SJ et al.; X-linked adrenoleukodystrophy (X-ALD) is characterized biochemically by decreased ability of cells to activate (via very-long-chain acyl-coenzyme A synthetase {VLCS}) and subsequently degrade very-long-chain fatty acids in peroxisomes . It is noteworthy that the gene defective in X-ALD encodes ALDP, a peroxisomal membrane protein unrelated to VLCS . We cloned human VLCS (hVLCS) and found that peroxisomes from X-ALD fibroblasts contained immunoreactive hVLCS, refuting the earlier hypothesis that ALDP is required to anchor VLCS to the peroxisomal membrane . Furthermore, hVLCS was topographically oriented facing the peroxisomal matrix in both control and X-ALD fibroblasts, contradicting the alternative hypothesis that ALDP is required to translocate VLCS into peroxisomes . However, overexpression of both hVLCS and ALDP in X-ALD fibroblasts synergistically increased very-long-chain fatty acid beta-oxidation, indicating that these proteins interact functionally. Cell, 1999 Aug 20, 98(4), 453 - 63 The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID; Wang SL et al.; Drosophila Reaper (RPR), Head Involution Defective (HID), and GRIM induce caspase-dependent cell death and physically interact with the cell death inhibitor DIAP1 . Here we show that HID blocks DIAP1's ability to inhibit caspase activity and provide evidence suggesting that RPR and GRIM can act similarly . Based on these results, we propose that RPR, HID, and GRIM promote apoptosis by disrupting productive IAP-caspase interactions and that DIAP1 is required to block apoptosis-inducing caspase activity . Supporting this hypothesis, we show that elimination of DIAP1 function results in global early embryonic cell death and a large increase in DIAP1-inhibitable caspase activity and that DIAP1 is still required for cell survival when expression of rpr, hid, and grim is eliminated. Trends Cell Biol, 1999 Oct, 9(10), 387 - 94 Septins: cytoskeletal polymers or signalling GTPases? Field CM, Kellogg D. Septins are a family of conserved proteins that have been implicated in a variety of cellular functions involving specialized regions of the cell cortex and changes in cell shape . The biochemistry and localization of septins suggest that they form a novel cytoskeletal system or that they function as scaffolds for the assembly of signalling complexes . This article discusses septin biochemistry and septin-interacting proteins, focusing on the missing link between the structure and biochemical properties of septin proteins, and on how they function at a molecular level in processes such as cytokinesis and yeast budding. J Biol Chem, 1999 Sep 17, 274(38), 27225 - 30 Wiskott-Aldrich syndrome protein induces actin clustering without direct binding to Cdc42; Kato M et al.; WASP (Wiskott-Aldrich syndrome protein) was identified as the gene product whose mutation causes the human hereditary disease Wiskott-Aldrich syndrome . WASP contains many functional domains and has been shown to induce the formation of clusters of actin filaments in a manner dependent on Cdc42 . However, there has been no report investigating what domain(s) is(are) important for the function . Here we present for the first time the results of detailed analyses on the domain-function relationship of WASP . First, the C-terminal verprolin-cofilin-acidic domain was shown to be essential for the regulation of actin cytoskeleton . In addition, we found that the clustering of WASP itself is distinct from actin clustering . The partial protein containing the region from the N-terminal pleckstrin homology domain to the basic residue-rich region also clustered especially around the nucleus as wild type WASP without inducing actin clustering . Finally, we obtained the quite unexpected result that a WASP mutant deficient in binding to Cdc42 still induced actin cluster formation, indicating that direct interaction between Cdc42 and WASP is not required for the regulation of actin cytoskeleton . This result may explain why no Wiskott-Aldrich syndrome patients have been identified with a missense mutation in the Cdc42-binding site. J Biol Chem, 1999 Sep 17, 274(38), 27010 - 7 A mutant form of human protein farnesyltransferase exhibits increased resistance to farnesyltransferase inhibitors; Del Villar K et al.; Protein farnesyltransferase (FTase) is a key enzyme responsible for the lipid modification of a large and important number of proteins including Ras . Recent demonstrations that inhibitors of this enzyme block the growth of a variety of human tumors point to the importance of this enzyme in human tumor formation . In this paper, we report that a mutant form of human FTase, Y361L, exhibits increased resistance to farnesyltransferase inhibitors, particularly a tricyclic compound, SCH56582, which is a competitive inhibitor of FTase with respect to the CAAX (where C is cysteine, A is an aliphatic amino acid, and X is the C-terminal residue that is preferentially serine, cysteine, methionine, glutamine or alanine) substrates . The Y361L mutant maintains FTase activity toward substrates ending with CIIS . However, the mutant also exhibits an increased affinity for peptides terminating with CIIL, a motif that is recognized by geranylgeranyltransferase I (GGTase I) . The Y361L mutant also demonstrates activity with Ha-Ras and Cdc42Hs proteins, substrates of FTase and GGTase I, respectively . In addition, the Y361L mutant shows a marked sensitivity to a zinc chelator HPH-5 suggesting that the mutant has altered zinc coordination . These results demonstrate that a single amino acid change at a residue at the active site can lead to the generation of a mutant resistant to FTase inhibitors . Such a mutant may be valuable for the study of the effects of FTase inhibitors on tumor cells. J Biol Chem, 1999 Sep 17, 274(38), 26850 - 9 Requirement of an E1A-sensitive coactivator for long-range transactivation by the beta-globin locus control region; Forsberg EC et al.; Four erythroid-specific DNase I-hypersensitive sites at the 5'-end of the beta-globin locus confer high-level transcription to the beta-globin genes . To identify coactivators that mediate long-range transactivation by this locus control region (LCR), we assessed the influence of E1A, an inhibitor of the CBP/p300 histone acetylase, on LCR function . E1A strongly inhibited transactivation of Agamma- and beta-globin promoters by the HS2, HS2-HS3, and HS1-HS4 subregions of the LCR in human K562 and mouse erythroleukemia cells . Short- and long-range transactivation mediated by the LCR were equally sensitive to E1A . The E1A sensitivity was apparent in transient and stable transfection assays, and E1A inhibited expression of the endogenous gamma-globin genes . Only sites for NF-E2 within HS2 were required for E1A sensitivity in K562 cells, and E1A abolished transactivation mediated by the activation domain of NF-E2 . E1A mutants defective in CBP/p300 binding only weakly inhibited HS2-mediated transactivation, whereas a mutant defective in retinoblastoma protein binding strongly inhibited transactivation . Expression of CBP/p300 potentiated HS2-mediated transactivation . Moreover, expression of GAL4-CBP strongly increased transactivation of a reporter containing HS2 with a GAL4 site substituted for the NF-E2 sites . Thus, we propose that a CBP/p300-containing coactivator complex is the E1A-sensitive factor important for LCR function. J Biol Chem, 1999 Sep 17, 274(38), 26705 - 12 Protein phosphatase 2C inactivates F-actin binding of human platelet moesin; Hishiya A et al.; During activation of platelets by thrombin phosphorylation of Thr(558) in the C-terminal domain of the membrane-F-actin linking protein moesin increases transiently, and this correlates with protrusion of filopodial structures . Calyculin A enhances phosphorylation of moesin by inhibition of phosphatases . To measure this moesin-specific activity, a nonradioactive enzyme-linked immunosorbent assay method was developed with the synthetic peptide Cys-Lys(555)-Tyr-Lys-Thr(P)-Leu-Arg(560) coupled to bovine serum albumin as the substrate and moesin phosphorylation state-specific polyclonal antibodies for the detection and quantitation of dephosphorylation . Calyculin A-sensitive and -insensitive protein-threonine phosphatase activities were detected in platelet lysates and separated by DEAE-cellulose chromatography . The calyculin A-sensitive enzyme was identified as a type 1 protein phosphatase . The calyculin A-insensitive enzyme activity was purified to homogeneity by phenyl- Sepharose, protamine-, and phosphonic acid peptide-agarose chromatography and characterized biochemically and immunologically as a 53-kDa protein(s) and a type 2C protein phosphatase (PP2C) . Phosphorylation of Thr(558) is necessary for F-actin binding of moesin in vitro . The purified enzyme, as well as bacterially made PP2Calpha and PP2Cbeta, efficiently dephosphorylate(s) highly purified platelet phospho-moesin . This reverses the activating effect of phosphorylation, and moesin no longer co-sediments with actin filaments . In vivo, regulation of these phosphatase activities are likely to influence dynamic interactions between the actin cytoskeleton and membrane constituents linked to moesin. Plant Mol Biol, 1999 Jul, 40(4), 729 - 36 Salt-specific regulation of the cytosolic O-acetylserine(thiol)lyase gene from Arabidopsis thaliana is dependent on abscisic acid; Barroso C et al.; The expression of Atcys-3A gene coding for cytosolic O-acetylserine(thiol)lyase, a key enzyme in cysteine biosynthesis, from Arabidopsis thaliana is significantly induced by exposure to salt and heavy-metal stresses . Addition of NaCl to mature plants induced a rapid accumulation of the mRNA throughout the leaf lamina and roots, and later on in stems, being mainly restricted to vascular tissues . The salt-specific regulation of Atcys-3A was also mediated by abscisic acid (ABA) since: (1) exogenous addition of ABA to the culture medium mimicked the salt-induced plant response by raising the level of Atcys-3A transcript, and (2) Arabidopsis mutants aba-1 and abi2-1 were not able to respond to NaCl . Our results suggest that a high rate of cysteine biosynthesis is required in Arabidopsis under salt stress necessary for a plant protection or adaptation mechanism . This hypothesis was supported by the observation that intracellular levels of cysteine and glutathione increased up to 3-fold after salt treatment. Hum Genet, 1999 Jul-Aug, 105(1-2), 98 - 103 The human CDC42 gene: genomic organization, evidence for the existence of a putative pseudogene and exclusion as a SJS1 candidate gene; Nicole S et al.; Schwartz-Jampel syndrome (SJS) is an autosomal recessive human disorder characterized by myotonia and osteoarticular deformities . Three types are distinguished based on age at onset: types 1A, 1B and 2 . We have previously localized the SJS1 gene, responsible for types 1A and 1B, on human chromosome 1p35-p36.1 in a region frequently rearranged in human tumours . The CDC42 gene, for which divergent localizations have previously been described (chromosomes 4, 7 and 20), has been mapped within the SJS1 critical interval by radiation hybrid and yeast/P1 artificial-chromosome-based physical map analyses . The CDC42 gene product is a small GTPase protein of the Rho family mediating a variety of signaling pathways including cytoskeletal rearrangements, cell-cycle progression and transformation . To search for mutations in SJS1 patients, we have determined the organization of the human CDC42 gene on chromosome 1p and found that it encodes for the placental and brain isoforms generated by alternative splicing . No mutations have been found in SJS1 patients, excluding CDC42 as the SJS1 gene . Interestingly, we have demonstrated that a CDC42-like transcript gene located on chromosome 4 does not contain introns and is similar to the placental isoform, suggesting that it is a processed pseudogene . The determination of the CDC42 gene structure described in this report should facilitate future studies of the potential role of CDC42 in human disorders. J Neurosci, 1999 Sep 15, 19(18), 7770 - 80 Neuronal interleukin-16 (NIL-16): a dual function PDZ domain protein; Kurschner C et al.; Interleukin (IL)-16 is a proinflammatory cytokine that has attracted widespread attention because of its ability to block HIV replication . We describe the identification and characterization of a large neuronal IL-16 precursor, NIL-16 . The N-terminal half of NIL-16 constitutes a novel PDZ domain protein sequence, whereas the C terminus is identical with splenocyte-derived mouse pro-IL-16 . IL-16 has been characterized only in the immune system, and the identification of NIL-16 marks a previously unsuspected connection between the immune and the nervous systems . NIL-16 is a cytosolic protein that is detected only in neurons of the cerebellum and the hippocampus . The N-terminal portion of NIL-16 interacts selectively with a variety of neuronal ion channels, which is similar to the function of many other PDZ domain proteins that serve as intracellular scaffolding proteins . Among the NIL-16-interacting proteins is the class C alpha1 subunit of a mouse brain calcium channel (mbC alpha1) . The C terminus of NIL-16 can be processed by caspase-3, resulting in the release of secreted IL-16 . Furthermore, in cultured cerebellar granule neurons undergoing apoptosis, NIL-16 proteolysis parallels caspase-3 activation . Cerebellar granule neurons express the IL-16 receptor CD4 . Exposure of these cells to IL-16 induces expression of the immediate-early gene, c-fos, via a signaling pathway that involves tyrosine phosphorylation . This suggests that IL-16 provides an autocrine function in the brain . Therefore, we hypothesize that NIL-16 is a dual function protein in the nervous system that serves as a secreted signaling molecule as well as a scaffolding protein. Mol Genet Metab, 1999 Sep, 68(1), 32 - 42 Human liver-specific very-long-chain acyl-coenzyme A synthetase: cDNA cloning and characterization of a second enzymatically active protein; Steinberg SJ et al.; Activation of fatty acids, catalyzed by acyl-coenzyme A (acyl-CoA) synthetases, is required for their subsequent metabolism . Peroxisomes and microsomes contain very-long-chain acyl-CoA synthetases (VLCSs) capable of activating fatty acids with a chain length of 22 or more carbons . Decreased peroxisomal VLCS activity is, in part, responsible for the biochemical pathology in X-linked adrenoleukodystrophy (X-ALD), illustrating the importance of VLCSs in cellular fatty acid homeostasis . We previously cloned two human genes encoding proteins homologous to rat peroxisomal VLCS; one (hVLCS) is the human ortholog to the rat VLCS gene and another (hVLCS-H1) encodes a related heart-specific protein . Here, we report the cloning of a third gene (hVLCS-H2) and characterization of its protein product . The hVLCS-H2 gene is located on human chromosome 19 and encodes a 690-amino-acid protein . The amino acid sequence of hVLCS-H2 is 44-45% identical and 67-69% similar to those of both hVLCS and hVLCS-H1 . COS-1 cells transiently overexpressing hVLCS-H2 activated the very-long-chain fatty acid lignocerate (C24:0) at a rate >1.5-fold higher than that of nontransfected cells (P < 0.002) . The hVLCS-H2-dependent activation of long- and branched-chain fatty acids following transient transfection was less striking . However, hVLCS-H2-dependent acyl-CoA synthetase activity with long- and very-long-chain fatty acid substrates was detected in COS-1 cells stably expressing hVLCS-H2 . For all substrates tested (C18:0, C20:0, C24:0, C26:0), the hVLCS-H2 catalyzed activity was significantly increased (P < 0.01 to P < 0.0001) . By both Northern analysis and reverse transcription polymerase chain reaction, hVLCS-H2 is expressed primarily in liver . Indirect immunofluorescence of COS-1 cells or human hepatoma-derived HepG2 cells expressing epitope-tagged hVLCS-H2 revealed that the protein was associated with the endoplasmic reticulum but not with peroxisomes . Thus, the primary role of hVLCS-H2 is likely to be in fatty acid elongation or complex lipid synthesis rather than in degradation . Cytokine, 1999 Sep, 11(9), 713 - 21 Interleukin 10 mitigates the development of the zymosan-induced multiple organ dysfunction syndrome in mice; Jansen MJ et al.; We investigated the effect of interleukin 10 on the development of zymosan-induced multiple organ dysfunction syndrome (MODS) and on plasma concentrations and production capacity of tumour necrosis factor (TNF)-alpha by peritoneal cells . Groups of C57BL/6 mice received a single intraperitoneal injection with zymosan, a cell wall component of Saccharomyces cerevisiae, at day 0 . Daily doses of human recombinant interleukin 10 (IL-10: 10 or 50 microg/kg) were given intraperitoneally either starting directly before administration of zymosan (day 0), or 5 or 8 days after administration of zymosan . The animals were monitored for survival, condition, body weight and temperature . On day 12 all surviving animals were killed to obtain plasma, organs and peritoneal cells . Plasma concentrations of TNF-alpha and lipopolysaccharide-stimulated production of TNF-alpha by peritoneal cells were measured; organ weights were registered as an indicator for organ damage . IL-10 improves survival and clinical condition and also reduces organ damage, but only at the highest dose used and only when started simultaneously with the administration of zymosan . Circulating TNF-alpha concentrations 12 days after zymosan are not affected by any of the IL-10 schedules used . However, lipopolysaccharide-stimulated production of TNF-alpha by peritoneal cells is increased, in a dose- and time-dependent fashion . The anti-inflammatory cytokine IL-10 is able to attenuate the development of MODS in this model, but only when given simultaneously with zymosan, and in high dosages . J Nat Prod, 1999 Aug, 62(8), 1173 - 4 A new labdane diterpenoid from Renealmia alpinia collected in the Suriname rainforest; Yang SW et al.; Continuation of a previous study on Renealmia alpinia resulted in the isolation of the new labdane diterpenoid 3, together with two known diterpenoids . The structure of the new diterpenoid was determined by a combination of NMR techniques and HRFABMS. Mol Endocrinol, 1999 Sep, 13(9), 1550 - 7 Coactivators for the orphan nuclear receptor RORalpha; Atkins GB et al.; A mutation in the nuclear orphan receptor RORalpha results in a severe impairment of cerebellar development by unknown mechanisms . We have shown previously that RORalpha contains a strong constitutive activation domain in its C terminus . We therefore searched for mammalian RORalpha coactivators using the minimal activation domain as bait in a two-hybrid screen . Several known and putative coactivators were isolated, including glucocorticoid receptor-interacting protein-1 (GRIP-1) and peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205) . These interactions were confirmed in vitro and require the intact activation domain of RORalpha although different requirements for interaction with GRIP-1 and PBP were detected . Even in the absence of exogenous ligand, RORalpha interacts with a complex or complexes of endogenous proteins, similar to those that bind to ligand-occupied thyroid hormone and vitamin D receptors . Both PBP and GRIP-1 were shown to be present in these complexes . Thus we have identified several potential RORalpha coactivators that, in contrast to the interactions with hormone receptors, interact with RORalpha in yeast, in bacterial extracts, and in mammalian cells in vivo and in vitro in the absence of exogenous ligand . GRIP-1 functioned as a coactivator for the RORalpha both in yeast and in mammalian cells . Thus, GRIP-1 is the first proven coactivator for RORalpha. Mol Microbiol, 1999 Sep, 33(5), 994 - 1003 Ambient pH signal transduction in Aspergillus: completion of gene characterization; Negrete-Urtasun S et al.; Completing the molecular analysis of the six pal genes of the ambient pH signal transduction pathway in Aspergillus nidulans, we report the characterization of palC and palH . The derived translation product of palH contains 760 amino acids with prediction of seven transmembrane domains in its N-terminal moiety . Remarkably, a palH frameshift mutant lacking just over half the PalH protein, including almost all of the long hydrophilic region C-terminal to the transmembrane domains, retains some PalH function . The palC-derived translation product contains 507 amino acids, and the null phenotype of a frameshift mutation indicates that at least one of the C-terminal 142 residues is essential for function . Uniquely among the A . nidulans pH-signalling pal genes, palC appears to have no Saccharomyces cerevisiae homologue, although it does have a Neurospora crassa expressed sequence tag homologue . In agreement with findings for the palA, palB and palI genes of this signalling pathway, levels of the palC and palH mRNAs do not appear to be pH regulated. Mol Biol Cell, 1999 Sep, 10(9), 2829 - 45 A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium; Lee S et al.; We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal . rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture . In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired . We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells . This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis . When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage . Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect . Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA . We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells . A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients . As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation. Appl Environ Microbiol, 1999 Sep, 65(9), 3873 - 9 Identification of an NADH-cytochrome b(5) reductase gene from an arachidonic acid-producing fungus, Mortierella alpina 1S-4, by sequencing of the encoding cDNA and heterologous expression in a fungus, Aspergillus oryzae; Sakuradani E et al.; Based on the sequence information for bovine and yeast NADH-cytochrome b(5) reductases (CbRs), a DNA fragment was cloned from Mortierella alpina 1S-4 after PCR amplification . This fragment was used as a probe to isolate a cDNA clone with an open reading frame encoding 298 amino acid residues which show marked sequence similarity to CbRs from other sources, such as yeast (Saccharomyces cerevisiae), bovine, human, and rat CbRs . These results suggested that this cDNA is a CbR gene . The results of a structural comparison of the flavin-binding beta-barrel domains of CbRs from various species and that of the M . alpina enzyme suggested that the overall barrel-folding patterns are similar to each other and that a specific arrangement of three highly conserved amino acid residues (i.e., arginine, tyrosine, and serine) plays a role in binding with the flavin (another prosthetic group) through hydrogen bonds . The corresponding genomic gene, which was also cloned from M . alpina 1S-4 by means of a hybridization method with the above probe, had four introns of different sizes . These introns had GT at the 5' end and AG at the 3' end, according to a general GT-AG rule . The expression of the full-length cDNA in a filamentous fungus, Aspergillus oryzae, resulted in an increase (4.7 times) in ferricyanide reduction activity involving the use of NADH as an electron donor in the microsomes . The M . alpina CbR was purified by solubilization of microsomes with cholic acid sodium salt, followed by DEAE-Sephacel, Mono-Q HR 5/5, and AMP-Sepharose 4B affinity column chromatographies; there was a 645-fold increase in the NADH-ferricyanide reductase specific activity . The purified CbR preferred NADH over NADPH as an electron donor . This is the first report of an analysis of this enzyme in filamentous fungi. Neuroscience, 1999, 93(3), 1179 - 87 Expression profile of the copper homeostasis gene, rAtox1, in the rat brain; Naeve GS et al.; In humans the regulation of cellular copper homeostasis is essential for proper organ development and function . A novel cytosolic protein, named Atox 1, was recently identified in yeast that functions in shuttling intracellular mononuclear copper {Cu(I)} to copper-requiring proteins . Atox 1 and its human homolog, hAtox1, are members of an emerging family of proteins termed copper chaperones that are involved in the maintenance of copper homeostasis . Northern blot analysis demonstrates that Atox 1 is widely expressed at varying levels in a variety of rat tissues including brain . Using in situ hybridization histochemistry, we characterized the expression profile for the rat homolog of Atox1 (rAtox1) in the normal adult rat brain . There is widespread expression within the brain that appears to be primarily neuronal . The highest levels of Atox1 message consists of distinct neuronal subtypes that are also characterized by their high levels of metals like copper, iron, and zinc, which include the pyramidal neurons of the cerebral cortex and hippocampus in addition to the neurons of the locus coeruleus . The high levels of a metal chaperone like Atox1 in subsets of neurons that also sequester metals suggests that Atox1 may be important in maintaining the functionality of metal requiring enzymes . A detailed analysis of the restricted expression profile for a novel copper chaperone, rAtox1, is described in the adult rat CNS . Further analysis shows that Atoxl expression is associated with neuronal populations that sequester copper. Eur J Cell Biol, 1999 Jul, 78(7), 453 - 62 A homologue of Sar1p localises to a novel trafficking pathway in malaria-infected erythrocytes; Albano FR et al.; We have identified a homologue of the GTP-binding protein, Sar1p, in Plasmodium falciparum . Sar1p is a small GTPase that is thought to play a crucial role in trafficking of proteins between the endoplasmic reticulum and the Golgi . The P.falciparum SAR1 gene is located on chromosome 4 and comprises two exons separated by a 508 bp intron . The deduced amino acid sequence of PfSar1p (GenBank accession number AF104306) shows 71% similarity (58% identity) to Sar1p from Saccharomyces cerevisiae . Expression of PfSar1p in erythrocytic stages of P . falciparum was confirmed by sequencing of a tryptic peptide derived from a polypeptide excised from an SDS-polyacrylamide gel . A recombinant protein corresponding to approximately 70% of the PfSar1p sequence was used to raise antibodies . The affinity-purified antiserum recognised a protein with an apparent molecular weight of 23 K in Western blots of malaria-infected erythrocytes but not in uninfected erythrocytes . PfSar1p was shown to be largely insoluble in non-ionic detergent and a low ionic strength buffer . Confocal immunofluorescence microscopy of malaria-infected erythrocytes was used to show that PfSar1p is located near the periphery of the parasite in discrete compartments, which appear to be distinct from the parasite endoplasmic reticulum . In addition, PfSar1p appears to be exported to structures outside the parasite in the erythrocyte cytoplasm . The export of PfSar1p to the erythrocyte cytosol is inhibited by treatment with brefeldin A . This provides the first evidence that the malaria parasite is capable of elaborating components of the classical vesicle-mediated trafficking machinery outside the boundaries of its own plasma membrane. FEBS Lett, 1999 Sep 3, 457(3), 452 - 4 Spatial gradients of cellular phospho-proteins; Brown GC et al.; If a protein is rapidly phosphorylated and dephosphorylated at separate cellular locations and protein diffusion is slow, then a spatial gradient of the phosphorylated form of the protein may develop within the cell . We have estimated the potential size of such gradients using measured values of protein diffusion coefficients and protein kinase and phosphatase activities . We analysed two different cellular geometries: (1) where the kinases is located on the plasma membrane of a spherical cell and the phospatase is distributed homogenously in the cytoplasm and (2) where the kinase is located on one planar membrane and the phosphatase on a second parallel planar membrane . The estimated gradients of phospho-proteins were potentially very large, which has important implications for cellular signalling. Nucleic Acids Res, 1999 Sep 15, 27(18), 3720 - 7 Identification of a mammalian RNA polymerase I holoenzyme containing components of the DNA repair/replication system; Hannan RD et al.; Traditional models for transcription initiation by RNA polymerase I include a stepwise assembly of basic transcription factors/regulatory proteins on the core promoter to form a preinitiation complex . In contrast, we have identified a preassembled RNA polymerase I (RPI) complex that contains all the factors necessary and sufficient to initiate transcription from the rDNA promoter in vitro . The purified RPI holoenzyme contains the RPI homolog of TFIID, SL-1 and the rDNA transcription terminator factor (TTF-1), but lacks UBF, an activator of rDNA transcription . Certain components of the DNA repair/replication system, including Ku70/80, DNA topoisomerase I and PCNA, are also associated with the RPI complex . We have found that the holo-enzyme supported specific transcription and that specific transcription was stimulated by the RPI transcription activator UBF . These results support the hypothesis that a fraction of the RPI exists as a preassembled, transcriptionally competent complex that is readily recruited to the rDNA promoter, i.e . as a holoenzyme, and provide important new insights into the mechanisms governing initiation by RPI. Nucleic Acids Res, 1999 Sep 15, 27(18), 3676 - 84 Crosslinking of proteins to DNA in human nuclei using a 60 femtosecond 266 nm laser; Lejnine S et al.; We developed appropriate conditions to use a laser with 60 femtosecond pulses, a frequency of 1 KHz and a wavelength of 266 nm to efficiently crosslink proteins to DNA in human nuclei for the purpose of using immunoprecipitation to study the binding of specific proteins to specific sequences of DNA under native conditions . Irradiation of nuclei for 30 min with 1-3 GW/cm(2)pulses crosslinked 10-12% of total protein to DNA . The efficiency of crosslinking was dose and protein specific . Histones H1 and H3 were crosslinked by 15 min of irradiation with 20-25% efficiency, at least 10 times more strongly than the other histones, consistent with experiments using conventional UV light . Irradiation for 15 min did not damage proteins, as assayed by SDS-PAGE of Ku-70 and histones . Although the same level of irradiation did not cause double-strand breaks, it did make the DNA partially insensitive to Eco RI restriction enzyme, probably through formation of thymidine dimers . Immuno-analysis of crosslinked nucleoprotein showed that Ku crosslinking to nuclear DNA is detectable only in the presence of breaks in the DNA, and that nucleosomes are bound to a significant fraction of the telomeric repeat (TTAGGG) (n). Genetics, 1999 Sep, 153(1), 95 - 105 The TamA protein fused to a DNA-binding domain can recruit AreA, the major nitrogen regulatory protein, to activate gene expression in Aspergillus nidulans; Small AJ et al.; The areA gene of Aspergillus nidulans encodes a GATA zinc finger transcription factor that activates the expression of a large number of genes subject to nitrogen metabolite repression . The amount and activity of the AreA protein under different nitrogen conditions is modulated by transcriptional, post-transcriptional, and post-translational controls . One of these controls of AreA activity has been proposed to involve the NmrA protein interacting with the DNA-binding domain and the extreme C terminus of AreA to inhibit DNA binding under nitrogen sufficient conditions . In contrast, mutational evidence suggests that the tamA gene has a positive role together with areA in regulating the expression of genes subject to nitrogen metabolite repression . This gene was identified by the selection of mutants resistant to toxic nitrogen source analogues, and a number of nitrogen metabolic activities have been shown to be reduced in these mutants . To investigate the role of this gene we have used constructs encoding the TamA protein fused to the DNA-binding domain of either the FacB or the AmdR regulatory proteins . These hybrid proteins have been shown to activate expression of the genes of acetate or GABA utilization, respectively, as well as the amdS gene . Strong activation was shown to require the AreA protein but was not dependent on AreA binding to DNA . The homologous areA gene of A . oryzae and nit-2 gene of Neurospora crassa can substitute for A . nidulans areA in this interaction . We have shown that the same C-terminal region of AreA and NIT-2 that is involved in the interaction with NmrA is required for the TamA-AreA interaction . However, it is unlikely that TamA requires the same residues as NmrA within the GATA DNA-binding domain of AreA. Hum Mol Genet, 1999, 8(10), 1893 - 900 Nonsense-mediated mRNA decay in health and disease; Frischmeyer PA et al.; All eukaryotes possess the ability to detect and degrade transcripts harboring premature signals for the termination of translation . Despite the ubiquitous nature of nonsense-mediated mRNA decay (NMD) and its demonstrated role in the modulation of phenotypes resulting from selected nonsense alleles, very little is known regarding its basic mechanism or the selective pressure for complete evolutionary conservation of this function . This review will present the current models of NMD that have been generated during the study of model organisms and mammalian cells . The physiological burden of nonsense transcripts and the emerging view that NMD plays a broad and critical role in the regulation of gene expression will also be discussed . Such issues are relevant to the proposal that pharmacological manipulation of NMD will find therapeutic application. Exp Cell Res, 1999 Sep 15, 251(2), 452 - 64 Effects of anti-PM-Scl 100 (Rrp6p exonuclease) antibodies on prenucleolar body dynamics at the end of mitosis; Fomproix N et al.; Prenucleolar bodies (PNBs) are transitory structures which serve as building blocks for nucleoli at the transition mitosis/interphase . The assembly of PNBs and their pathway are not clearly understood . To better understand these events, the behavior of the PNB-containing PM-Scl 100 protein was compared with that of other PNB proteins . This nucleolar protein was chosen because its yeast homologue, Rrp6p exonuclease {1}, is known to participate in late events in 5.8 S rRNA (ribosomal RNA) processing . There was a heterogeneous distribution of nucleolar proteins in different classes of PNBs . The PM-Scl 100 colocalized predominantly with protein B23 . The PM-Scl-100-containing PNBs were translocated at later times to nucleoli as opposed to the fibrillarin-containing PNBs . Microinjections of antibodies directed against PM-Scl 100 during mitosis inhibited targeting of PM-Scl 100 to the nucleolus . However fibrillarin and protein B23 still participated in nucleolar assembly in early G1 . We conclude that there are different kinds of PNBs whose translocation to the nucleoli follow ordered kinetics . Interestingly, proteins involved in late steps of processing as PM-Scl 100 are translocated late, suggesting that they are not cotranscriptionally associated with the rRNA precursors . Biochemistry, 1999 Aug 31, 38(35), 11425 - 32 Redesign of cytochrome c peroxidase into a manganese peroxidase: role of tryptophans in peroxidase activity; Gengenbach A et al.; Trp191Phe and Trp51Phe mutations have been introduced into an engineered cytochrome c peroxidase (CcP) containing a Mn(II)-binding site reported previously (MnCcP; see Yeung, B . K.-S., et al . (1997) Chem . Biol . 5, 215-221) . The goal of the present study is to elucidate the role of tryptophans in peroxidase activity since CcP contains both Trp51 and Trp191 while manganese peroxidase (MnP) contains phenylalanine residues at the corresponding positions . The presence of Trp191 in CcP allows formation of a unique high-valent intermediate containing a ferryl oxo and tryptophan radical called compound I' . The absence of a tryptophan residue at this position in MnP is the main reason for the formation of an intermediate called compound I which contains a ferryl oxo and porphyrin pi-cation radical . In this study, we showed that introduction of the Trp191Phe mutation to MnCcP did not improve MnP activity (specific activity: MnCcP, 0.750 micromol min-1 mg-1; MnCcP(W191F), 0.560 micromol min-1 mg-1 . k(cat)/K(m): MnCcP, 0.0517 s-1 mM-1; MnCcP(W191F), 0.0568 s-1 mM-1) despite the fact that introduction of the same mutation to WTCcP caused the formation of a transient compound I (decay rate, 60 s-1) . However, introducing both the Trp191Phe and Trp51Phe mutations not only resulted in a longer lived compound I in WTCcP (decay rate, 18 s-1), but also significantly improved MnP activity in MnCcP (MnCcP(W51F, W191F): specific activity, 8.0 micromol min-1 mg-1; k(cat)/K(m), 0 . 599 s-1 mM-1) . The increase in activity can be attributed to the Trp51Phe mutation since MnCcP(W51F) showed significantly increased MnP activity relative to MnCcP (specific activity, 3.2 micromol min-1 mg-1; k(cat)/K(m), 0.325 s-1 mM-1) . As with MnP, the activity of MnCcP(W51F, W191F) was found to increase with decreasing pH . Our results demonstrate that, while the Trp191Phe and Trp51Phe mutations both play important roles in stabilizing compound I, only the Trp51Phe mutation contributes significantly to increasing the MnP activity because this mutation increases the reactivity of compound II, whose oxidation of Mn(II) is the rate-determining step in the reaction mechanism. Prostaglandins Leukot Essent Fatty Acids, 1999 May-Jun, 60(5-6), 323 - 8 Human very long-chain acyl-CoA synthetase and two human homologs: initial characterization and relationship to fatty acid transport protein; Watkins PA et al.; Several human genes with a high degree of homology to rat very long-chain acyl-CoA synthetase (rVLCS) and mouse fatty acid transport protein (mFATP) were identified . Full-length cDNA clones were obtained for three genes, and predicted amino acid sequences were generated . Initial characterization indicated that one gene was most likely hVLCS, the human ortholog of rVLCS . The other two (hVLCS-H1 and hVLCS-H2) were more closely related to rVLCS than to mFATP . Phylogenetic analysis of amino acid sequences confirmed that hVLCS-H1 and hVLCS-H2 were evolutionarily closer to VLCSs than FATPs . Alignment of predicted amino acid sequences of human, rat and mouse VLCSs and FATPs revealed the existence of two highly conserved motifs . While one motif is also present in long-chain acyl-CoA synthetases, the other serves to distinguish the VLCS/FATP family from the long-chain synthetase family . Elucidation of the biochemical functions of all VLCS/FATP family members should provide new insights into cellular fatty acid metabolism. Anticancer Res, 1999 May-Jun, 19(3A), 1821 - 6 Replication error in colorectal carcinoma: association with loss of heterozygosity at mismatch repair loci and clinicopathological variables; Johannsdottir JT et al.; Instability of microsatellite DNA or replication error (RER) is characteristic of tumours caused by mismatch repair (MMR) deficiency . Germline mutations in MMR genes are associated with Hereditary non-polyposis colorectal carcinoma (HNPCC) and somatic mutations in these genes are also found in a substantial fraction of colorectal cancers (CRC) . In this study we concurrently screened colorectal tumours for the RER phenotype and loss of heterozygosity (LOH) at MMR gene loci . The RER phenotype was evident in 47/197 (24%) tumours . RER was more commonly detected in young patients (< 50 years) and in tumours located in the proximal colon . RER was positively associated with LOH at the hMSH2/hMSH6 loci on chromosome 2p, where LOH was observed in 46% of the RER+ tumours . LOH at hMLH1 and hPMS1 loci was more frequent in the younger patients (< 50 years) . RER was not associated with clinicopathological parameters, such as Duke's stage and tumour differentiation (grade) . The RER phenotype was associated with better overall survival, but there was a trend towards significance when multivariate analysis was used . This indicates that loss of MMR genes generate a less aggressive phenotype, and raises the question about RER being a useful indicator of prognosis for CRC patients. Anticancer Res, 1999 May-Jun, 19(3A), 1729 - 35 Overexpression of cDNA encoding FANCC, SPHAR, MPG, SNM1 or HA 3611 does not render CHO cells more resistant to DNA crosslinking agents; Grombacher T et al.; DNA crosslinking agents (DCA) are commonly used cytostatic drugs, whose efficiency in tumor therapy is limited due to the appearance of drug resistant tumor cells . In an effort to modulate the resistance of cells to DCA, we transfected into Chinese hamster cells various cDNAs whose loss of function was previously shown to render cells more sensitive to crosslinking agents . We show that overexpression of FANCC, SPHAR, MPG, SNM1 or HA 3611 (a human homologue of the yeast crosslink DNA repair gene SNM1) does not alter the level of resistance of CHO cells to clinically relevant DCA, such as mafosfamide, melphalan and mitomycin C . Therefore, DCA resistance frequently observed in tumor cells is not likely to be the result of up-regulation of either one of these genes, but a more complex phenomenon . Also, the data suggest that protection of normal cells from toxic side effects of DCA cannot easily be accomplished by transfer of either one of these genes. EMBO J, 1999 Sep 1, 18(17), 4816 - 22 Different import pathways through the mitochondrial intermembrane space for inner membrane proteins; Leuenberger D et al.; Earlier work on the protein import system of yeast mitochondria has identified two soluble 70 kDa protein complexes in the intermembrane space . One complex contains the essential proteins Tim9p and Tim10p and mediates transport of cytosolically-made metabolite carrier proteins from the outer to the inner membrane . The other complex contains the non-essential proteins Tim8p and Tim13p as well as loosely associated Tim9p; its function was unclear, but it interacted structurally or functionally with the Tim9p-Tim10p complex . We now show that the two 70 kDa complexes each mediate the import of a different subset of integral inner membrane proteins and that they can transfer these proteins to one of three different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as yet uncharacterized insertion site . Yeast mitochondria thus use multiple pathways for escorting hydrophobic inner membrane proteins across the aqueous intermembrane space. EMBO J, 1999 Sep 1, 18(17), 4669 - 78 The Drosophila STE20-like kinase misshapen is required downstream of the Frizzled receptor in planar polarity signaling; Paricio N et al.; The Drosophila misshapen (msn) gene is a member of the STE20 kinase family . We show that msn acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings . Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation . Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling . This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes . We also show that Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure . Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation . To address the nature of this redundancy, we provide evidence for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn. Curr Biol, 1999 Aug 26, 9(16), 903 - 6 Protein kinase A is required for chromosomal DNA replication; Costanzo V et al.; Passage through mitosis resets cells for a new round of chromosomal DNA replication {1} . In late mitosis, the pre-replication complex - which includes the origin recognition complex (ORC), Cdc6 and the minichromosome maintenance (MCM) proteins - binds chromatin as a pre-requisite for DNA replication . S-phase-promoting cyclin-dependent kinases (Cdks) and the kinase Dbf4-Cdc7 then act to initiate replication . Before the onset of replication Cdc6 dissociates from chromatin . S-phase and M-phase Cdks block the formation of a new pre-replication complex, preventing DNA over-replication during the S, G2 and M phases of the cell cycle {1} . The nuclear membrane also contributes to limit genome replication to once per cell cycle {2} . Thus, at the end of M phase, nuclear membrane breakdown and the collapse of Cdk activity reset cells for a new round of chromosomal replication . We showed previously that protein kinase A (PKA) activity oscillates during the cell cycle in Xenopus egg extracts, peaking in late mitosis . The oscillations are induced by the M-phase-promoting Cdk {3} {4} . Here, we found that PKA oscillation was required for the following phase of DNA replication . PKA activity was needed from mitosis exit to the formation of the nuclear envelope . PKA was not required for the assembly of ORC2, Cdc6 and MCM3 onto chromatin . Inhibition of PKA activity, however, blocked the release of Cdc6 from chromatin and subsequent DNA replication . These data suggest that PKA activation in late M phase is required for the following S phase. Curr Biol, 1999 Aug 26, 9(16), R606 - 9 Transcription: Common cofactors and cooperative recruitment; Wu WH et al.; Mammalian counterparts of the yeast SRB/MED transcriptional 'mediator' complex have recently been identified . These complexes define a common cofactor requirement for diverse transcriptional activators and underscore the conserved nature of the transcriptional machinery among eukaryotic organisms. Curr Biol, 1999 Jul 29-Aug 12, 9(15), R562 - 4 Chromosome segregation: dual control ensures fidelity; Taylor SS; A mitotic checkpoint arrests cell cycle progression in response to spindle damage . It now appears that this checkpoint has two separate arms, one that prevents anaphase and a second that prevents cytokinesis and DNA re-replication. Curr Biol, 1999 Jul 29-Aug 12, 9(15), R575 - 6 The nucleolus: nucleolar space for RENT; Cockell MM et al.; Recent studies indicate that the nucleolus is not just a site of ribosome biogenesis . Intriguing links have been found between nucleolar components and the machinery that regulates the cell cycle. Eur J Biochem, 1999 Aug, 263(3), 736 - 45 Alternative promoters direct tissue-specific expression of the mouse protein phosphatase 2Cbeta gene; Ohnishi M et al.; Type 2C protein phosphatases (PP2Cs), a class of ubiquitous and evolutionally conserved serine/threonine protein phosphatases, are encoded in at least four distinct genes and implicated in the regulation of various cellular functions . Of these four PP2C genes, the expression of the PP2Cbeta gene has been reported to be tissue-specific and development-dependent . To understand more precisely the regulatory mechanism of this expression, we have isolated and characterized overlapping mouse genomic lambda clones . A comparison of genomic sequences with PP2Cbeta cDNA sequences provided information on the structure and localization of intron/exon boundaries and indicated that PP2Cbeta isoforms with different 5' termini were generated by alternative splicing of its pre-mRNA . The 5'-flanking region of exon 1 had features characteristic of a housekeeping gene: it was GC-rich, lacked TATA boxes and CAAT boxes in the standard positions, and contained potential binding sites for the transcription factor SP1 . In the 5'-flanking region of exon 2, several consensus sequences were found, such as a TATA-like sequence and negative regulatory element box-1, -2 and -3 . Subsequent analysis by transient transfection assay with a reporter gene showed that these regions act as distinct promoters . Analysis of PP2Cbeta transcripts by reverse transcriptase-PCR showed that exon-1 transcripts were expressed ubiquitously in all of the tissues examined, whereas exon-2 transcripts were predominantly expressed in the testis, intestine and liver . These results suggest that the alternative usage of two promoters within the PP2Cbeta gene regulates tissue-specific expression of PP2Cbeta mRNA. Proc Natl Acad Sci U S A, 1999 Aug 31, 96(18), 10438 - 43 Dynamic visualization of nervous system in live Drosophila; Sun B et al.; We have constructed transgenic Drosophila melanogaster lines that express green fluorescent protein (GFP) exclusively in the nervous system . Expression is controlled with transcriptional regulatory elements present in the 5' flanking DNA of the Drosophila Na(+), K(+)-ATPase beta-subunit gene Nervana2 (Nrv2) . This regulatory DNA is fused to the yeast transcriptional activator GAL4, which binds specifically to a sequence motif termed the UAS (upstream activating sequence) . Drosophila lines carrying Nrv2-GAL4 transgenes have been genetically recombined with UAS-GFP (S65T) transgenes (Nrv2-GAL4+UAS-GFP) inserted on the same chromosomes . We observe strong nervous system-specific fluorescence in embryos, larvae, pupae, and adults . The GFP fluorescence is sufficiently bright to allow dynamic imaging of the nervous system at all of these developmental stages directly through the cuticle of live Drosophila . These lines provide an unprecedented view of the nervous system in living animals and will be valuable tools for investigating a number of developmental, physiological, and genetic neurobiological problems. Oncogene, 1999 Aug 12, 18(32), 4616 - 25 Suppression of the poly(ADP-ribose) polymerase activity by DNA-dependent protein kinase in vitro; Ariumi Y et al.; It has been suggested that DNA-dependent protein kinase (DNA-PK) is a central component of DNA double-strand-break repair . The mechanism of DNA-PK action, however, has not been fully understood . Poly(ADP-ribose) polymerase (PARP) is another nuclear enzyme which has high affinity to DNA ends . In this study, we analysed the interaction between these two enzymes . First, DNA-PK was found to suppress the PARP activity and alters the pattern of poly(ADP-ribosyl)ation . Although DNA-PK phosphorylates PARP in a DNA-dependent manner, this modification is unlikely to be responsible for the suppression of PARP activity, since this suppression occurs even in the absence of ATP . Conversely, PARP was found to ADP-ribosylate DNA-PK in vitro . However, the auto-phosphorylation activity of DNA-PK was not influenced by this modification . In a competitive electrophoretic mobility shift assay, Ku 70/80 complex, the DNA binding component of DNA-PK, was found to have higher affinity to a short fragment of DNA than does PARP . Furthermore, co-immunoprecipitation analysis suggested direct or close association between Ku and PARP . Thus, DNA-PK suppresses PARP activity, probably through direct binding and/or sequestration of DNA-ends which serve as an important stimulator for both enzymes. Oncogene, 1999 Aug 12, 18(32), 4598 - 605 Transcriptional regulation by targeted recruitment of cyclin-dependent CDK9 kinase in vivo; Majello B et al.; The CDK9 kinase in association with Cyclin T is a component of the transcription positive-acting complex pTEFb which facilitates the transition from abortive to productive transcription elongation by phosphorylating the carboxyl-terminal domain of RNA polymerase II . The Cyclin T1/CDK9 complex is implicated in Tat transactivation, and it has been suggested that Tat functions by recruiting this complex to RNAPII through cooperative binding to RNA . Here, we demonstrate that targeted recruitment of Cyclin T1/CDK9 kinase complex to specific promoters, through fusion to a DNA-binding domain of either Cyclin T1 or CDK9 kinase, stimulates transcription in vivo . Transcriptional enhancement was dependent on active CDK9, as a catalytically inactive form had no transcriptional effect . We determined that, unlike conventional activators, DNA-bound CDK9 does not activate enhancerless TATA-promoters unless TBP is overexpressed, suggesting that CDK9 acts in vivo at a step subsequent to TFIID recruitment DNA-bound . Finally, we determined that CDK9-mediated transcriptional activation is mediated by preferentially stimulating productive transcription elongation. Genes Dev, 1999 Aug 15, 13(16), 2159 - 76 A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication; Pasero P et al.; Using a reconstituted DNA replication assay from yeast, we demonstrate that two kinase complexes are essential for the promotion of replication in vitro . An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is required in trans to stimulate initiation in G(1)-phase nuclei, whereas the Dbf4/Cdc7 kinase complex must be provided by the template nuclei themselves . The regulatory subunit of Cdc7p, Dbf4p, accumulates during late G(1) phase, becomes chromatin associated prior to Clb/Cdc28 activation, and assumes a punctate pattern of localization that is similar to, and dependent on, the origin recognition complex (ORC) . The association of Dbf4p with a detergent-insoluble chromatin fraction in G(1)-phase nuclei requires ORC but not Cdc6p or Clb/Cdc28 kinase activity, and correlates with competence for initiation . We propose a model in which Dbf4p targets Cdc7p to the prereplication complex prior to the G(1)/S transition, by a pathway parallel to, but independent of, the Cdc6p-dependent recruitment of MCMs. Genes Dev, 1999 Aug 15, 13(16), 2118 - 33 Nuclear export of the small ribosomal subunit requires the ran-GTPase cycle and certain nucleoporins; Moy TI et al.; After their assembly in the nucleolus, ribosomal subunits are exported from the nucleus to the cytoplasm . After export, the 20S rRNA in the small ribosomal subunit is cleaved to yield 18S rRNA and the small 5' ITS1 fragment . The 5' ITS1 RNA is normally degraded by the cytoplasmic Xrn1 exonuclease, but in strains lacking XRN1, the 5' ITS1 fragment accumulates in the cytoplasm . Using the cytoplasmic localization of the 5' ITS1 fragment as an indicator for the export of the small ribosomal subunit, we have identified genes that are required for ribosome export . Ribosome export is dependent on the Ran-GTPase as mutations in Ran or its regulators caused 5' ITS1 to accumulate in the nucleoplasm . Mutations in the genes encoding the nucleoporin Nup82 and in the NES exporter Xpo1/Crm1 also caused the nucleoplasmic accumulation of 5' ITS1 . Mutants in a subset of nucleoporins and in the nuclear transport factors Srp1, Kap95, Pse1, Cse1, and Mtr10 accumulate the 5' ITS1 in the nucleolus and affect ribosome assembly . In contrast, we did not detect nuclear accumulation of 5' ITS1 in 28 yeast strains that have mutations in other genes affecting nuclear trafficking. Genes Dev, 1999 Aug 15, 13(16), 2059 - 71 ECSIT is an evolutionarily conserved intermediate in the Toll/IL-1 signal transduction pathway; Kopp E et al.; Activation of NF-kappaB as a consequence of signaling through the Toll and IL-1 receptors is a major element of innate immune responses . We report the identification and characterization of a novel intermediate in these signaling pathways that bridges TRAF6 to MEKK-1 . This adapter protein, which we have named ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), is specific for the Toll/IL-1 pathways and is a regulator of MEKK-1 processing . Expression of wild-type ECSIT accelerates processing of MEKK-1, whereas a dominant-negative fragment of ECSIT blocks MEKK-1 processing and activation of NF-kappaB . These results indicate an important role for ECSIT in signaling to NF-kappaB and suggest that processing of MEKK-1 is required for its function in the Toll/IL-1 pathway. J Biol Chem, 1999 Sep 3, 274(36), 25927 - 32 Cell cycle regulation of human CDC6 protein . Intracellular localization, interaction with the human mcm complex, and CDC2 kinase-mediated hyperphosphorylation; Fujita M et al.; The binding of mammalian MCM complexes to chromatin is cell cycle-regulated and under CDC2 kinase negative control . Here, we investigated the properties of mammalian CDC6 protein, a candidate regulator of MCM . The levels of CDC6 were relatively constant during the HeLa cell cycle . In asynchronous cells, CDC6 was mainly detected in the nuclei with immunostaining, but some CDC6 was not extractable with nonionic detergent . In contrast to the chromatin-bound MCM, this fraction of CDC6 was resistant to DNase I treatment, suggesting that it binds to the detergent- and nuclease-resistant nuclear structure . In S phase cells, CDC6 became detectable in the cytoplasm with immunostaining; however, the level of the bound CDC6 was unchanged . In G(2)/M phase cells, the level of the bound CDC6 was still maintained, which was hyperphosphorylated by CDC2 kinase . These data suggest that some CDC6 protein is associated with the specific nuclear structure throughout the cell cycle and that major binding sites on chromatin differ between MCM and CDC6 . However, co-immunoprecipitation assays with chemical cross-linking indicated that a small part of the chromatin-bound MCM is present close to the bound CDC6. J Biol Chem, 1999 Sep 3, 274(36), 25807 - 13 Evidence that transcription factor IIB is required for a post-assembly step in transcription initiation; Cho EJ et al.; Mutation of glutamate 62 to lysine in yeast transcription factor (TF) IIB (Sua7) causes a cold-sensitive phenotype . This mutant also leads to preferential transcription of downstream start sites on some promoters in vivo . To explore the molecular nature of these phenotypes, the TFIIB E62K mutant was characterized in vitro . The mutant interacts with TATA-binding protein normally . In three different assays, the mutant can also interact with RNA polymerase II and recruit it and the other basal transcription factors to a promoter . Despite the ability to assemble a transcription complex, the TFIIB E62K protein is severely defective in transcription in vitro . Therefore, the role of TFIIB must be more than simply bridging TATA-binding protein and polymerase at the promoter . We propose that the region around Glu-62 in yeast TFIIB plays a role in start site selection, perhaps mediating a conformational change in the polymerase or the DNA during the search for initiation sites . This step may be related to the yeast-specific spacing between TATA elements and start sites since mutations of the corresponding glutamate in mammalian TFIIB do not produce a similar effect. J Biol Chem, 1999 Sep 3, 274(36), 25461 - 70 Helical interactions and membrane disposition of the 16-kDa proteolipid subunit of the vacuolar H(+)-ATPase analyzed by cysteine replacement mutagenesis; Harrison MA et al.; Theoretical mechanisms of proton translocation by the vacuolar H(+)-ATPase require that a transmembrane acidic residue of the multicopy 16-kDa proteolipid subunit be exposed at the exterior surface of the membrane sector of the enzyme, contacting the lipid phase . However, structural support for this theoretical mechanism is lacking . To address this, we have used cysteine mutagenesis to produce a molecular model of the 16-kDa proteolipid complex . Transmembrane helical contacts were determined using oxidative cysteine cross-linking, and accessibility of cysteines to the lipid phase was determined by their reactivity to the lipid-soluble probe N-(1-pyrenyl)maleimide . A single model for organization of the four helices of each monomeric proteolipid was the best fit to the experimental data, with helix 1 lining a central pore and helix 2 and helix 3 immediately external to it and forming the principal intermolecular contacts . Helix 4, containing the crucial acidic residue, is peripheral to the complex . The model is consistent not only with theoretical proton transport mechanisms, but has structural similarity to the dodecameric ring complex formed by the related 8-kDa proteolipid of the F(1)F(0)-ATPase . This suggests some commonality between the proton translocating mechanisms of the vacuolar and F(1)F(0)-ATPases. J Biol Chem, 1999 Sep 3, 274(36), 25197 - 200 Association of frabin with the actin cytoskeleton is essential for microspike formation through activation of Cdc42 small G protein; Umikawa M et al.; We have recently isolated a novel actin filament-binding protein, named frabin . Frabin has one actin filament-binding domain (ABD), one Dbl homology domain (DHD), first pleckstrin homology domains (PHD) adjacent to DHD, one cysteine rich-domain (CRD), and second PHD from the N terminus to the C terminus in this order . Full-length frabin induces microspike formation and c-Jun N-terminal kinase (JNK) activation . We found here that the fragment of frabin containing DHD and first PHD stimulated guanine nucleotide exchange of Cdc42Hs small G protein, but not that of RhoA or Rac1 small G protein . However, this fragment of frabin did not induce microspike formation, and ABD was additionally necessary for microspike formation . Frabin having ABD was associated with the actin cytoskeleton, whereas frabin lacking ABD was diffusely distributed in the cytoplasm . In contrast, ABD was not necessary for JNK activation but CRD and second PHD were additionally necessary for this activation . These results indicate that the association of frabin with the actin cytoskeleton is essential for microspike formation but not for JNK activation and that different domains of frabin are involved in microspike formation and JNK activation through Cdc42 activation. Cancer Res, 1999 Aug 15, 59(16), 4012 - 7 Ku autoantigen affects the susceptibility to anticancer drugs; Kim SH et al.; The Ku70/80 autoantigens (Ku) are the DNA-binding components of a DNA-dependent protein kinase (PK) involved in DNA double strand breaks repairing a V(D)J recombination . Because apoptosis is associated with DNA fragmentation and, consequently, creation of double strand breaks, and a variety of DNA-damaging drugs kill tumor cells by apoptosis, we tested the impact of Ku deficiency on the sensitivity of anticancer drugs . Ku-null mutant cell lines Ku70-/- and Ku80-/- were highly sensitive to anticancer drugs, compared with their wild-type cells . Ku-deficient cells were more sensitive to bleomycin-induced DNA fragmentation and exhibited a higher level of c-jun NH2-kinase/stress-activated PK activity than wild-type cells, whereas R7080-6 cells overexpressing both human Ku70 and Ku80 were resistant to bleomycin-induced apoptosis and exhibited a lower level of c-jun NH2-kinase/stress-activated PK activity . The Ku-protein level and Ku DNA binding activity were decreased after treatment with bleomycin, adriamycin, or vincristine, and the decreases were blocked by the treatment of z-DEVD-fmk, a specific inhibitor of caspase-3, suggesting that loss of Ku DNA binding is, in part, due to a caspase-mediated decrease in Ku protein levels . By contrast, HSF1 DNA-binding activity was increased by the treatment of these anticancer drugs and, subsequently, mitochondrial heat shock protein HSP75 was specifically induced . Our data suggest that Ku can affect the susceptibility to anticancer drug-induced apoptosis. Cancer Res, 1999 Aug 15, 59(16), 3883 - 8 Common nonsense mutations in RAD52; Bell DW et al.; RAD51, RAD52, and RAD54 encode proteins that are critical to the repair of double-strand DNA breaks by homologous recombination . The physical interactions among the products of RAD51, BRCA1, and BRCA2 have suggested that the BRCA1 and BRCA2 breast cancer susceptibility genes may function, at least in part, in this DNA damage repair pathway . Given the observation that different genes within a common functional pathway may be targeted by mutations in human cancers, we analyzed RAD51, RAD52, and RAD54 for the presence of germ-line mutations in 100 cases with early-onset breast cancer and for somatic mutations in 15 human breast cancer cell lines . Two premature stop codons, Ser346ter and Tyr415ter, were identified in germ-line RAD52 alleles from 5% of early-onset breast cancer cases . Together, these two heterozygous mutations were also found in 8% of a healthy control population, indicating that they do not confer an increased risk for breast cancer . A rare germ-line missense mutation was identified in RAD54, whereas no sequence variants were found in RAD51 . None of the three RAD genes demonstrated somatic mutations in breast cancer cell lines . We conclude that, despite their potential functional association with the BRCA gene products, RAD51, RAD52, and RAD54 are not themselves targeted by mutations in human breast cancer . The presence of common nonsense mutations in RAD52 within the population may have significance for other conditions associated with potential alterations in DNA damage repair pathways. J Cell Biochem, 1999 Oct 1, 75(1), 82 - 92 Identification of an autonomous transactivation domain in helix H3 of the vitamin D receptor; Kraichely DM et al.; The vitamin D receptor (VDR) contains an alpha-helical, ligand-inducible activation function (AF-2) at the COOH-terminus of the ligand-binding domain (LBD) . In this study, a second distinct activation domain was identified in the VDR LBD . Using a yeast-based system to screen a random mutant library of GAL4-VDR (93-427), a mutant GAL4-VDR fusion protein with constitutive transcriptional activity was isolated . Sequence analysis identified a C to T transition that introduced a stop codon at glutamine 239 eliminating a large portion of the LBD, including the AF-2 domain . The GAL4-VDR (93-238) mutant exhibited ligand-independent transactivation activity both in yeast and in mammalian cells . Deletion analysis defined a minimal activation domain within helix H3 between D195 and I 238 in the VDR . An aspartic acid residue (D232) within helix H3 was essential for the autonomous transactivation activity since altering this residue to an alanine or an asparagine dramatically reduced its transactivation potential . Expression of the minimal helix H3 activation domain interfered with ligand-activated transcription by full-length VDR suggesting that helix H3 interacts with limiting cellular factors important for VDR-activated transcription . Consequently, we have identified a novel activation domain in helix H3 of the VDR that apparently plays an important role in 1,25-(OH)(2)D(3)-activated transcription . Biochem Biophys Res Commun, 1999 Aug 27, 262(2), 565 - 9 Inhibitory effects of vitamin A and vitamin K on rat cytochrome P4501A1-dependent monooxygenase activity; Inouye K et al.; The inhibitory effects of vitamins A and K toward P4501A1-dependent 7-ethoxycoumarin O-deethylation were examined in the reconstituted system containing the microsomal fraction prepared from the recombinant Saccharomyces cerevisiae cells producing rat P4501A1 and yeast NADPH-P450 reductase . On vitamins A, all-trans-retinol, all-trans-retinal, all-trans-retinoic acid and retinol-palmitate showed competitive inhibition with K(i) values of 0.068, 0.079, 2.6 and 2.0 microM, respectively . Judging from the K(i) values, the inhibitory effects of those vitamins A appear to have physiological significance on the basis of their contents in liver, lung and kidney . On vitamins K, vitamin K(1) showed competitive inhibition with K(i) value of 24 microM, while vitamin K(2) showed noncompetitive inhibition with K(i) value of 60 microM . Judging from these K(i) values together with the contents of these vitamins K in liver, the inhibitory effects of the vitamins K are not as significant as those of vitamins A . These results suggest that the ingestion of enough amounts of vitamins A from foods might lead to the inhibition of the activity of P4501A1 which is known to be induced by smoking, drugs such as omeprazole and lansoprazole, and environmental pollutants like dioxins . Arch Biochem Biophys, 1999 Sep 1, 369(1), 157 - 62 N-Ethylmaleimide-modified Hsp70 inhibits protein folding; Hermawan A et al.; Hsp70 molecular chaperones facilitate protein folding and translocation by binding to hydrophobic regions of nascent or unfolded proteins, thereby preventing their aggregation . N-Ethylmaleimide (NEM) inhibits the ATPase and protein translocation-stimulating activities of the yeast Hsp70 Ssa1p by modifying its three cysteine residues, which are located in its ATPase domain . NEM alters the conformation of Ssa1p and disrupts the coupling between its nucleotide- and polypeptide-binding domains . Ssa1p and the yeast DnaJ homolog Ydj1p constitute a protein folding machinery of the yeast cytosol . Using firefly luciferase as a model protein to study chaperone-dependent protein refolding, we have found that NEM also inhibits the protein folding activity of Ssa1p . Interestingly, the NEM-modified protein (NEM-Ssa1p) is a potent inhibitor of protein folding . NEM-Ssa1p can prevent the aggregation of luciferase and stimulate the ATPase activity of Ssa1p suggesting that it acts as an inhibitor by binding to nonnative forms of luciferase and by competing with them for the polypeptide binding site of Ssa1p . NEM-Ssa1p inhibits Ssa1p/Ydj1p-dependent protein refolding at different stages indicating that the chaperones bind and release nonnative forms of luciferase multiple times before folding is completed . Radiat Environ Biophys, 1999 Jul, 38(2), 133 - 8 Repair of cellular radiation damage in space under microgravity conditions; Pross HD et al.; The influence of microgravity on the repair of x-ray-induced DNA double-strand breaks was studied in the temperature-conditional repair mutant rad54-3 of diploid yeast Saccharomyces cerevisiae . Cells were exposed on the ground and kept at a low temperature until microgravity conditions were achieved . In orbit, they were incubated at the permissive temperature to allow repair . Before re-entry they were again cooled down and kept at a low temperature until final analysis . The experiment, which was flown on the shuttle Atlantis on flight STS-76 (SMM-03), showed that repair of pre-formed DNA double-strand breaks in yeast is not impaired by microgravity. Am J Respir Cell Mol Biol, 1999 Sep, 21(3), 337 - 46 Selective expression of RT6 superfamily in human bronchial epithelial cells; Balducci E et al.; RT6 proteins are glycosylphosphatidylinositol (GPI)-linked alloantigens that are localized to cytotoxic T lymphocytes and that have nicotinamide adenine dinucleotide glycohydrolase and adenosine diphosphate (ADP)-ribosyltransferase activities . In view of the importance of GPI-linked surface proteins in mediating interactions of cells with their milieu, and the varied functions of airway cells in inflammation, we undertook the present study to determine whether human homologues of the RT6 superfamily of ADP-ribosyltransferases (ART) are expressed in pulmonary epithelial cells . We hypothesized that these surface proteins or related family members may be present in cells that interact with inflammatory cells, and that they may thereby be involved in intercellular signaling . Using in situ analysis and Northern blot analysis, we identified ART1 messenger RNA (mRNA) in airway epithelial cells . As expected for GPI-anchored proteins, the localization of ART1 at the apical surface of ciliated epithelial cells was demonstrated by staining with polyclonal anti-ART1 antibody, and was confirmed by loss of this immunoreactivity after treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), which selectively cleaves GPI anchors and releases proteins from the plasma membrane . Using in situ hybridization with specific ART3 and ART4 oligonucleotides, we also identified two additional members of the RT6 superfamily in epithelial cells . In accord with these findings, we identified ART3 and ART4 mRNAs through reverse transcription- polymerase chain reaction of polyadenine-positive RNA from human trachea . Interestingly, these proteins appeared to be preferentially localized to the airway epithelium . The localized expression of these members of the RT6 superfamily in human pulmonary epithelial cells may reflect a role for them in cell-cell signaling during immune responses within the airway. Chromosoma, 1999 Aug, 108(4), 243 - 9 Identification and characterization of MmORC4 and MmORC5, two subunits of the mouse origin of replication recognition complex; Springer J et al.; Two new members of the mouse origin recognition complex (ORC) have been cloned that are closely related to Saccharomyces cerevisiae ORC4 and ORC5 as well as to their human homolog . Both MmORC4p and MmORC5p have a putative nucleotide triphosphate binding motif . Transcription of MmORC4 and MmORC5 is not suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation . The transcription levels of both ORC genes are constantly high in all phases of the cell cycle . A screen based on the two-hybrid approach suggests that the product of the ORC4 gene interacts with the ORC2, but not with the ORC1 protein . The conservation of structure among members of the ORC4- and ORC5-related family of proteins suggests that these proteins play a key role in the initiation of DNA replication in all eukaryotes. Biochemistry, 1999 Aug 24, 38(34), 11026 - 39 Effect of DNA polymerases and high mobility group protein 1 on the carrier ligand specificity for translesion synthesis past platinum-DNA adducts; Vaisman A et al.; Translesion synthesis past Pt-DNA adducts can affect both the cytotoxicity and mutagenicity of the platinum adducts . We have shown previously that the extent of replicative bypass in vivo is influenced by the carrier ligand of platinum adducts . The specificity of replicative bypass may be determined by the DNA polymerase complexes that catalyze translesion synthesis past Pt-DNA adducts and/or by DNA damage-recognition proteins that bind to the Pt-DNA adducts and block translesion replication . In the present study, primer extension on DNA templates containing site-specifically placed cisplatin, oxaliplatin, JM216, or chlorodiethylenetriamine-Pt adducts revealed that the eukaryotic DNA polymerases beta, zeta, gamma, and human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) had a similar specificity for translesion synthesis past Pt-DNA adducts (dien >> oxaliplatin >/= cisplatin > JM216) . Primer extension assays performed in the presence of high mobility group protein 1 (HMG1), which is known to recognize cisplatin-damaged DNA, revealed that inhibition of translesion synthesis by HMG1 also depended on the carrier ligand of the Pt-DNA adduct (cisplatin > oxaliplatin = JM216 >> dien) . These data were consistent with the results of gel-shift experiments showing similar differences in the affinity of HMG1 for DNA modified with the different platinum adducts . Our studies show that both DNA polymerases and damage-recognition proteins can impart specificity to replicative bypass of Pt-DNA adducts . This information may serve as a model for further studies of translesion synthesis. Endocr J, 1999 Apr, 46(2), 279 - 84 Polymorphism of homopolymeric glutamines in coactivators for nuclear hormone receptors; Hayashi Y et al.; Some of the recently identified coactivators which interact with members of nuclear hormone receptors contain a stretch of homopolymeric glutamines (poly-Q) . Length of poly-Q in several genes are known to be polymorphic in healthy subjects, and extraordinary expansion of poly-Q in specific genes is known to cause neurodegenerative disorders . In the present study, we investigated whether such polymorphism can be observed in two coactivators, CBP (CREB {cyclic AMP responsive element binding protein}-binding protein) and AIB1/ACTR (amplified in breast cancer-1/ACTR, also called RAC3/TRAM-1) . The genomic regions encoding the poly-Q were amplified by means of PCR using fluorescence labeled primer and analyzed by an automatic sequencer . While contiguous glutamine residues inAIB1/ACTR ranged from 26 to 32 with a heterozygosity of 54%, no polymorphism could be observed in poly-Q of CBP among 54 unrelated subjects . These results suggest that the residue in CBP may play a critical role in the function so that individuals with CBP containing different sizes of poly-Q might have been eliminated . It has been reported that AIB1/ACTR is overexpressed in some of the cell lines derived from breast cancer . If the length of poly-Q alters the stability of AIB1/ACTR and/or potency to enhance hormone action through nuclear receptors, the length of poly-Q is likely to be one of the genetic factors affecting not only susceptibility to breast cancers but also the sensitivity to hormones . This polymorphism should also be tested in patients with neurodegenerative disorders of unknown cause. J Cell Biol, 1999 Aug 23, 146(4), 791 - 800 Regulation of APC activity by phosphorylation and regulatory factors; Kotani S et al.; Ubiquitin-dependent proteolysis of Cut2/Pds1 and Cyclin B is required for sister chromatid separation and exit from mitosis, respectively . Anaphase-promoting complex/cyclosome (APC) specifically ubiquitinates Cut2/Pds1 at metaphase-anaphase transition, and ubiquitinates Cyclin B in late mitosis and G1 phase . However, the exact regulatory mechanism of substrate-specific activation of mammalian APC with the right timing remains to be elucidated . We found that not only the binding of the activators Cdc20 and Cdh1 and the inhibitor Mad2 to APC, but also the phosphorylation of Cdc20 and Cdh1 by Cdc2-Cyclin B and that of APC by Polo-like kinase and cAMP-dependent protein kinase, regulate APC activity . The cooperation of the phosphorylation/dephosphorylation and the regulatory factors in regulation of APC activity may thus control the precise progression of mitosis. Virus Genes, 1999, 18(3), 243 - 64 Identification of a gene cluster within the genome of Chilo iridescent virus encoding enzymes involved in viral DNA replication and processing; Muller K et al.; The nucleotide sequence of the genome of Chilo iridescent virus (CIV) between the genome coordinates 0.974 and 0.101 comprising 27,079 bp was determined . Computer-assisted analysis of the DNA sequence of this particular region of the CIV genome revealed the presence of 42 potential open reading frames (ORFs) with coding capacities for polypeptides ranging from 50 to 1,273 amino acid residues . The analysis of the amino acid sequences deduced from the individual ORFs resulted in the identification of 10 potential viral genes that show significant homology to functionally characterized proteins of other species . A cluster of five viral genes that encode enzymes involved in the viral DNA replication was identified including the DNA topoisomerase II (A039L,1,132 amino acids (aa)), the DNA polymerase (ORF A031L,1,273 aa), a helicase (ORF A027L, 530 aa), a nucleoside triphosphatase I (ORF A025L, 1,171 aa), and an exonuclease II (ORF A019L, 624aa), all ORFs possessing the same genomic orientation . The DNA polymerase of CIV showed the highest homology (24.8% identity) to the DNA polymerase of lymphocystis disease virus lymphocystis disease virus 1 (LCDV-1), a member of the family Iridoviridae, indicating the close relatedness of the two viruses . In addition, four putative gene products were found to be significantly homologous to previously identified hypothetical proteins of CIV. Methods, 1999 Jul, 18(3), 329 - 34 Minichromosome maintenance as a genetic assay for defects in DNA replication; Tye BK; Minichromosome maintenance (mcm) is an effective genetic assay for mutants defective in DNA replication . Two classes of mcm mutants have been identified using this screen: those that differentially affect the activities of certain autonomously replicating sequences (ARSs) and those that uniformly affect the activities of all ARSs . The ARS-specific MCM genes are essential for the initiation of DNA replication . Among these are members of the MCM2-7 family that encode subunits of the preinitiation complex and MCM10, whose gene product interacts with members of the Mcm2-7 proteins . Among the ARS-nonspecific MCM gene products are chromosome transmission factors . Refinement of this genetic assay as a screening tool and further analysis of existing mcm mutants may reveal new replication initiation proteins . Nucleic Acids Res, 1999 Aug 15, 27(16), 3348 - 54 Mammalian mitochondrial extracts possess DNA end-binding activity; Coffey G et al.; Mammalian mitochondrial protein extracts possess DNA end-binding (DEB) activity . Protein binding to a 394 bp double-stranded DNA molecule was measured using an electrophoretic mobility shift assay . Mitochondrial DEB activity was highly specific for linear DNA . Inclusion of a vast excess of non-radioactive circular DNA did not disrupt binding to radioactive f394 . In contrast, binding was abolished by the inclusion of linear competitor DNA . In mammals, nuclear DEB activity is due to Ku, a hetero-dimer composed of the Ku70 and Ku86 proteins . To determine whether mitochondrial DEB activity was also due to Ku, protein extracts were prepared from the Chinese hamster XR-V15B cell line, which lacks this protein . As anticipated, nuclear extracts prepared from these cells lacked DEB activity . In contrast, mitochondrial extracts prepared from these cells had wild-type levels of DEB activity, demonstrating that this latter activity is not a consequence of nuclear contamination . Although the nuclear and mitochondrial DEB activities are independent of each other, they are nevertheless closely related, since mitochondrial DEB activity was 'supershifted' by both anti-Ku70 and anti-Ku86 antisera . The nuclear DEB protein Ku plays an essential role in nuclear DNA double-strand break repair . The DEB activity described herein may therefore play a similar role in mitochondrial DNA repair. Nucleic Acids Res . 1999 Aug 1;27(15):e9. Improved mutation detection in GC-rich DNA fragments by combined DGGE and CDGE; Wu Y et al.; Denaturing gradient gel electrophoresis (DGGE) has proven to be a powerful pre-screening method for the detection of DNA variants . If such variants occur, however, in DNA fragments that are very rich in G and C, they may escape detection . To overcome this limitation, we tested a novel gel system which combines DGGE and constant denaturant gel electrophoresis (CDGE), as it might have the advantages of both methods . Indeed, this combination had the advantages of both methods, good separation of hetero-duplex molecules and prevention of total strand dissociation, and it proved successful in the detection of DNA variants in several GC-rich fragments. Mol Cell Biol, 1999 Sep, 19(9), 6441 - 7 Trithorax and ASH1 interact directly and associate with the trithorax group-responsive bxd region of the Ultrabithorax promoter; Rozovskaia T et al.; Trithorax (TRX) and ASH1 belong to the trithorax group (trxG) of transcriptional activator proteins, which maintains homeotic gene expression during Drosophila development . TRX and ASH1 are localized on chromosomes and share several homologous domains with other chromatin-associated proteins, including a highly conserved SET domain and PHD fingers . Based on genetic interactions between trx and ash1 and our previous observation that association of the TRX protein with polytene chromosomes is ash1 dependent, we investigated the possibility of a physical linkage between the two proteins . We found that the endogenous TRX and ASH1 proteins coimmunoprecipitate from embryonic extracts and colocalize on salivary gland polytene chromosomes . Furthermore, we demonstrated that TRX and ASH1 bind in vivo to a relatively small (4 kb) bxd subregion of the homeotic gene Ultrabithorax (Ubx), which contains several trx response elements . Analysis of the effects of ash1 mutations on the activity of this regulatory region indicates that it also contains ash1 response element(s) . This suggests that ASH1 and TRX act on Ubx in relatively close proximity to each other . Finally, TRX and ASH1 appear to interact directly through their conserved SET domains, based on binding assays in vitro and in yeast and on coimmunoprecipitation assays with embryo extracts . Collectively, these results suggest that TRX and ASH1 are components that interact either within trxG protein complexes or between complexes that act in close proximity on regulatory DNA to maintain Ubx transcription. Mol Cell Biol, 1999 Sep, 19(9), 6367 - 78 Transcriptional activation by NF-kappaB requires multiple coactivators; Sheppard KA et al.; Nuclear factor-kappaB (NF-kappaB) plays a role in the transcriptional regulation of genes involved in inflammation and cell survival . In this report we demonstrate that NF-kappaB recruits a coactivator complex that has striking similarities to that recruited by nuclear receptors . Inactivation of either cyclic AMP response element binding protein (CREB)-binding protein (CBP), members of the p160 family of coactivators, or the CBP-associated factor (p/CAF) by nuclear antibody microinjection prevents NF-kappaB-dependent transactivation . Like nuclear receptor-dependent gene expression, NF-kappaB-dependent gene expression requires specific LXXLL motifs in one of the p160 family members, and enhancement of NF-kappaB activity requires the histone acetyltransferase (HAT) activity of p/CAF but not that of CBP . This coactivator complex is differentially recruited by members of the Rel family . The p50 homodimer fails to recruit coactivators, although the p50-p65 heterodimeric form of the transcription factor assembles the integrator complex . These findings provide new mechanistic insights into how this family of dimeric transcription factors has a differential effect on gene expression. Mol Cell Biol, 1999 Sep, 19(9), 6269 - 75 The Drosophila melanogaster DmRAD54 gene plays a crucial role in double-strand break repair after P-element excision and acts synergistically with Ku70 in the repair of X-ray damage; Kooistra R et al.; The RAD54 gene has an essential role in the repair of double-strand breaks (DSBs) via homologous recombination in yeast as well as in higher eukaryotes . A Drosophila melanogaster strain deficient in the RAD54 homolog DmRAD54 is characterized by increased X-ray and methyl methanesulfonate (MMS) sensitivity . In addition, DmRAD54 is involved in the repair of DNA interstrand cross-links, as is shown here . However, whereas X-ray-induced loss-of-heterozygosity (LOH) events were completely absent in DmRAD54(-/-) flies, treatment with cross-linking agents or MMS resulted in only a slight reduction in LOH events in comparison with those in wild-type flies . To investigate the relative contributions of recombinational repair and nonhomologous end joining in DSB repair, a DmRad54(-/-)/DmKu70(-/-) double mutant was generated . Compared with both single mutants, a strong synergistic increase in X-ray sensitivity was observed in the double mutant . No similar increase in sensitivity was seen after treatment with MMS . Apparently, the two DSB repair pathways overlap much less in the repair of MMS-induced lesions than in that of X-ray-induced lesions . Excision of P transposable elements in Drosophila involves the formation of site-specific DSBs . In the absence of the DmRAD54 gene product, no male flies could be recovered after the excision of a single P element and the survival of females was reduced to 10% compared to that of wild-type flies . P-element excision involves the formation of two DSBs which have identical 3' overhangs of 17 nucleotides . The crucial role of homologous recombination in the repair of these DSBs may be related to the very specific nature of the breaks. Mol Cell Biol, 1999 Sep, 19(9), 6260 - 8 Involvement of DNA end-binding protein Ku in Ty element retrotransposition; Downs JA et al.; Saccharomyces cerevisiae Ty elements are retrotransposons whose life cycles are strikingly similar to those of retroviruses . They transpose via an RNA intermediate that is converted to linear double-stranded cDNA and then inserted into the host genome . Although Ty integration is mediated by the element-encoded integrase, it has been proposed that host factors are involved in this process . Here, we show that the DNA end-binding protein Ku, which functions in DNA double-strand break repair, potentiates retrotransposition . Specifically, by using a galactose-inducible Ty1 system, we found that in vivo, Ty1 retrotransposition rates were substantially reduced in the absence of Ku . In contrast, this phenotype was not observed with yeast strains containing mutations in other genes that are involved in DNA repair . We present evidence that Ku associates with Ty1 viruslike particles both in vitro and in vivo . These results provide an additional role for Ku and suggest that it might function in the life cycles of retroelements in other systems. Mol Cell Biol, 1999 Sep, 19(9), 6130 - 9 Analysis of primary structural determinants that distinguish the centromere-specific function of histone variant Cse4p from histone H3; Keith KC et al.; Cse4p is a variant of histone H3 that has an essential role in chromosome segregation and centromere chromatin structure in budding yeast . Cse4p has a unique 135-amino-acid N terminus and a C-terminal histone-fold domain that is more than 60% identical to histone H3 and the mammalian centromere protein CENP-A . Cse4p and CENP-A have biochemical properties similar to H3 and probably replace H3 in centromere-specific nucleosomes in yeasts and mammals, respectively . In order to identify regions of Cse4p that distinguish it from H3 and confer centromere function, a systematic site-directed mutational analysis was performed . Nested deletions of the Cse4p N terminus showed that this region of the protein contains at least one essential domain . The C-terminal histone-fold domain of Cse4p was analyzed by changing Cse4p amino acids that differ between Cse4p and H3 to the analogous H3 residues . Extensive substitution of contiguous Cse4p residues with H3 counterparts resulted in cell lethality . However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated rates of mitotic chromosome loss . The results indicate that residues critical for wild-type Cse4p function and high-fidelity chromosome transmission are distributed across the entire histone-fold domain . Our findings are discussed in the context of the known structure of H3 within the nucleosome and compared with previous results reported for CENP-A. Mol Cell Biol, 1999 Sep, 19(9), 5981 - 90 Phosphorylation-independent inhibition of Cdc28p by the tyrosine kinase Swe1p in the morphogenesis checkpoint; McMillan JN et al.; The morphogenesis checkpoint in budding yeast delays cell cycle progression in G(2) when the actin cytoskeleton is perturbed, providing time for cells to complete bud formation prior to mitosis . Checkpoint-induced G(2) arrest involves the inhibition of the master cell cycle regulatory cyclin-dependent kinase, Cdc28p, by the Wee1 family kinase Swe1p . Results of experiments using a nonphosphorylatable CDC28(Y19F) allele suggested that the checkpoint stimulated two inhibitory pathways, one that promoted phosphorylation at tyrosine 19 (Y19) and a poorly characterized second pathway that did not require Cdc28p Y19 phosphorylation . We present the results from a genetic screen for checkpoint-defective mutants that led to the repeated isolation of the dominant CDC28(E12K) allele that is resistant to Swe1p-mediated inhibition . Comparison of this allele with the nonphosphorylatable CDC28(Y19F) allele suggested that Swe1p is still able to inhibit CDC28(Y19F) in a phosphorylation-independent manner and that both the Y19 phosphorylation-dependent and -independent checkpoint pathways in fact reflect Swe1p inhibition of Cdc28p . Remarkably, we found that a Swe1p mutant lacking catalytic activity could significantly delay the cell cycle in vivo during a physiological checkpoint response, even when expressed at single copy . The finding that a Wee1 family kinase expressed at physiological levels can inhibit a nonphosphorylatable cyclin-dependent kinase has broad implications for many checkpoint studies using such mutants in other organisms. Mol Cell Biol, 1999 Sep, 19(9), 5952 - 9 Histone acetyltransferase complexes can mediate transcriptional activation by the major glucocorticoid receptor activation domain; Wallberg AE et al.; Previous studies have shown that the Ada adapter proteins are important for glucocorticoid receptor (GR)-mediated gene activation in yeast . The N-terminal transactivation domain of GR, tau1, is dependent upon Ada2, Ada3, and Gcn5 for transactivation in vitro and in vivo . Using in vitro techniques, we demonstrate that the GR-tau1 interacts directly with the native Ada containing histone acetyltransferase (HAT) complex SAGA but not the related Ada complex . Mutations in tau1 that reduce tau1 transactivation activity in vivo lead to a reduced binding of tau1 to the SAGA complex and conversely, mutations increasing the transactivation activity of tau1 lead to an increased binding of tau1 to SAGA . In addition, the Ada-independent NuA4 HAT complex also interacts with tau1 . GAL4-tau1-driven transcription from chromatin templates is stimulated by SAGA and NuA4 in an acetyl coenzyme A-dependent manner . Low-activity tau1 mutants reduce SAGA- and NuA4-stimulated transcription while high-activity tau1 mutants increase transcriptional activation, specifically from chromatin templates . Our results demonstrate that the targeting of native HAT complexes by the GR-tau1 activation domain mediates transcriptional stimulation from chromatin templates. Mol Cell Biol, 1999 Sep, 19(9), 5847 - 60 Functional analysis of the SIN3-histone deacetylase RPD3-RbAp48-histone H4 connection in the Xenopus oocyte; Vermaak D et al.; We investigated the protein associations and enzymatic requirements for the Xenopus histone deacetylase catalytic subunit RPD3 to direct transcriptional repression in Xenopus oocytes . Endogenous Xenopus RPD3 is present in nuclear and cytoplasmic pools, whereas