Mol Cell Biol, 2001 Jan, 21(2), 390 - 9 Copper-modulated gene expression and senescence in the filamentous fungus Podospora anserina; Borghouts C et al.; We have previously shown that the control of cellular copper homeostasis by the copper-modulated transcription factor GRISEA has an important impact on the phenotype and lifespan of Podospora anserina . Here we demonstrate that copper depletion leads to the induction of an alternative respiratory pathway and to an increase in lifespan . This response compensates mitochondrial dysfunctions via the expression of PaAox, a nuclear gene coding for an alternative oxidase . It resembles the retrograde response in Saccharomyces cerevisiae . In P . anserina, this pathway appears to be induced by specific impairments of the copper-dependent cytochrome c oxidase . It is not induced as the result of a general decline of mitochondrial functions during senescence . We cloned and characterized PaAox . Transcript levels are decreased when cellular copper, superoxide, and hydrogen peroxide levels are raised . Copper also controls transcript levels of PaSod2, the gene encoding the mitochondrial manganese superoxide dismutase (PaSOD2) . PaSod2 is a target of transcription factor GRISEA . During the senescence of wild-type strain s, the activity of PaSOD2 decreases, whereas the activity of the cytoplasmic copper/zinc superoxide dismutase (PaSOD1) increases . Collectively, the data explain the postponed senescence of mutant grisea as a defined consequence of copper depletion, ultimately leading to a reduction of oxidative stress . Moreover, they suggest that during senescence of the wild-type strain, copper is released from mitochondria . The involved mechanism is unknown . However, it is striking that the permeability of mitochondrial membranes in animal systems changes during apoptosis and that mitochondrial proteins with an important impact on this type of cellular death are released. J Virol, 2001 Jan, 75(2), 782 - 8 Characterization of the AdoMet-dependent guanylyltransferase activity that is associated with the N terminus of bamboo mosaic virus replicase; Li YI et al.; Bamboo mosaic virus (BaMV), a member of the potexvirus group, infects primarily members of the Bambusoideae . Open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that has long been postulated to be a replicase involved in the replication and formation of the cap structure at the 5' end of the viral genome . To identify and characterize the enzymatic activities associated with the N-terminal domain of the BaMV ORF1 protein, the intact replicase and two C-terminally truncated proteins were expressed in Saccharomyces cerevisiae . All three versions of BaMV ORF1 proteins could be radiolabeled by {alpha-(32)P}GTP, which is a characteristic of guanylyltransferase activity . The presence of S-adenosylmethionine (AdoMet) was essential for this enzymatic activity . Thin-layer chromatography analysis suggests that the radiolabeled moiety linked to the N-terminal domain of the BaMV ORF1 protein is m(7)GMP . The N-terminal domain also exhibited methyltransferase activity that catalyzes the transfer of the {(3)H}methyl group from AdoMet to GTP or guanylylimidodiphosphate . Therefore, during cap structure formation in BaMV, methylation of GTP may occur prior to transguanylation as for alphaviruses and brome mosaic virus . This study establishes the association of RNA capping activity with the N-terminal domain of the replicase of potexviruses and further supports the idea that the reaction sequence of RNA capping is conserved throughout the alphavirus-like superfamily of RNA viruses. J Virol, 2001 Jan, 75(2), 638 - 44 Ty1 proteolytic cleavage sites are required for transposition: all sites are not created equal; Merkulov GV et al.; The retroviral protease is a key enzyme in a viral multienzyme complex that initiates an ordered sequence of events leading to virus assembly and propagation . Viral peptides are initially synthesized as polyprotein precursors; these precursors undergo a number of proteolytic cleavages executed by the protease in a specific and presumably ordered manner . To determine the role of individual protease cleavage sites in Ty1, a retrotransposon from Saccharomyces cerevisiae, the cleavage sites were systematically mutagenized . Altering the cleavage sites of the yeast Ty1 retrotransposon produces mutants with distinct retrotransposition phenotypes . Blocking the Gag/PR site also blocks cleavage at the other two cleavage sites, PR/IN and IN/RT . In contrast, mutational block of the PR/IN or IN/RT sites does not prevent cleavage at the other two sites . Retrotransposons with mutations in each of these sites have transposition defects . Mutations in the PR/IN and IN/RT sites, but not in the Gag/PR site, can be complemented in trans by endogenous