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