RbAp48 and SIN3 are predominantly nuclear . We cloned Xenopus RbAp48 and SIN3 and show that expression of RPD3, but not RbAp48 or SIN3, leads to an increase in nuclear and cytoplasmic histone deacetylase activity and transcriptional repression of the TRbetaA promoter . This repression requires deacetylase activity and nuclear import of RPD3 mediated by a carboxy-terminal nuclear localization signal . Exogenous RPD3 is not incorporated into previously described oocyte deacetylase and ATPase complexes but cofractionates with a component of the endogenous RbAp48 in the oocyte nucleus . We show that RPD3 associates with RbAp48 through N- and C-terminal contacts and that RbAp48 also interacts with SIN3 . Xenopus RbAp48 selectively binds to the segment of the N-terminal tail immediately proximal to the histone fold domain of histone H4 in vivo . Exogenous RPD3 may be targeted to histones through interaction with endogenous RbAp48 to direct transcriptional repression of the Xenopus TRbetaA promoter in the oocyte nucleus . However, the exogenous RPD3 deacetylase functions to repress transcription in the absence of a requirement for association with SIN3 or other targeted corepressors. Acta Biochim Pol, 1999, 46(1), 77 - 89 Nuclear receptors, their coactivators and modulation of transcription; Manteuffel-Cymborowska M; Nuclear receptors are ligand-dependent transcription factors which can also be activated in the absence of their lipophilic ligands by signaling substances acting on cell membrane receptors . This ligand-independent activation indicates the importance of nuclear receptor phosphorylation for their function . Nuclear receptor-mediated transcription of target genes is further increased by interactions with recruited coactivators forming a novel family of nuclear proteins . CBP/p300, a coactivator of different classes of transcription factors, including the tumor suppressor protein p53, plays a special role acting as a bridging protein between inducible transcription factors and the basal transcription apparatus, and as an integrator of diverse signaling pathways . Coactivators of nuclear receptors and associated proteins forming a multicomponent complex have an intrinsic histone acetylase activity in contrast to nuclear receptor and heterodimer Mad-Max corepressors, which recruit histone deacetylase . Similarly the Rb protein interacts with histone deacetylase to repress transcription of cell cycle regulatory genes . Targeted histone acetylation/deacetylation results in remodeling of chromatin structure and correlates with activation/repression of transcription . Recent data point to the important role of coactivator proteins associated with inducible transcription factors in transcription regulation, and in the integration of multiple signal transduction pathways within the nucleus. J Mol Biol, 1999 Aug 27, 291(4), 997 - 1013 Proteasome beta-type subunits: unequal roles of propeptides in core particle maturation and a hierarchy of active site function; Jager S et al.; The 26 S proteasome is a large eukaryotic protease complex acting in ubiquitin-mediated degradation of abnormal and many short-lived, regulatory proteins . Its cylinder-shaped 20 S proteolytic core consists of two sets, each of seven different alpha and beta-type subunits arranged into two outer alpha-rings surrounding two inner beta-rings . The beta-rings form a central chamber with a total of six proteolytically active centers located in the beta1, beta2 and beta5 subunits . Activation of these subunits occurs during late assembly stages through intramolecular precursor autolysis removing propeptides attached to Thr1, which then serves as N-terminal nucleophile in substrate hydrolysis . This maturation entails intermolecular cleavage of propeptides residing in two of the non-active beta-type subunits, beta6 and beta7 . In yeast, deletion of the beta5/Pre2 propeptide was shown to be lethal by preventing assembly of the core particle, while its expression as a separate entity restored growth . We investigated the role of the yeast beta1/Pre3, beta2/Pup1 and beta7/Pre4 propeptides by expressing the mature subunit moieties without propeptides as C-terminal fusions to ubiquitin . In all cases, viable strains could be generated . Deletion of the beta1/Pre3 and beta7/Pre4 propeptides did not affect cell growth, but deletion of the beta2/Pup1 propeptide led to poor growth, which was partially restored by co-expression of the free propeptide . Gain of proteolytic activity of beta1/Pre3 and beta2/Pup1 was abolished or drastically reduced, respectively, if their respective propeptides were not N-terminally bound . We detected N -alpha-acetylation at Thr1 of beta1/Pre3 as cause for its inactivation . Thus, one role for the propeptides of active beta-type subunits might be to protect the mature subunits catalytic Thr1 alpha-amino group from acetylation . The beta2/Pup1 propeptide was, in addition, required for efficient 20 S proteasome maturation, as revealed by the accumulation of beta7/Pre4 precursor and intermediate processing forms upon expression of mature beta2/Pup1 . Finally, growth phenotypes resulting from expression of active site mutated beta-type subunits uncoupled from their propeptides allowed us to deduce the hierarchy of the importance of individual subunit activities for proteasomal function as follows: beta5/Pre2>>beta2/Pup1>/=beta1/Pre3 . J Mol Biol, 1999 Aug 27, 291(4), 965 - 76 A calorimetric study of the folding-unfolding of an alpha-helix with covalently closed N and C-terminal loops; Taylor JW et al.; The thermal melting of a dicyclic 29-residue peptide, having helix-stabilizing side-chain to side-chain covalent links at each terminal, has been studied by circular dichroism spectropolarimetry (CD) and differential scanning calorimetry (DSC) . The CD spectra for this dicyclic peptide indicate that it is monomeric, almost fully alpha-helical at -10 degrees C, and undergoes a reversible transition from the folded to the disordered state with increasing temperature . The temperature dependencies of the ellipticity at 222 nm and the excess heat capacity measured calorimetrically are well fit by a two-state model, which indicates a cooperative melting transition that is complete within the temperature ranges of these experiments (from -10 degrees C to 100 degrees C) . This allows a complete analysis of the thermodynamics of helix formation . The helix unfolding is found to proceed with a small positive heat-capacity increment, consistent with the solvation of some non-polar groups upon helix unfolding . It follows that the hydrogen bonds are not the only factors responsible for the formation of the alpha-helix, and that hydrophobic interactions are also playing a role in its stabilization . At 30 degrees C, the calorimetric enthalpy and entropy values are estimated to be 650(+/-50) cal mol(-1)and 2.0(+/-0.2) cal K(-1)mole(-1), respectively, per residue of this peptide . Comparison with the thermodynamic characteristics obtained for the unfolding of double-stranded alpha-helical coiled-coils shows that at that temperature the enthalpic contribution of non-polar groups to the stabilization of the alpha-helix is insignificant and the estimated transition enthalpy can be assigned to the hydrogen bonds . With increasing temperature, the increasing magnitude of the negative enthalpy of hydration of the exposed polar groups should decrease the helix-stabilizing enthalpy of the backbone hydrogen bonds . However, the helix-stabilizing negative entropy of hydration of these groups should also increase in magnitude with increasing temperature, offsetting this effect . J Exp Zool, 1999 Sep 1, 284(4), 379 - 91 Distinct parameters are involved in controlling the number of rounds of cell division in each tissue during ascidian embryogenesis; Yamada A et al.; We counted cell numbers during embryogenesis of the ascidian, Halocynthia roretzi, every hour . Cell numbers were determined by counting the numbers of nuclei in squashed embryos . The cell number of a larva just after hatching was approximately 3000 . Our study addresses the question of what factors control the number of rounds of cell division during development . Three kinds of egg fragments were prepared by cutting unfertilized eggs to alter the volume of cytoplasm and the amount of DNA . After the egg fragments were fertilized, the cell numbers were estimated at the hatching stage . The cell numbers of the resulting larvae differed from those of normal larvae . Precursor blastomeres of various tissues were then isolated from normal and manipulated embryos, and cultured as partial embryos . The cell numbers of the resulting partial embryos were counted to estimate the number of cell divisions in each larval tissue . The results suggested that the number of cell divisions is controlled by a distinct mechanism in each tissue . We propose that the number of rounds of cell division during ascidian embryogenesis is controlled by three mechanisms: the first depending on the volume of cytoplasm; the second on the nucleo-cytoplasmic ratio; and the third depending on neither of these parameters . J . Exp . Zool . 284:379-391, 1999 . Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9648 - 53 Wiskott-Aldrich syndrome protein regulates podosomes in primary human macrophages; Linder S et al.; Wiskott-Aldrich syndrome protein (WASp) is a hematopoietic-specific, multidomain protein whose mutation is responsible for the immunodeficiency disorder Wiskott-Aldrich syndrome . WASp contains a binding motif for the Rho GTPase CDC42Hs as well as verprolin/cofilin-like actin-regulatory domains, but no specific actin structure regulated by CDC42Hs-WASp has been identified . We found that WASp colocalizes with CDC42Hs and actin in the core of podosomes, a highly dynamic adhesion structure of human blood-derived macrophages . Microinjection of constitutively active V12CDC42Hs or a constitutively active WASp fragment consisting of the verprolin/cofilin-like domains led to the disassemly of podosomes . Conversely, macrophages from patients expressing truncated forms of WASp completely lacked podosomes . These findings indicate that WASp controls podosome assembly and, in cooperation with CDC42Hs, podosome disassembly in primary human macrophages. Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9574 - 9 A mechanism for Rb/p130-mediated transcription repression involving recruitment of the CtBP corepressor; Meloni AR et al.; Previous work has demonstrated the critical role for transcription repression in quiescent cells through the action of E2F-Rb or E2F-p130 complexes . Recent studies have shown that at least one mechanism for this repression involves the recruitment of histone deacetylase . Nevertheless, these studies also suggest that other events likely contribute to E2F/Rb-mediated repression . Using a yeast two-hybrid screen to identify proteins that specifically interact with the Rb-related p130 protein, we demonstrate that p130, as well as Rb, interacts with a protein known as CtIP . This interaction depends on the p130 pocket domain, which is important for repression activity, as well as an LXCXE sequence within CtIP, a motif previously shown to mediate interactions of viral proteins with Rb . CtIP interacts with CtBP, a protein named for its ability to interact with the C-terminal sequences of adenovirus E1A . Recent work has demonstrated that the Drosophila homologue of CtBP is a transcriptional corepressor for Hairy, Knirps, and Snail . We now show that both CtIP and CtBP can efficiently repress transcription when recruited to a promoter by the Gal4 DNA binding domain, thereby identifying them as corepressor proteins . Moreover, the full repression activity of CtIP requires a PLDLS domain that is also necessary for the interaction with CtBP . We propose that E2F-mediated repression involves at least two events, either the recruitment of a histone deacetylase or the recruitment of the CtIP/CtBP corepressor complex. J Mol Biol, 1999 Aug 20, 291(3), 715 - 25 Proteins can adopt totally different folded conformations; Damaschun G et al.; The three-dimensional structure of a protein is determined by interactions between its amino acids and by interactions of the amino acids with molecules of the environment . The great influence of the latter interactions is demonstrated for the enzyme phosphoglycerate kinase from yeast (PGK) . In the native state, PGK is a compact, bilobal molecule; 35% and 13% of its amino acids are organised in the form of alpha-helices and beta-sheets, respectively . The molecules unfold at acidic pH and low ionic strength forming random-walk structures with a persistence length of 3 nm . More than 90% of the amino acid residues of the ensemble have phi,psi-angles corresponding to those of a straight beta-chain . Upon addition of 50% (v/v) trifluoroethanol to the acid-unfolded protein, the entire molecule is transformed into a rod-like, flexible alpha-helix . Addition of anions, such as chloride or trichloroacetate, to the acid-unfolded protein leads to the formation of amyloid-like fibres over a period of many hours when the anion concentration exceeds a critical limit . Half of the amino acid residues are then organised in beta-sheets . Both of the non-natively folded states of PGK contain more regular secondary structure than the native one . The misfolding starts in both cases from the acid-unfolded state, in which the molecules are essentially more expanded than in other denatured states, e.g . those effected by temperature or guanidine hydrochloride . Int J Cancer, 1999 Sep 24, 83(1), 80 - 2 Analysis of the RAD54 gene on chromosome 1p as a potential tumor-suppressor gene in parathyroid adenomas; Carling T et al.; Parathyroid adenomas causing primary hyperparathyroidism (pHPT) frequently exhibit allelic loss of DNA markers on the short arm of chromosome 1, indicating the presence of one or more tumor-suppressor genes on 1p . Since the development of pHPT is enhanced in individuals exposed to ionizing radiation to the neck, it could be anticipated that genes involved in DNA repair and recombination may be special targets for mutation in parathyroid tumorigenesis, whether irradiation-associated or not . RAD54 is a member of a family of genes involved in such functions, and RAD54 knockout mice show increased sensitivity to ionizing radiation . The localization of the RAD54 gene to 1p32 has therefore elevated it to a most compelling candidate parathyroid tumor-suppressor gene . Twelve parathyroid adenomas demonstrating allelic loss at chromosome 1p were selected from 55 parathyroid adenomas previously analyzed for loss of heterozygosity using polymorphic microsatellite markers . All 18 exons of the RAD54 gene were fully analyzed by automated sequencing for detection of point mutations or micro-deletions in each parathyroid adenoma . No mutational aberrations were detected in the RAD54 gene, strongly suggesting that complete somatic inactivation of RAD54 is infrequently, if ever, associated with the development of parathyroid adenomas . Whether genes controlling DNA repair and recombination are involved in parathyroid neoplasia remains to be determined . Plant Cell, 1999 Aug, 11(8), 1419 - 32 The conserved KNOX domain mediates specificity of tobacco KNOTTED1-type homeodomain proteins; Sakamoto T et al.; Overproduction of the tobacco KNOTTED1-type homeodomain proteins NTH1, NTH15, and NTH23 in transgenic tobacco plants causes mild, severe, and no morphological alterations, respectively . The deduced amino acid sequences of the homeodomains and adjacent ELK domains are highly conserved, and the N-terminal KNOX domains also are moderately conserved . To investigate the contributions of both the conserved and divergent regions to the severity of morphological alterations, we generated chimeric proteins by exchanging different regions of NTH1, NTH15, and NTH23 . The severity of the abnormal phenotype was dependent upon the synergistic action of both the N terminus, containing the KNOX domain, and the C terminus, containing the ELK homeodomain . Detailed analysis focusing on the C terminus revealed that the C-terminal half of the ELK domain is more effective in inducing the abnormal phenotypes than are the homeodomains . For the N terminus, severe morphological alterations were induced by exchanging a part of the KNOX domain of NTH1 with the corresponding region of NTH15 . This limited region in the KNOX domain of all homeodomain proteins includes a predicted alpha-helical region, but only that in NTH15 is predicted to form a typical amphipathic structure . We discuss the possibility, based on these results, that the secondary structure of the KNOX domain is important for the induction of abnormal morphology in transgenic tobacco plants. EMBO J, 1999 Aug 16, 18(16), 4571 - 8 tRNA prefers to kiss; Scarabino D et al.; Six RNA aptamers that bind to yeast phenylalanine tRNA were identified by in vitro selection from a random-sequence pool . The two most abundantly represented aptamers interact with the tRNA anticodon loop, each through a sequence block with perfect Watson-Crick complementarity to the loop . It was possible to truncate one of these aptamers to a simple hairpin loop that forms a classical 'kissing complex' with the anticodon loop . Three other aptamers have nearly complete complementarity to the anticodon loop . The sixth aptamer has two sequence blocks, one complementary to the tRNA T loop and the other to the D loop; this aptamer binds better to a mutant tRNA that disrupts the normal D-loop/T-loop tertiary interaction than to the wild-type tRNA . Selection of complements to tRNA loops occurred despite an attempt to direct binding to tertiary structural features of tRNA . This serves as a reminder of how special the RNA-RNA interactions are that are not based on complementarity . Nonetheless, these aptamers must present the tRNA complement in some special structural context; the simple single-strand complement of the anticodon loop did not bind tRNA effectively. EMBO J, 1999 Aug 16, 18(16), 4361 - 71 Metal ion transporters and homeostasis; Nelson N; Transition metals are essential for many metabolic processes and their homeostasis is crucial for life . Aberrations in the cellular metal ion concentrations may lead to cell death and severe diseases . Metal ion transporters play a major role in maintaining the correct concentrations of the various metal ions in the different cellular compartments . Recent studies of yeast mutants revealed key elements in metal ion homeostasis, including novel transport systems . Several of the proteins discovered in yeast are highly conserved, and defects in some of the yeast mutants could be complemented by their human homologs . The studies of yeast metal ion transporters helped to unravel the molecular mechanism of macrophage defense against bacterial infection and hereditary diseases. Curr Opin Cell Biol, 1999 Aug, 11(4), 453 - 9 Membrane tethering in intracellular transport; Waters MG et al.; Studies of various membrane trafficking steps over the past year indicate that membranes are tethered together prior to the interaction of v-SNAREs and t-SNAREs across the membrane junction . The tethering proteins identified to date are quite large, being either fibrous proteins or multimeric protein complexes . The tethering factors employed at different steps are evolutionarily unrelated, yet their function seems to be closely tied to the more highly conserved Rab GTPases . Tethering factors may collaborate with Rabs and SNAREs to generate targeting specificity in the secretory pathway. J Bioenerg Biomembr, 1999 Apr, 31(2), 143 - 51 Mutations in the voltage-dependent anion channel of the mitochondrial outer membrane cause a dominant nonlethal growth impairment; Angeles R et al.; Point mutations at K234 and K236 in the yeast voltage-dependent anion channel 1 (VDAC1) of the mitochondrial outer membrane have been shown to markedly impair the membrane insertion of this protein (Smith et al., 1995; Angeles et al., 1998) . Mutants of this type were expressed in vivo in a strain of yeast with a disruption in the VDAC1 gene . Expression of the various VDAC1 forms was under the control of a Gall promoter . Wild-type VDAC1 expression fully complemented the slow growth phenotype caused by the disruption . VDAC1 mutants in which K234 and K236 were replaced by arginine, glutamate, or glutamine caused a more severe negative effect on growth . This effect appeared to be dominant since the mutant VDAC1 forms suppressed growth in a yeast strain that retained its VDAC1 gene . This apparent dominant negative effect on growth did not seem to be specific for any stage of the cell cycle . However, the growth defect was not lethal as the affected cells still could accumulate the vital stain, FUN1 . Expression of a mutant in which K234 had been replaced by glutamate had more serious negative growth effects than did a similar mutation at K236 . Expression of delta71-116 VDAC1 complemented the VDAC1 disruption; however, expression of the same deletion mutant in which the lysines corresponding to K234 and K236 were mutated to glutamate severely impaired growth . These results have shown that a deficiency of lysine at positions 234 and 236 in VDAC I causes a nonlethal growth defect that is more severe than deletion of 45 amino acids from VDACI or disruption of the VDAC1 gene . They also indicate that there is a hierarchy in the importance of these lysines with mutations at K234 being the more serious. J Bioenerg Biomembr, 1999 Apr, 31(2), 137 - 42 Mutation of K234 and K236 in the voltage-dependent anion channel 1 impairs its insertion into the mitochondrial outer membrane; Angeles R et al.; Previous in vitro studies indicated that mutation of both K234 and K236 to arginine, glutamine, or glutamic acid impaired the ability of the voltage-dependent anion channel (VDAC1) to insert into the outer membrane of the mitochondria (Smith et al . 1995) . These same mutants were expressed in a strain of Saccharomyces cerevisiae with a disruption in the VDAC1 gene . The mutant VDAC1 forms were found in the mitochondria suggesting that they were correctly sorted to the outer membrane . However, only very small amounts of the mutants were inserted into the mitochondrial membranes . Mitochondria isolated from the strains expressing the mutants were capable of catalyzing the translocation of both wild-type VDAC1 and pre-alcohol dehydrogenase III indicating that the translocation apparatus was functional . These results confirm the previously drawn conclusion that K234 and K236 are part of a membrane insertion motif . The failure of the mutant VDAC1 forms to insert did not cause VDAC1 precursors to accumulate in the soluble cell cytoplasm or in the microsomal fraction . The apparent lack of a "precursor pool" suggested that a post-transcriptional control mechanism might limit the amounts of VDAC1 precursors in the cell . Such a control mechanism is consistent with the observation that the amount of VDAC1 was very similar after epichromosomal (gene in a 2u plasmid controlled by a Gal1 promoter) and chromosomal expression (endogenous gene controlled by the endogenous promoter). Biochem Biophys Res Commun, 1999 Aug 19, 262(1), 157 - 62 Phosphorylation by p44 MAP Kinase/ERK1 stimulates CBP histone acetyl transferase activity in vitro; Ait-Si-Ali S et al.; The transcriptional coactivator CBP displays an intrinsic histone acetyl transferase (HAT) activity which seems to participate in transcriptional activation through the destabilization of nucleosome structure . CBP is involved in the activity of several transcription factors that are nuclear endpoints of intracellular signal transduction pathways . In some instances, the transcription factors are phosphorylated upon cell activation, which induces their interaction with CBP . CBP itself is a phosphoprotein and can be phosphorylated by cycle-dependent kinases or by MAP kinases . Here we show that CBP phosphorylation by p44 MAP kinase/ERK1 results in the stimulation of its HAT enzymatic activity . The p44 MAP kinase/ERK1 phosphorylation sites are located in the C-terminal part of the protein, outside of the HAT domain . These sites are required for enzymatic stimulation, suggesting that phosphorylation by p44 MAP kinase/ERK1 induces a conformational change of the CBP molecule . Our data suggest that, in some instances, CBP itself might be a target for signal transduction pathways . Genome Res, 1999 Aug, 9(8), 681 - 8 Large-scale gene expression data analysis: a new challenge to computational biologists; Zhang MQ; The use of high-density DNA arrays to monitor gene expression at a genome-wide scale constitutes a fundamental advance in biology . In particular, the expression pattern of all genes in Saccharomyces cerevisiae can be interrogated using microarray analysis where cDNAs are hybridized to an array of each of the approximately 6000 genes in the yeast genome . In this survey I review three recent experiments related to transcriptional regulation and discuss the great challenge for computational biologists trying to extract functional information from such large-scale gene expression data. Neurochem Res, 1999 Jun, 24(6), 739 - 50 Heterogeneous long chain acyl-CoA synthetases control distribution of individual fatty acids in newly-formed glycerolipids of neuronal cells undergoing neurite outgrowth; Li J et al.; Using PC12 cells undergoing neurite outgrowth, we studied the activation of various fatty acids, of different chain lengths and degrees of saturation, by long chain acyl-CoA synthetases (LCASs) . Cells treated with nerve growth factor (NGF) were labeled with {3H}glycerol, {3H}oleic acid (OA) or {3H}arachidonic acid (AA) in the presence of other unlabeled fatty acids of endogenous or exogenous origin . Triacsin C (4.8 microM), an inhibitor of acyl-CoA synthetase, decreased the incorporation of exogenous {3H}OA into glycerolipids by 30-90%, and increased by about 60% the accumulation of free {3H}OA in the cells . However it did not affect the incorporation of endogenous fatty acids nor of exogenous {3H}AA into phospholipids, suggesting that LCASs which activate exogenous AA and at least some endogenous fatty acids are relatively insensitive to this drug . Activities of the LCAS that is specific for AA (ACS), or of the non-specific LCAS which activates OA and other fatty acids (OCS), were much higher in microsomal and cytoplasmic fractions than in mitochondria or nuclei . The Vmax and Km values of ACS and OCS in microsomes were 12 and 0.7 nmol/min/mg protein and 70 and 37 microM, respectively; and in cytoplasm, 6 and 0.6 nmol/min/mg protein and 38 and 60 microM, respectively . Triacsin C (2-33 microM) did not affect ACS activity in microsomal or cytoplasmal fractions, but inhibited OCS activities dose-dependently and competitively: IC50 and apparent Ki values were 13.5 microM and 14 microM in microsomes, and 3.8 microM and 4 microM in cytoplasm . NGF stimulated the activities of the LCASs, and, consistently, the incorporation of the various fatty acids into glycerolipids . These data indicate that LCASs are heterogeneous with respect to their intracellular locations, substrate specificities, kinetic characteristics and sensitivities to triacsin C; and that this heterogeneity affects the extents to which individual fatty acids are utilized to form glycerolipids. Eur Biophys J, 1999, 28(5), 369 - 79 Dynamical structure of transfer RNA studied by normal mode analysis; Matsumoto A et al.; The internal motion of yeast phenylalanine transfer RNA is studied by normal mode analysis in extended dihedral angle space in which the flexibility of five-membered ribose rings is treated faithfully by introducing a variable for its pseudo-rotational motion . Analysis of global molecular motion reveals that the molecule is very soft . We show that this softness comes not from the property of the "material" comprising the molecule but from its slender shape . Analysis of thermal distance fluctuations reveals that this molecule can be regarded as consisting dynamically of three blocks . Thermal fluctuations of the mainchain dihedral angles show rigidity of the anticodon region . They also show flexibility of regions around non-stacking bases . Base-stacking interactions cause suppression of the correlated functions of mainchain dihedral angles beyond a ribose ring . We analyze the thermal fluctuation of parameters describing the positions of two adjacent bases . Fluctuations of relative translational parameters in the anticodon and acceptor stem regions are found to be larger that those in other stem regions . The relative translational motions cause the two stem regions to undergo global twisting and bending motions . We show that the role of pseudo-rotational motion of sugars is important in regions around bases which are involved in nonregular interactions. Cancer Res, 1999 Aug 1, 59(15), 3689 - 97 Tethering a type IB topoisomerase to a DNA site by enzyme fusion to a heterologous site-selective DNA-binding protein domain; Beretta GL et al.; Topoisomerase IB (Top1) has essential functions in higher eukaryotes, but effective anticancer agents can transform it into a lethal DNA-cleaving toxin . Fusion of the yeast Gal4 DNA-binding protein domain (amino acids 1-105; Gal4DBD) to the NHz terminus of full-length human Top1 results in a GalTop chimera that maintains basic properties of the two parent proteins . DNA cleavage and binding activities of GalTop were then compared to Top1 to establish whether the fusion protein had altered site specificity . Under conditions of reduced binding of Top1 to DNA, Gal4DBD was able to selectively anchor the chimera on a template containing a Gal4 consensus motif, thus bringing Top1 to cleave 20-40-bp sequences close to the Gal4 motif with high specificity . Footprinting analyses showed that the chimera protected a DNA region that was wider than that protected by a Gal4DBD protein fragment, consistent with the cleavage results . The data demonstrate that a Top1 can be targeted to a specific DNA site by protein fusion to a heterologous DNA-binding domain . Such hybrid topoisomerase-derived enzymes may be useful for directing Top1 activity to specific genomic loci in living cells. Biochim Biophys Acta, 1999 Aug 17, 1433(1-2), 87 - 102 Engineering and biochemical characterization of the rat microsomal cytochrome P4501A1 fused to ferredoxin and ferredoxin-NADP(+) reductase from plant chloroplasts; Lacour T et al.; Fusion proteins of rat cytochrome P4501A1 with maize ferredoxin I (Fd) and pea ferredoxin NADP(+) reductase (FNR), the last electron transfer proteins of the photosynthetic channel in plant chloroplasts, were obtained by gene fusion in the yeast expression vector pAAH5N . The encoded fusion proteins P4501A1-Fd, P4501A1-FNR, P4501A1-Fd-FNR and P4501A1-FNR-Fd were produced in microsomes of the yeast Saccharomyces cerevisiae AH22 . Enzymatic assays were carried out in vitro with the isolated microsomes . P4501A1-Fd-FNR and P4501A1-FNR-Fd were found to catalyze P450-monooxygenase activities towards 7-ethoxycoumarin and the herbicide chlortoluron . P4501A1-Fd-FNR was the most efficient enzyme as measured in vitro in ferricyanide and cytochrome c reductions, as well as P450-monooxygenase assays . Apparent K(m) and k(cat) of P4501A1-Fd-FNR were 70 microM and 7800 min(-1) for NADPH, 13.2 microM and 51.1 min(-1) for 7-ethoxycoumarin, and 21.3 microM and 23 . 8 min(-1) for the herbicide chlortoluron, respectively . Fd in P4501A1-Fd-FNR fusion enzyme was found to be a limiting factor compared to P4501A1 fused to the yeast NADPH-cytochrome P450 reductase, an artificial enzyme described previously . The efficiency of electron transfer in the P4501A1 fusion proteins and a possible in vivo molecular coupling of Fd and FNR with microsomal cytochrome P4501A1 produced in plant chloroplasts are discussed. Mech Dev, 1999 Aug, 86(1-2), 183 - 91 Retroviral promoter-trap insertion into a novel mammalian septin gene expressed during mouse neuronal development; Xiong JW et al.; We have characterized a retroviral promoter-trap insertion into a novel mammalian septin gene, Sep3 . Its predicted amino acid sequence shares significant homology to that of Saccharomyces cerevisiae CDC3, CDC10, CDC11, CDC12, the Drosophila genes Pnut, Sep1, Sep2, and the mammalian genes BH5, CDC10, Nedd5, Diff6, and Sep2, which are implicated in cytokinesis and cell polarity . Sep3 encodes a protein of 465 amino acids, and contains an evolutionary conserved ATP/GTP-binding motif, two coiled-coil domains, and a highly hydrophobic domain at the C terminus . Alkaline phosphatase reporter gene expression in transgenic embryos was first detected at E8.5 in the neural fold, and high levels of expression continued throughout embryogenesis in the neural tube and brain . In addition, a low level of transient expression was detected in the somites, gut, and branchial arches of mouse embryos . Overall, reporter gene expression recapitulated Sep3 mRNA expression during mouse embryogenesis . In adults, Sep3 transcripts were only detected in the brain and testis . Zoo blot analysis revealed that Sep3-related sequences exist in several vertebrate species including zebrafish, frog, chicken, mouse and human . Consistent with the retroviral insertion into the 3' UTR of the Sep3 gene, no obvious phenotypes associated with the promoter trap were detected in transgenic embryos or adult mice . In summary, we report the first isolation of a novel full-length Sep3 cDNA and extensive characterization of its expression during mouse embryogenesis and in adult tissues. Nucleic Acids Res, 1999 Sep 1, 27(17), 3494 - 502 Mapping of protein-protein interactions within the DNA-dependent protein kinase complex; Gell D et al.; In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks . Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively . Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown . We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction . Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related . On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined . We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs . Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms . These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80. Nucleic Acids Res, 1999 Sep 1, 27(17), 3433 - 7 Electron microscopic analysis reveals that replication factor C is sequestered by single-stranded DNA; Keller RC et al.; Replication factor C (RF-C) is a eukaryotic heteropentameric protein required for DNA replication and repair processes by loading proliferating cell nuclear antigen (PCNA) onto DNA in an ATP-dependent manner . Prior to loading PCNA, RF-C binds to DNA . This binding is thought to be restricted to a specific DNA structure, namely to a primer/template junction . Using the electron microscope we have examined the affinity of human heteropentameric RF-C and the DNA-binding region within the large subunit of RF-C from Drosophila melanogaster (dRF-Cp140) to heteroduplex DNA . The electron microscopic data indicate that both human heteropentameric RF-C and the DNA-binding region within dRF-Cp140 are sequestered by single-stranded DNA . No preferential affinity for the 3' or 5' transition points from single- to double-stranded DNA was evident. Science, 1999 Aug 13, 285(5430), 1084 - 7 Control of the terminal step of intracellular membrane fusion by protein phosphatase 1; Peters C et al.; Intracellular membrane fusion is crucial for the biogenesis and maintenance of cellular compartments, for vesicular traffic between them, and for exo- and endocytosis . Parts of the molecular machinery underlying this process have been identified, but most of these components operate in mutual recognition of the membranes . Here it is shown that protein phosphatase 1 (PP1) is essential for bilayer mixing, the last step of membrane fusion . PP1 was also identified in a complex that contained calmodulin, the second known factor implicated in the regulation of bilayer mixing . The PP1-calmodulin complex was required at multiple sites of intracellular trafficking; hence, PP1 may be a general factor controlling membrane bilayer mixing. Mol Cell, 1999 Jul, 4(1), 123 - 8 A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme; Wittschieben BO et al.; The elongator complex is a major component of the RNA polymerase II (RNAPII) holoenzyme responsible for transcriptional elongation in yeast . Here we identify Elp3, the 60-kilodalton subunit of elongator/RNAPII holoenzyme, as a highly conserved histone acetyltransferase (HAT) capable of acetylating core histones in vitro . In vivo, ELP3 gene deletion confers typical elp phenotypes such as slow growth adaptation, slow gene activation, and temperature sensitivity . These results suggest a role for a novel, tightly RNAPII-associated HAT in transcription of DNA packaged in chromatin. Mol Cell, 1999 Jul, 4(1), 85 - 95 Crystal structure of the vesicular transport protein Sec17: implications for SNAP function in SNARE complex disassembly; Rice LM et al.; SNAP proteins play an essential role in membrane trafficking in eukaryotic cells . They activate and recycle SNARE proteins by serving as adaptors between SNAREs and the cytosolic chaperone NSF . We have determined the crystal structure of Sec17, the yeast homolog of alpha-SNAP, to 2.9 A resolution . Sec17 is composed of an N-terminal twisted sheet of alpha-helical hairpins and a C-terminal alpha-helical bundle . The N-terminal sheet has local similarity to the tetratricopeptide repeats from protein phosphatase 5 but has a different overall twist . Sec17 also shares structural features with HEAT and clathrin heavy chain repeats . Possible models of SNAP:SNARE binding suggest that SNAPs may function as lever arms, transmitting forces generated by conformational changes in NSF/Sec18 to drive disassembly of SNARE complexes. Mol Cell, 1999 Jul, 4(1), 63 - 73 A conserved motif present in a class of helix-loop-helix proteins activates transcription by direct recruitment of the SAGA complex; Massari ME et al.; The class I helix-loop-helix (HLH) proteins, which include E2A, HEB, and E2-2, have been shown to be required for lineage-specific gene expression during T and B lymphocyte development . Additionally, the E2A proteins function to regulate V(D)J recombination, possibly by allowing access of variable region segments to the recombination machinery . The mechanisms by which E2A regulates transcription and recombination, however, are largely unknown . Here, we identify a novel motif, LDFS, present in the vertebrate class I HLH proteins as well as in a yeast HLH protein that is essential for transactivation . We provide both genetic and biochemical evidence that the highly conserved LDFS motif stimulates transcription by direct recruitment of the SAGA histone acetyltransferase complex. Genes Dev, 1999 Aug 1, 13(15), 1994 - 2004 Uncoupling of the hnRNP Npl3p from mRNAs during the stress-induced block in mRNA export; Krebber H et al.; Npl3p, the major mRNA-binding protein of the yeast Saccharomyces cerevisiae shuttles between the nucleus and the cytoplasm . A single amino acid change in the carboxyl terminus of Npl3p (E409 --> K) renders the mutant protein largely cytoplasmic because of a delay in its import into the nucleus . This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p . Conversely, using this mutant, we show that Npl3p and mRNA export out of the nucleus is significantly slowed in cells bearing mutations in XPO1/CRM1, which encodes the export receptor for NES-containing proteins and in RAT7, which encodes an essential nucleoporin . Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus . The stress-induced export of Npl3p is independent of both the activity of Xpo1p and the continued selective export of heat-shock mRNAs that occurs following stress . UV-cross-linking experiments show that Npl3p is bound to mRNA under normal conditions, but is no longer RNA associated in stressed cells . Taken together, we suggest that the uncoupling of Npl3p and possibly other mRNA-binding proteins from mRNAs in the nucleus provides a general switch that regulates mRNA export . By this model, under normal conditions Npl3p is a major component of an export-competent RNP complex . However, under conditions of stress, Npl3p no longer associates with the export complex, rendering it export incompetent and thus nuclear. Genes Dev, 1999 Aug 1, 13(15), 1983 - 93 Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center; Siatecka M et al.; A U5 snRNP protein, hPrp8, interacts closely with the GU dinucleotide at the 5' splice site (5'SS), forming a specific UV-inducible cross-link . To test if this physical contact between the 5'SS and the carboxy-terminal region of Prp8 reflects a functional recognition of the 5'SS during spliceosome assembly, we mutagenized the corresponding region of yeast Prp8 and screened the resulting mutants for suppression of 5'SS mutations in vivo . All of the isolated prp8 alleles not only suppress 5'SS but also 3'SS mutations, affecting the second catalytic step . Suppression of the 5'SS mutations by prp8 alleles was also tested in the presence of U1-7U snRNA, a predicted suppressor of the U+2A mutation . As expected, U1-7U efficiently suppresses prespliceosome formation, and the first, but not the second, step of U+2A pre-mRNA splicing . Independently, Prp8 functionally interacts with both splice sites at the later stage of splicing, affecting the efficiency of the second catalytic step . The striking proximity of two of the prp8 suppressor mutations to the site of the 5'SS:hPrp8 cross-link suggests that some protein:5'SS contacts made before the first step may be subsequently extended to accommodate the 3'SS for the second catalytic step . Together, these results strongly implicate Prp8 in specific interactions at the catalytic center of the spliceosome. Genes Dev, 1999 Aug 1, 13(15), 1970 - 82 Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome; Collins CA et al.; The highly conserved spliceosomal protein Prp8 is known to cross-link the critical sequences at both the 5' (GU) and 3' (YAG) ends of the intron . We have identified prp8 mutants with the remarkable property of suppressing exon ligation defects due to mutations in position 2 of the 5' GU, and all positions of the 3' YAG . The prp8 mutants also suppress mutations in position A51 of the critical ACAGAG motif in U6 snRNA, which has been observed previously to cross-link position 2 of the 5' GU . Other mutations in the 5' splice site, branchpoint, and neighboring residues of the U6 ACAGAG motif are not suppressed . Notably, the suppressed residues are specifically conserved from yeast to man, and from U2- to U12-dependent spliceosomes . We propose that Prp8 participates in a previously unrecognized tertiary interaction between U6 snRNA and both the 5' and 3' ends of the intron . This model suggests a mechanism for positioning the 3' splice site for catalysis, and assigns a fundamental role for Prp8 in pre-mRNA splicing. Genes Dev, 1999 Aug 1, 13(15), 1936 - 49 Pds1 and Esp1 control both anaphase and mitotic exit in normal cells and after DNA damage; Tinker-Kulberg RL et al.; The separation of sister chromatids in anaphase is followed by spindle disassembly and cytokinesis . These events are governed by the anaphase-promoting complex (APC), which triggers the ubiquitin-dependent proteolysis of key regulatory proteins: anaphase requires the destruction of the anaphase inhibitor Pds1, whereas mitotic exit requires the destruction of mitotic cyclins and the inactivation of Cdk1 . We find that Pds1 is not only an inhibitor of anaphase, but also blocks cyclin destruction and mitotic exit by a mechanism independent of its effects on sister chromatid separation . Pds1 is also required for the mitotic arrest and inhibition of cyclin destruction that occurs after DNA damage . Even in anaphase cells, where Pds1 levels are normally low, DNA damage stabilizes Pds1 and prevents cyclin destruction and mitotic exit . Pds1 blocks cyclin destruction by inhibiting its binding partner Esp1 . Mutations in ESP1 delay cyclin destruction; overexpression of ESP1 causes premature cyclin destruction in cells arrested in metaphase by spindle defects and in cells arrested in metaphase and anaphase by DNA damage . The effects of Esp1 are dependent on Cdc20 (an activating subunit of the APC) and on several additional proteins (Cdc5, Cdc14, Cdc15, Tem1) that form a regulatory network governing mitotic exit . We speculate that the inhibition of cyclin destruction by Pds1 may contribute to the ordering of late mitotic events by ensuring that mitotic exit is delayed until after anaphase is initiated . In addition, the stabilization of Pds1 after DNA damage provides a mechanism to delay both anaphase and mitotic exit while DNA repair occurs. Br J Haematol, 1999 Jul, 106(1), 55 - 63 A monoclonal antibody, 3G12, reacts with a novel surface molecule, Hal-1, with high expression in CD30-positive anaplastic large cell lymphomas; Asanuma H et al.; We established a monoclonal antibody, 3G12 (IgG1), with antiproliferative effects on a human T-cell leukaemia cell line, SUP-T13 . Among haematolymphoid cell lines, 3G12 reacted with most T-cell lines, Epstein-Barr transformed B-cell lines, some myelomonocytic cell lines and, most strongly with an anaplastic large cell lymphoma (ALCL) cell line, Karpas 299 . The cell panel reactive with 3G12 was similar, but not identical, to that of the anti-CD30 antibody Ber-H2 . 3G12 induced Fas-independent apoptosis in SUP-T13 and it also induced growth-inhibition in a limited number of other cell lines, but not Karpas 299 . Immunohistochemical studies on paraffin-embedded tissue specimens demonstrated that 3G12 reacted with most CD30-positive ALCL cases and some T-cell lymphomas and some Hodgkin's lymphomas, but not with B-cell lymphomas or non-haematogeneic tumours . The immunoprecipitation study with 3G12 demonstrated a major band of 200 kD and a minor band of 100 kD, which were different from CD30 . Thus 3G12 defines a novel antigen that shares a similarity to CD30 in terms of distribution among haemopoietic cells . The data suggest that the 3G12-defined antigen, designated Hal-1, is important as a marker for ALCL and may play a role in its pathogenesis. Plant Physiol, 1999 Aug, 120(4), 1049 - 56 Functional characterization and expression analysis of the amino acid permease RcAAP3 from castor bean; Neelam A et al.; A polymerase chain reaction-based library screening procedure was used to isolate RcAAP3, an amino acid permease cDNA from castor bean (Ricinus communis) . RcAAP3 is 1.7 kb in length, with an open reading frame that encodes a protein with a calculated molecular mass of 51 kD . Hydropathy analysis indicates that the RcAAP3 protein is highly hydrophobic in nature with nine to 11 putative transmembrane domains . RcAAP3-mediated uptake of citrulline in a yeast transport mutant showed saturable kinetics with a K(m) of 0.4 mM . Transport was higher at acidic pH and was inhibited by the protonophore carbonylcyanide-m-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism . Citrulline uptake was strongly inhibited (72%) by the permeable sulfydryl reagent N-ethylmaleimide, but showed lower sensitivity (30% inhibition) to the nonpermeable reagent p-chloromercuribenzenesulfonic acid . Diethylpyrocarbonate, a histidine modifier, inhibited citrulline uptake by 80% . A range of amino acids inhibited citrulline uptake, suggesting that RcAAP3 may be a broad substrate permease that can transport neutral and basic amino acids with a lower affinity for acidic amino acids . Northern analysis indicated that RcAAP3 is widely expressed in source and sink tissues of castor bean, and that the pattern of expression is distinct from RcAAP1 and RcAAP2. Acta Histochem, 1999 Jul, 101(3), 271 - 9 Analysis of cell surface properties using derivatized agarose beads; Salbilla BA et al.; An assay has been developed to analyse cell surface properties using agarose beads derivatized with amino acids, sugars, proteins, and other molecules . The assay is simple and rapid and is useful to identify new cell surface markers . Various species and strains of yeast, paramecium, and Euglena were tested for their ability to bind to over 100 types of derivatized beads . A variety of specificity studies were performed in order to understand the nature of cell-bead binding . Our results indicate that cell-bead binding is often specific enough to distinguish between configurational isomers and spacer sizes and can be blocked by addition of specific molecules to the incubation medium . In some cases, different species or strains differed only by their binding to a single bead type . This simple and rapid assay may help to uncover new cell surface receptors and may lead to the development of clinically useful compounds for therapeutic applications. Biochem Biophys Res Commun, 1999 Aug 11, 261(3), 802 - 7 Defining functional domains of Ku80: DNA end binding and survival after radiation; Osipovich O et al.; The Ku heterodimeric protein (Ku80/Ku70) is an essential component of the double-strand break DNA repair pathway in mammalian cells . We have recently defined a central region within Ku80 that is required for heterodimerization with Ku70 . We now identified a core region within Ku80 (amino acids 210 to 531) that is necessary for binding of Ku to DNA ends . Interaction with Ku70 and DNA end binding are important for Ku80 function in vivo, since Ku80 mutants lacking DNA end binding activity were unable to restore radiation resistance in Ku80 deficient fibroblast cell lines . However, Ku80 mutants were identified that retained DNA end binding activity but were unable to restore radiation survival, thus pointing to additional functional properties of Ku80 . An N-terminal deletional mutant of Ku80 was able to suppress wild type Ku80 function for radiation survival in several cell lines, thus demonstrating dominant negative function . Biochemistry, 1999 Aug 10, 38(32), 10474 - 9 Analysis of the substrate specificity of human sulfotransferases SULT1A1 and SULT1A3: site-directed mutagenesis and kinetic studies; Brix LA et al.; Sulfonation is an important metabolic process involved in the excretion and in some cases activation of various endogenous compounds and xenobiotics . This reaction is catalyzed by a family of enzymes named sulfotransferases . The cytosolic human sulfotransferases SULT1A1 and SULT1A3 have overlapping yet distinct substrate specificities . SULT1A1 favors simple phenolic substrates such as p-nitrophenol, whereas SULT1A3 prefers monoamine substrates such as dopamine . In this study we have used a variety of phenolic substrates to functionally characterize the role of the amino acid at position 146 in SULT1A1 and SULT1A3 . First, the mutation A146E in SULT1A1 yielded a SULT1A3-like protein with respect to the Michaelis constant for simple phenols . The mutation E146A in SULT1A3 resulted in a SULT1A1-like protein with respect to the Michaelis constant for both simple phenols and monoamine compounds . When comparing the specificity of SULT1A3 toward tyramine with that for p-ethylphenol (which differs from tyramine in having no amine group on the carbon side chain), we saw a 200-fold preference for tyramine . The kinetic data obtained with the E146A mutant of SULT1A3 for these two substrates clearly showed that this protein preferred substrates without an amine group attached . Second, changing the glutamic acid at position 146 of SULT1A3 to a glutamine, thereby neutralizing the negative charge at this position, resulted in a 360-fold decrease in the specificity constant for dopamine . The results provide strong evidence that residue 146 is crucial in determining the substrate specificity of both SULT1A1 and SULT1A3 and suggest that there is a direct interaction between glutamic acid 146 in SULT1A3 and monoamine substrates. J Virol, 1999 Sep, 73(9), 7262 - 70 RhoA interacts with the fusion glycoprotein of respiratory syncytial virus and facilitates virus-induced syncytium formation; Pastey MK et al.; The fusion glycoprotein (F) of respiratory syncytial virus (RSV), which mediates membrane fusion and virus entry, was shown to bind RhoA, a small GTPase, in yeast two-hybrid interaction studies . The interaction was confirmed in vivo by mammalian two-hybrid assay and in RSV-infected HEp-2 cells by coimmunoprecipitation . Furthermore, the interaction of F with RhoA was confirmed in vitro by enzyme-linked immunosorbent assay and biomolecular interaction analysis . Yeast two-hybrid interaction studies with various deletion mutants of F and with RhoA indicate that the key binding domains of these proteins are contained within, or overlap, amino acids 146 to 155 and 67 to 110, respectively . The biological significance of this interaction was studied in RSV-infected HEp-2 cells that were stably transfected to overexpress RhoA . There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that RhoA can facilitate RSV-induced syncytium formation. Semin Cell Dev Biol, 1999 Apr, 10(2), 215 - 25 Retinoid receptors in health and disease: co-regulators and the chromatin connection; Minucci S et al.; Retinoid receptors behave as ligand-dependent transcriptional regulators, repressing transcription in the absence of ligand and activating transcription in its presence . The different effects on transcription are carried out through recruitment of co-regulators: unliganded receptors bind corepressors (NCoR and SMRT) that are found within a complex containing histone deacetylase (HDAC) activity, whereas liganded receptors recruit coactivators with histone acetylase activity (HATs) . Chromatin remodeling activities have also shown to be required, suggesting a hierarchy of promoter structure modifications in RA target genes carried out by multiple coregulatory complexes . In this review, we examine the experimental evidence for the model just sketched . We focus on recent findings highlighting new molecular details in receptor-coregulator interactions, including the discovery and initial characterisation of novel complexes with multiple chromatin modifying activities . Finally, we look at the role of aberrant recruitment of the NCoRHDAC complex by altered retinoid receptors in the pathogenesis of acute promyelocytic leukemia . These results point to a crucial role for control of transcription factor coregulator interactions in the regulation of cellular processes, and suggest new molecular targets for cancer therapy . Semin Cell Dev Biol, 1999 Apr, 10(2), 205 - 14 MAP kinase-mediated signalling to nucleosomes and immediate-early gene induction; Thomson S et al.; Extracellular signals are transduced into the nucleus through a variety of signalling mechanisms to elicit changes in patterns of gene expression . This review is focused in the MAP kinase cascades and the part they play in the induction of the immediate-early (IE) genes . We discuss the MAP kinases and their downstream effectors that are known to phosphorylate substrates in the nucleus . In addition to phosphorylating specific transcription factors, MAP kinases and their downstream kinases are implicated in eliciting rapidly targeted alterations in the chromatin environment of specific genes by modulating the phosphorylation and/or acetylation of nucleosomal and chromatin proteins . Semin Cell Dev Biol, 1999 Apr, 10(2), 197 - 203 Histone acetylation in signal transduction by growth regulatory signals; Magnaghi-Jaulin L et al.; Cell fate is determined by extracellular signals which are transmitted to the nucleus and result in the transcriptional regulation of specific subsets of genes . Transcriptional regulation has been recently linked to enzymatic activities which are able to acetylate or deacetylate core histone tails . A number of transcriptional co-regulators are histone acetyl-transferases or histone deacetylases . Here, we discuss the involvement of these enzymes in critical steps of cell proliferation or cell differentiation control Semin Cell Dev Biol, 1999 Apr, 10(2), 189 - 95 The mammalian SWI/SNF complex and the control of cell growth; Muchardt C et al.; The mammalian SWI/SNF complex is a chromatin remodelling complex that uses the energy of ATP hydrolysis to facilitate access of transcription factors to regulatory DNA sequences . This complex, that was initially described as a co-factor for nuclear receptors, has recently been associated with the control of cell growth . Two of the subunits known as BRG-1 and brm can associate with the Retinoblastoma tumour suppressor gene product and co-operate with this protein for repression of E2F activity . In addition, expression of brm is frequently down-regulated upon cellular transformation and re-introduction of this protein into fibroblasts transformed by activated ras induces partial reversion of the transformed phenotype . Finally, the hSNF5/INI1 gene, encoding another subunit of the SWI/SNF complex, is subject to bi-allelic mutations in rhabdoid tumours, a very aggressive form of paediatric cancers . These observations provide a novel link between malignant transformation and chromatin remodelling machineries . Semin Cell Dev Biol, 1999 Apr, 10(2), 169 - 77 Histone acetyltransferase complexes; Grant PA et al.; Modification of histone amino terminal tails by acetylation has long been linked to the transcriptional capacity of genes in chromatin and to various aspects of chromatin dynamics . Over the last few years a flurry of reports have described the purification and identification of a large number of histone acetyltransferases . Many of these enzymes had previously been described as transcriptional regulators and have frequently been isolated as part of larger multisubunit protein complexes . This review describes the association of acetyltransferases with partner proteins and the additional functional attributes of such complexes beyond catalytic function . Semin Cell Dev Biol, 1999 Apr, 10(2), 131 - 7 A molecular basis for auxin action; Leyser O et al.; The plant hormone auxin is central in the regulation of growth and development, however, the molecular basis for its action has remained enigmatic . In the absence of a molecular model, the wide range of responses elicited by auxin have been difficult to explain . Recent advances using molecular genetic approaches in Arabidopsis have led to the isolation of a number of key genes involved in auxin action . Of particular importance are genes involved in channelling polar auxin transport through the plant . In addition a model for auxin signal transduction, centred on regulated protein degradation, has been developed . Bioessays, 1999 Aug, 21(8), 685 - 96 Should we kill the messenger? The role of the surveillance complex in translation termination and mRNA turnover; Czaplinski K et al.; Eukaryotes have evolved conserved mechanisms to rid cells of faulty gene products that can interfere with cell function . mRNA surveillance is an example of a pathway that monitors the translation termination process and promotes degradation of transcripts harboring premature translation termination codons . Studies on the mechanism of mRNA surveillance in yeast and humans suggest a common mechanism where a "surveillance complex" monitors the translation process and determines whether translation termination has occurred at the correct position within the mRNA . A model will be presented that suggests that the surveillance complex assesses translation termination by monitoring the transition of an RNP as it is converted from a nuclear to a cytoplasmic form during the initial rounds of translation . Dev Genet, 1999, 25(2), 168 - 79 Enhancer of split {E(spl)(D)} is a gro-independent, hypermorphic mutation in Drosophila; Nagel AC et al.; Enhancer of split {E(spl)} refers to a gene complex in Drosophila melanogaster, which contains a number of target genes of the Notch signaling pathway . The complex was originally identified by a dominant mutation E(spl)(D) that displays allele-specific interactions with a recessive mutation in the Notch locus called split (N(spl)) . The spl phenotype is characterized by smaller eyes with irregularly spaced ommatidia, and it is strongly enhanced by E(spl)(D) . This enhancement is correlated with a truncation of one of the E(spl) bHLH genes, m8, causing an increased stability of the mutant transcripts and an altered C-terminus in the mutant M8* protein . Concurrently, an insertion of a middle repetitive element in the adjacent groucho (gro) gene was observed . In this work, three different E(spl)(D) revertants (BE22, BE25, BX37), which have lost the ability to enhance N(spl) completely, were analyzed at the molecular level . In each case, the structure of the mutant M8* protein was affected, suggesting a specific involvement of the aberrant protein in the enhancement of the spl phenotype . This hypothesis is supported by the finding that a perfect phenocopy of spl enhancement can be achieved with hybrid constructs, where the altered C-terminus of M8* was fused to other E(spl) bHLH proteins . Thus, the ability to interact with N(spl) is not restricted to M8* but instead can be induced by an appropriate mutation in other E(spl) bHLH genes within the context of N(spl) . In a N(spl) background, E(spl)(D) behaves like a hyperactive M8 mutation . However, the mutant M8* protein has lost the ability of binding to the corepressor Gro, which is an essential feature for normal E(spl) activity . Yet, other protein interactions, notably those with other bHLH proteins of either E(spl) or proneural family, are still observed . These findings suggest that the structural changes associated with the E(spl)(D) mutant protein are the primary cause for the phenotypic interactions with the recessive Notch mutation N(spl) . Gene Expr, 1999, 7(4-6), 293 - 300 The cellular response to protein misfolding in the endoplasmic reticulum; Welihinda AA et al.; In eukaryotic cells, accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER) leads to a stress response . Cells respond to ER stress by upregulating the synthesis of ER resident protein chaperones, thus increasing the folding capacity in this organelle . In addition, this response also activates pathways to induce programmed cell death . The stress-induced chaperone synthesis is regulated at the level of transcription . In Saccharomyces cerevisiae, the transmembrane protein, Ire1p, with both serine/threonine kinase and site-specific endoribonuclease activities is implicated as the sensor of unfolded proteins in the ER that transmits the signal from the ER to activate transcription in the nucleus . Activation of the unfolded protein response (UPR) pathway also requires the bZIP transcription factor, Haclp . Although HACl is transcribed constitutively, the mRNA is poorly translated . Upon accumulation of unfolded proteins, Ire1p generates a new processed form of HACl mRNA that is efficiently translated by removal of a 252 base sequence . Using the yeast-interaction trap system we identified additional components of the UPR . A yeast transcriptional coactivator complex, Gcn5p/Ada, which is composed of Gcn5p, Ada2p, Ada3p, and Ada5p, was identified that interacts with Ire1p and Hac1p . Deletion of GCN5, ADA2, and/or ADA3 reduces, and deletion of ADA5 completely abrogates, the transcriptional induction in response to misfolded protein in the ER . A protein phosphatase, Ptc2p, was also identified as a negative regulator of the UPR that directly interacts with and dephosphorylates activated Ire1p . Recently, two mammalian homologues of Ire1p, IRE1 and IRE2, were identified . hIre1p, is preferentially localized to the nuclear envelope and requires a functional nuclease activity to transmit the UPR . These results indicate that some features of the UPR are conserved from yeast to humans and may be composed of a multicomponent complex that is regulated by phosphorylation status and is associated with the nuclear envelope to regulate processes including transcriptional induction and mRNA processing . We propose that activation of Ire1p induces splicing of HACl mRNA as well as engages and targets the Gcn5/Ada/Hac1 protein complex to genes that are transcriptionally activated in response to unfolded protein in the ER . The transcriptional activation is facilitated by targeting the histone acetylase, Gcn5p in yeast, to promote histone acetylation at chromatin encoding ER stress-responsive genes . In addition, activation of Ire1p leads to increased lipid biosynthesis, thereby allowing ER expansion to accommodate increasing lumenal constituents . Under conditions of more severe stress, cells activate an Ire1p-dependent death pathway that is mediated through induction of GADD153/CHOP. Gene Expr, 1999, 7(4-6), 283 - 91 A role for RNA metabolism in inducing the heat shock response; Carlson T et al.; Yeast HSF is constitutively trimeric and DNA bound . Heat shock is thought to activate HSF by inducing a conformational change . We have developed an assay in which we can follow a conformational change of HSF that correlates with activity and thus appears to be the active conformation . This conformational change requires two HSF trimers bound cooperatively to DNA . The conformational change can be induced in whole cell extracts, and is thus amenable to biochemical analysis . We have purified a factor that triggers the conformational change . The factor is sensitive to dialysis, insensitive to NEM, and is not extractable by phenol . It is small, and apparently not a peptide . Mass spectroscopy identifies a novel guanine nucleotide that tracks with activity on columns . This novel nucleotide, purchased from Sigma, induces the conformational change (although this does not prove the identity of the activating factor unambiguously, because Sigma's preparation is contaminated with other compounds) . What is the source of this nucleotide in cells? Activity can be generated by treating extracts with ribonuclease; this implicates RNA degradation as a source of HSF-activating activity . The heat shock response is primarily responsible for monitoring the levels of protein chaperones; how can RNA degradation be involved? Synthetic lethal interactions link HSF activity to ribosome biogenesis, suggesting a possible model . Ribosomal proteins are produced in large quantities, and in excess of rRNA; unassembled r-proteins are rapidly degraded (t1/2 approximately 3 min) . Unassembled r-proteins aggregate readily . It is likely that unassembled r-proteins represent a major target of chaperones in vivo, and for proteasome-dependent degradation . Interference with rRNA processing (e.g., by heat shock) requires hsp70s to handle the aggregation-prone r-proteins, and proteasome proteins to help degrade the unassembled r-proteins before they aggregate . A nucleotide signal could be generated from the degradation products of the rRNA itself. J Mol Biol, 1999 Aug 6, 291(1), 59 - 70 The cloning and expression of Pfacs1, a Plasmodium falciparum fatty acyl coenzyme A synthetase-1 targeted to the host erythrocyte cytoplasm; Matesanz F et al.; Plasmodium is unable to carry out de novo fatty acid synthesis and has to obtain these compounds from their host for subsequent activation by thioesterification with coenzyme A . This activity is catalyzed by a fatty acyl-CoA synthetase enzyme (EC 6.2.1.3) . Here, we describe a novel gene from P . falciparum whose recombinant purified product from baculovirus-transfected insect cell line had the enzymatic activity of a long-chain fatty acyl-CoA synthetase . It was named pf acs1, since it belongs to a multi-member gene family as revealed by the sequence of several clones and a multi-band pattern in Southern blots . The sequence specifies a product of 820 amino acid residues . It was transcribed and expressed in infected erythrocytes having an apparent molecular mass of 100 kDa . Immuno-labeling of infected erythrocytes with a specific antibody against the carboxy-terminal part of the PfACS1 localized the product early after the erythrocyte invasion in vesicle-like structures budding off the parasitoforous membrane toward the red cell cytoplasm . Its unique carboxy- terminal structure of 70 extra amino acid residues, longer than any other reported acyl-CoA synthetase, is probably related to its localization in the cytoplasm of the host erythrocyte . The phylogenetic relationship among other AMP-forming enzymes, placed PfACS1 closer to Saccharomyces cerevisiae, sharing significant amino acid identities, especially in the conserved signature motif that modulates fatty acid substrate specificity and ATP/AMP-binding domains . Taking into account the importance of this enzymatic activity for the parasite, its extra-cellular location inside the infected erythrocyte, and the divergence with respect to the homologous human enzymes, it may be an important protein as a potential target candidate for chemotherapeutic antimalaria drugs . J Biol Chem, 1999 Aug 13, 274(33), 23229 - 34 Quantitative characterization of furin specificity . Energetics of substrate discrimination using an internally consistent set of hexapeptidyl methylcoumarinamides; Krysan DJ et al.; Furin, an essential mammalian proprotein processing enzyme of the kexin/furin family of subtilisin-related eukaryotic processing proteases, is implicated in maturation of substrates involved in development, signaling, coagulation, and pathogenesis . We examined the energetics of furin specificity using a series of peptidyl methylcoumarinamide substrates . In contrast to previous reports, we found that furin can cleave such substrates with kinetics comparable to those observed with extended peptides and physiological substrates . With the best of these hexapeptidyl methylcoumarinamides, furin displayed k(cat)/K(m) values greater than 10(6) M(-1) s(-1) . Furin exhibited striking substrate inhibition with hexapeptide but not tetrapeptide substrates, an observation of significance to the evaluation of peptide-based furin inhibitors . Quantitative comparison of furin and Kex2 recognition at P(1), P(2), and P(4) demonstrates that whereas interactions at P(1) make comparable contributions to catalysis by the two enzymes, furin exhibited a approximately 10-fold lesser dependence on P(2) recognition but a 10-100-fold greater dependence on P(4) recognition . Furin has recently been shown to exhibit P(6) recognition and we found that this interaction contributes approximately 1.4 kcal/mol toward catalysis independent of the nature of the P(4) residue . We have also shown that favorable residues at P(2) and P(6) will compensate for less than optimal residues at either P(1) or P(4) . The quantitative analysis of furin and Kex2 specificity sharply distinguish the nature of substrate recognition by the processing and degradative members of subtilisin-related proteases. Solid State Nucl Magn Reson, 1999 Jul, 14(2), 117 - 36 Long-distance rotational echo double resonance measurements for the determination of secondary structure and conformational heterogeneity in peptides; Arshava B et al.; The utility of rotational echo double resonance (REDOR) NMR spectroscopy for determining the conformations of linear peptides has been examined critically using a series of crystalline and amorphous samples . The focus of the present work was the evaluation of long-distance (> 5 A) interactions using 13C-15N dephasing . Detailed studies of specifically labeled melanostatin and synthetic analogs of the alpha-factor yeast mating hormone show that nitrogen-dephased, carbon-observe REDOR measurements are reliable for distances up to 6.0 A, and that dipolar interactions can be detected for distances up to 7 A . By contrast, nitrogen-observe REDOR gives reliable results only for distances shorter than 5.0 A . To measure distances accurately, REDOR data must be corrected for the effects of natural-abundance spins . These corrections are particularly important for measuring long distances, which are of the greatest value for determining peptide secondary structure . We have developed a spherical shell model for calculating the effect of these background spins . The REDOR studies also indicate that in a lyophilized powder, the tridecapeptide alpha-factor mating pheromone from Saccharomyces cerevisiae (WHWLQLKPGQPMY) probably exists as a distribution of different turn structures around the KPGQ region . This finding revises previous solid-state NMR studies on this peptide, which concluded alpha-factor assumes a distorted type-I beta-turn in the Pro-Gly central region of the molecule {J.R . Garbow, M . Breslav, O . Antohi, F . Naider, Biochemistry, 33 (1994) 10094}. Science, 1999 Aug 6, 285(5429), 882 - 6 Molecular identification of a eukaryotic, stretch-activated nonselective cation channel; Kanzaki M et al.; Calcium-permeable, stretch-activated nonselective cation (SA Cat) channels mediate cellular responses to mechanical stimuli . However, genes encoding such channels have not been identified in eukaryotes . The yeast MID1 gene product (Mid1) is required for calcium influx in the yeast Saccharomyces cerevisiae . Functional expression of Mid1 in Chinese hamster ovary cells conferred sensitivity to mechanical stress that resulted in increases in both calcium conductance and the concentration of cytosolic free calcium . These increases were dependent on the presence of extracellular calcium and were reduced by gadolinium, a blocker of SA Cat channels . Single-channel analyses with cell-attached patches revealed that Mid1 acts as a calcium-permeable, cation-selective stretch-activated channel with a conductance of 32 picosiemens at 150 millimolar cesium chloride in the pipette . Thus, Mid1 appears to be a eukaryotic, SA Cat channel. Mol Biol Cell, 1999 Aug, 10(8), 2519 - 30 Probing the molecular environment of membrane proteins in vivo; Wittke S et al.; The split-Ubiquitin (split-Ub) technique was used to map the molecular environment of a membrane protein in vivo . Cub, the C-terminal half of Ub, was attached to Sec63p, and Nub, the N-terminal half of Ub, was attached to a selection of differently localized proteins of the yeast Saccharomyces cerevisiae . The efficiency of the Nub and Cub reassembly to the quasi-native Ub reflects the proximity between Sec63-Cub and the Nub-labeled proteins . By using a modified Ura3p as the reporter that is released from Cub, the local concentration between Sec63-Cub-RUra3p and the different Nub-constructs could be translated into the growth rate of yeast cells on media lacking uracil . We show that Sec63p interacts with Sec62p and Sec61p in vivo . Ssh1p is more distant to Sec63p than its close sequence homologue Sec61p . Employing Nub- and Cub-labeled versions of Ste14p, an enzyme of the protein isoprenylation pathway, we conclude that Ste14p is a membrane protein of the ER . Using Sec63p as a reference, a gradient of local concentrations of different t- and v-SNARES could be visualized in the living cell . The RUra3p reporter should further allow the selection of new binding partners of Sec63p and the selection of molecules or cellular conditions that interfere with the binding between Sec63p and one of its known partners. J Gen Physiol, 1999 Aug, 114(2), 203 - 13 Sulfonylurea and K(+)-channel opener sensitivity of K(ATP) channels . Functional coupling of Kir6.2 and SUR1 subunits; Koster JC et al.; The sensitivity of K(ATP) channels to high-affinity block by sulfonylureas and to stimulation by K(+) channel openers and MgADP (PCOs) is conferred by the regulatory sulfonylurea receptor (SUR) subunit, whereas ATP inhibits the channel through interaction with the inward rectifier (Kir6.2) subunit . Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) profoundly antagonized ATP inhibition of K(ATP) channels expressed from cloned Kir6.2+SUR1 subunits, but also abolished high affinity tolbutamide sensitivity . By stabilizing the open state of the channel, PIP(2) drives the channel away from closed state(s) that are preferentially affected by high affinity tolbutamide binding, thereby producing an apparent loss of high affinity tolbutamide inhibition . Mutant K(ATP) channels (Kir6 . 2{DeltaN30} or Kir6.2{L164A}, coexpressed with SUR1) also displayed an "uncoupled" phenotype with no high affinity tolbutamide block and with intrinsically higher open state stability . Conversely, Kir6 . 2{R176A}+SUR1 channels, which have an intrinsically lower open state stability, displayed a greater high affinity fraction of tolbutamide block . In addition to antagonizing high-affinity block by tolbutamide, PIP(2) also altered the stimulatory action of the PCOs, diazoxide and MgADP . With time after PIP(2) application, PCO stimulation first increased, and then subsequently decreased, probably reflecting a common pathway for activation of the channel by stimulatory PCOs and PIP(2) . The net effect of increasing open state stability, either by PIP(2) or mutagenesis, is an apparent "uncoupling" of the Kir6.2 subunit from the regulatory input of SUR1, an action that can be partially reversed by screening negative charges on the membrane with poly-L-lysine. Oncogene, 1999 Jul 22, 18(29), 4182 - 90 The zinc finger protein A20 interacts with a novel anti-apoptotic protein which is cleaved by specific caspases; De Valck D et al.; A20 is a Cys2/Cys2 zinc finger protein which is induced by a variety of inflammatory stimuli and which has been characterized as an inhibitor of cell death by a yet unknown mechanism . In order to clarify its molecular mechanism of action, we used the yeast two-hybrid system to screen for proteins that interact with A20 . A cDNA fragment was isolated which encoded a portion of a novel protein (TXBP151), which was recently found to be a human T-cell leukemia virus type-I (HTLV-I) Tax-binding protein . The full-length 2386 bp TXBP151 mRNA encodes a protein of 86 kDa . Like A20, overexpression of TXBP151 could inhibit apoptosis induced by tumour necrosis factor (TNF) in NIH3T3 cells . Moreover, transfection of antisense TXBP151 partially abolished the anti-apoptotic effect of A20 . Furthermore, apoptosis induced by TNF or CD95 (Fas/APO-1) was associated with proteolysis of TXBP151 . This degradation could be inhibited by the broad-spectrum caspase inhibitor zVAD-fmk or by expression of the cowpox virus-derived inhibitor CrmA, suggesting that TXBP151 is a novel substrate for caspase family members . TXBP151 was indeed found to be specifically cleaved in vitro by members of the caspase-3-like subfamily, viz . caspase-3, caspase-6 and caspase-7 . Thus TXBP151 appears to be a novel A20-binding protein which might mediate the anti-apoptotic activity of A20, and which can be processed by specific caspases. Oncogene, 1999 Jul 8, 18(27), 3970 - 8 p53 compound heterozygosity in a severely affected child with Li-Fraumeni syndrome; Quesnel S et al.; The Li-Fraumeni Syndrome (LFS) is a rare, dominantly inherited syndrome that features high risk of cancers in childhood and early adulthood . Affected families tend to develop bone and soft tissue sarcomas, breast cancers, brain tumors, leukemias, and adrenocortical carcinomas . In some kindreds, the genetic abnormality associated with this cancer phenotype is a heterozygous germline mutation in the p53 tumor suppressor gene . Recently, we identified one patient who presented in early childhood with multiple primary cancers and who harbored three germline p53 alterations (R156H and R267Q on the maternal allele and R290H on the paternal allele) . To classify the biologic effects of these alterations, functional properties of each of the p53 mutants were examined using in vitro assays of cellular growth suppression and transcriptional activation . Each amino acid substitution conferred partial or complete loss of wild-type p53 function, but the child completed normal embryonic development . This observation has not been previously reported in a human, but is consistent with observations of normal embryogenesis in p53-deficient mice. J Natl Cancer Inst, 1999 Aug 4, 91(15), 1288 - 94 Chromatin remodeling and transcriptional regulation; Luo RX et al.; Extensive studies in the past few years have begun to demonstrate that chromosome structure plays a critical role in transcriptional regulation . Two highly conserved mechanisms for altering chromosome structure have been identified: 1) post-translational modification of histones and 2) adenosine triphosphate (ATP)-dependent chromosome remodeling . Acetylation of histone lysine residues has been known for three decades to be associated with transcriptional activation . Recent discoveries, however, show that a number of transcriptional regulators are histone acetylases or histone deacetylases . Specific DNA-binding transcription factors recruit histone acetylases and deacetylases to promoters to activate or repress transcription . These results strongly support the notion that histone acetylation and deacetylation play an important role in transcriptional regulation . Recent findings have also provided insight into the molecular mechanisms by which ATP-dependent chromosome-remodeling activities participate in transcriptional regulation . Furthermore, some ATP-dependent chromosome-remodeling activities have been shown to complex with histone deacetylases . In the complexes studied to date, the ATP-dependent chromosome-remodeling activity enhances the histone deacetylase activity . Therefore, the two mechanisms appear to work in concert to achieve precise control of transcription . Disruption of chromosome remodeling has been linked to a number of diseases, and a complete understanding of the complex chromosome-remodeling machinery may lead to the development of new therapies. DNA Cell Biol, 1999 Jul, 18(7), 593 - 603 Cloning, mapping, and expression of ial, a novel Drosophila member of the Ipl1/aurora mitotic control kinase family; Reich A et al.; The members of the Ipl1-aurora like kinase (IARK) subfamily are conserved serine/threonine kinases that play a key role in the control of chromosome segregation, centrosome separation, and cytokinesis from yeast to mammals . We report on the isolation of a new Drosophila member of the family, designated Ipl1-aurora-like kinase (ial) Phylogenetic analysis of kinase domains established that ial is more divergent from known mammalian IARKs than is aurora . Mapping based on examination of chromosomal aberrations, together with mapping within contigs identified by the Drosophila Genome Project, placed the gene at 32B on the left arm of the second chromosome . Discrete single-gene mutations in this region, including all known relevant P-element disruptions, were examined and proven not to be mutations in ial . Characterization of spatial and temporal expression of ial and its gene product showed that it manifests itself in patterns which can be consistent with a role in cell cycle control. Biochem J, 1999 Aug 15, 342 ( Pt 1), 27 - 32 Production in vitro by the cytochrome P450 CYP94A1 of major C18 cutin monomers and potential messengers in plant-pathogen interactions: enantioselectivity studies; Pinot F et al.; The major C(18) cutin monomers are 18-hydroxy-9,10-epoxystearic and 9,10,18-trihydroxystearic acids . These compounds are also known messengers in plant-pathogen interactions . We have previously shown that their common precursor 9,10-epoxystearic acid was formed by the epoxidation of oleic acid in Vicia sativa microsomes (Pinot, Salaun, Bosch, Lesot, Mioskowski and Durst (1992) Biochem . Biophys . Res . Commun . 184, 183-193) . Here we determine the chirality of the epoxide produced as (9R,10S) and (9S,10R) in the ratio 90:10 respectively . We further show that microsomes from yeast expressing the cytochrome P450 CYP94A1 are capable of hydroxylating the methyl terminus of 9,10-epoxystearic and 9,10-dihydroxystearic acids in the presence of NADPH to form the corresponding 18-hydroxy derivatives . The reactions were not catalysed by microsomes from yeast transformed with a void plasmid or in absence of NADPH . After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid with microsomes of yeast expressing CYP94A1, the chirality of the residual epoxide was shifted to 66:34 in favour of the (9S,10R) enantiomer . Both enantiomers were incubated separately and V(max)/K(m) values of 16 and 3.42 ml/min per nmol of P450 for (9R, 10S) and (9S,10R) respectively were determined, demonstrating that CYP94A1 is enantioselective for the (9R,10S) enantiomer, which is preferentially formed in V . sativa microsomes . Compared with the epoxide, the diol 9,10-dihydroxystearic acid was a much poorer substrate for the omega-hydroxylase, with a measured V(max)/K(m) of 0.33 ml/min per nmol of P450 . Our results indicate that the activity of CYP94A1 is strongly influenced by the stereochemistry of the 9, 10-epoxide and the nature of substituents on carbons 9 and 10, with V(max)/K(m) values for epoxide>>oleic acid>diol. Am J Trop Med Hyg, 1999 Jul, 61(1), 131 - 40 Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples; Mookherjee S et al.; As resistance to chloroquine spreads in sub-Saharan Africa, pyrimethamine plus sulfadoxine (PSD) is increasingly used as a first-line treatment for falciparum malaria . Populations of Plasmodium falciparum (Pf) resistant to PSD have been selected quickly in other regions . The resistance is strongly correlated with point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), the two targets of the drug . It is critical to identify drug-resistant Pf-DHFR alleles that are present at a low frequency in these populations since alleles that confer drug resistance will be quickly selected by PSD use . It is difficult to identify these rare alleles by standard molecular techniques . We have designed a yeast expression system that facilitates the identification and rapid analysis of Pf-DHFR alleles that confer PSD resistance, even when they are present at very low frequency in polyclonal patient samples . We analyzed samples from patients in Kilifi, Kenya collected between 1992 and 1995 . We determined the prevalence of the drug-sensitive and drug-resistant alleles in patient samples analyzed in parallel by an allele-specific enzyme digestion (ASED) assay . We identified a pyrimethamine-resistant allele (S108N) present at a frequency of < 1% in a sample that was scored as only S108 by ASED . In addition, a novel pyrimethamine-resistant allele (1164M) was isolated twice, once each from two different patient samples . This approach will allow determination of the prevalence of Pf-DHFR alleles that confer pyrimethamine resistance in particular regions, and the rapid identification of novel alleles that confer drug resistance. FEBS Lett, 1999 Jul 9, 454(3), 257 - 61 A family of structurally related RING finger proteins interacts specifically with the ubiquitin-conjugating enzyme UbcM4; Martinez-Noel G et al.; The ubiquitin-conjugating enzyme UbcM4 was previously shown to be necessary for normal mouse development . As a first step in identifying target proteins or proteins involved in the specificity of UbcM4-mediated ubiquitylation, we have isolated seven cDNAs encoding proteins that specifically interact with UbcM4 but with none of the other Ubcs tested . This interaction was observed in yeast as well as in mammalian cells . With one exception, all UbcM4-interacting proteins (UIPs) belong to a family of proteins that contain a RING finger motif . As they are structurally related to RING finger proteins that have recently been shown to play an essential role in protein ubiquitylation and degradation, the possibility is discussed that UIPs are involved in the specific recognition of substrate proteins of UbcM4. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9130 - 5 Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication; Aparicio OM et al.; Chromosomes are replicated in characteristic, temporal patterns during S phase . We have compared the timing of association of replication proteins at early- and late-replicating origins of replication . Minichromosome maintenance proteins assemble simultaneously at early- and late-replicating origins . In contrast, Cdc45p association with late origins is delayed relative to early origins . DNA polymerase alpha association is similarly delayed at late origins and requires Cdc45p function . Activation of the S phase checkpoint inhibits association of Cdc45p with late-firing origins . These studies suggest that Cdc45p is poised to serve as a key regulatory target for both the temporal and checkpoint-mediated regulation of replication origins. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9124 - 9 Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism; Galan JM et al.; Ubiquitin-dependent degradation of regulatory proteins controls many cellular processes, including cell cycle progression, morphogenesis, and signal transduction . Skp1p-cullin-F-box protein (SCF) complexes are ubiquitin ligases composed of a core complex including Skp1p, Cdc53p, one of multiple F-box proteins that are thought to provide substrate specificity to the complex, and the ubiquitin-conjugating enzyme, Cdc34p . It is not understood how SCF complexes are regulated and how physiological conditions alter their levels . Here we show that three F-box proteins, Grr1p, Cdc4p, and Met30p, are unstable components of the SCF, and are themselves degraded in a ubiquitin- and proteasome-dependent manner in vivo . Ubiquitination requires all the core components of the SCF and an intact F-box, suggesting that ubiquitination occurs within the SCF complex by an autocatalytic mechanism . Cdc4p and Grr1p are intrinsically unstable, and their steady-state levels did not fluctuate through the cell cycle . Taken together, our results suggest that ubiquitin-dependent degradation of F-box proteins allows rapid switching among multiple SCF complexes, thereby enabling cells to adapt quickly to changing physiological conditions and progression through different phases of the cell cycle. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9083 - 8 MSE55, a Cdc42 effector protein, induces long cellular extensions in fibroblasts; Burbelo PD et al.; Cdc42 is a member of the Rho GTPase family that regulates multiple cellular activities, including actin polymerization, kinase-signaling activation, and cell polarization . MSE55 is a nonkinase CRIB (Cdc42/Rac interactive-binding) domain-containing molecule of unknown function . Using glutathione S-transferase-capture experiments, we show that MSE55 binds to Cdc42 in a GTP-dependent manner . MSE55 binding to Cdc42 required an intact CRIB domain, because a MSE55 CRIB domain mutant no longer interacted with Cdc42 . To study the function of MSE55 we transfected either wild-type MSE55 or a MSE55 CRIB mutant into mammalian cells . In Cos-7 cells, wild-type MSE55 localized at membrane ruffles and increased membrane actin polymerization, whereas expression of the MSE55 CRIB mutant showed fewer membrane ruffles . In contrast to these results, MSE55 induced the formation of long, actin-based protrusions in NIH 3T3 cells as detected by immunofluorescence and live-cell video microscopy . MSE55-induced protrusion formation was blocked by expression of dominant-negative N17Cdc42, but not by expression of dominant-negative N17Rac . These findings indicate that MSE55 is a Cdc42 effector protein that mediates actin cytoskeleton reorganization at the plasma membrane. Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9015 - 20 Genetic elimination of known pheromones reveals the fundamental chemical bases of mating and isolation in Drosophila; Savarit F et al.; Overexpression of the UAS-tra transgene in Drosophila melanogaster females led to the complete elimination of their cuticular pheromones . According to current models of Drosophila behavior, these flies should induce no courtship . In fact, they are still attractive to conspecific males . Three classes of stimuli are shown to induce courtship, with different effects on male behavior: (i) known pheromones produced by control females, (ii) stimuli produced by living control and transgenic flies, and (iii) as-yet-undetermined pheromones present on both control and transgenic flies . Only the latter class of pheromones are required for mating . They appear to represent a layer of ancestral attractive substances present in D . melanogaster and its sibling species; known cuticular pheromones modulate this attractivity positively or negatively . The absence of inhibitory pheromones leads to high levels of interspecific mating, suggesting an important role for these cuticular hydrocarbons in isolation between species. Eur J Cell Biol, 1999 Jun, 78(6), 375 - 81 Two distinct domains in hsc70 are essential for the interaction with the synaptic vesicle cysteine string protein; Stahl B et al.; The cysteine string protein (csp) is a synaptic vesicle protein found to be essential for normal neurotransmitter release . The precise function of csp in the synaptic vesicle cycle is still enigmatic . By interacting with the heat-shock cognate hsc70, a cochaperone-chaperone complex with an unknown function is formed . We report here that the formation of this complex is mediated by two distinct domains in hsc70 . The ATPase domain and the substrate-binding domain must cooperate to create a binding site for csp . The C-terminal domain of hsc70 seems to function as a regulator for the formation of the cochaperone-chaperone complex . We also show that the interaction of csp with heat-shock proteins is confined to hsc70 and hsp70 . Other heat-shock proteins, like hsp60 and hsp90, do not interact with csp.
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