Ty1 copies . Hence, the digestion of the Gag/PR site and release of the protease N terminus is a prerequisite for processing at the remaining sites; cleavage of PR/IN is not required for the cleavage of IN/RT, and vice versa . Of the three cleavage sites in the Gag-Pol precursor, the Gag/PR site is processed first . Thus, Ty1 Gag-Pol processing proceeds by an ordered pathway. J Clin Endocrinol Metab, 2000 Dec, 85(12), 4912 - 5 The endocrine activities of 8-prenylnaringenin and related hop (Humulus lupulus L.) flavonoids; Milligan SR et al.; The female flowers of the hop plant have long been used as a preservative and a flavoring agent in beer, but they are now being included in some herbal preparations for women for "breast enhancement." This study investigated the relative estrogenic, androgenic and progestogenic activities of the known phytoestrogen, 8-prenylnaringenin, and structurally related hop flavonoids . 6-Prenylnaringenin, 6,8-diprenylnaringenin and 8-geranylnaringenin exhibited some estrogenicity, but their potency was less than 1% of that of 8-prenylnaringenin . 8-Prenylnaringenin alone competed strongly with 17ss-estradiol for binding to both the alpha- and ss-estrogen receptors . None of the compounds (xanthohumol, isoxanthohumol, 8-prenyl-naringenin, 6-prenylnaringenin, 3'-geranylchalconaringenin, 6-geranylnaringenin, 8-geranylnaringenin, 4'-O:-methyl-3'-prenylchalconaringenin and 6,8-diprenylnaringenin) nor polyphenolic hop extracts showed progestogenic or androgenic bioactivity . These results indicate that the endocrine properties of hops and hop products are due to the very high estrogenic activity of 8-prenylnaringenin and concern must be expressed about the unrestricted use of hops in herbal preparations for women. J Cell Biol, 2000 Dec 25, 151(7), 1501 - 12 Cell cycle programs of gene expression control morphogenetic protein localization; Lord M et al.; Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression . Almost without exception, the significance of cell cycle periodic gene expression has not been tested . Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process . The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated . BUD3 is expressed close to the onset of mitosis . BUD10 is expressed in late G1 . Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined . We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function . In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function . Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location . Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms. J Cell Biol, 2000 Dec 25, 151(7), 1381 - 90 p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks; Schultz LB et al.; p53 binding protein 1 (53BP1), a protein proposed to function as a transcriptional coactivator of the p53 tumor suppressor, has BRCT domains with high homology to the Saccharomyces cerevisiae Rad9p DNA damage checkpoint protein . To examine whether 53BP1 has a role in the cellular response to DNA damage, we probed its intracellular localization by immunofluorescence . In untreated primary cells and U2OS osteosarcoma cells, 53BP1 exhibited diffuse nuclear staining; whereas, within 5-15 min after exposure to ionizing radiation (IR), 53BP1 localized at discreet nuclear foci . We propose that these foci represent sites of processing of DNA double-strand breaks (DSBs), because they were induced by IR and chemicals that cause DSBs, but not by ultraviolet light; their peak number approximated the number of DSBs induced by IR and decreased over time with kinetics that parallel the rate of DNA repair; and they colocalized with IR-induced Mre11/NBS and gamma-H2AX foci, which have been previously shown to localize at sites of DSBs . Formation of 53BP1 foci after irradiation was not dependent on ataxia-telangiectasia mutated (ATM), Nijmegen breakage syndrome (NBS1), or wild-type p53 . Thus, the fast kinetics of 53BP1 focus formation after irradiation and the lack of dependency on ATM and NBS1 suggest that 53BP1 functions early in the cellular response to DNA DSBs. Carcinogenesis, 2000 Dec, 21(12), 2281 - 5 Candidate mutator genes in mismatch repair-deficient thymic lymphomas: no evidence of mutations in the DNA polymerase delta gene; Campbell MR et al.; DNA mismatch repair (MMR) proteins recognize nucleotides that are incorrectly paired . Deficiencies in MMR lead to increased genomic instability reflected in an increased mutation frequency and predisposition to tumorigenesis . Mice lacking the MMR gene, Msh2, develop thymic lymphomas that exhibit much higher mutational frequencies than other Msh2(-/-) tumours and Msh2(-/-) normal thymic tissue, suggesting that an additional mutator may have been acquired in a tissue-specific manner . Clustered mutations observed exclusively in the thymic lymphomas suggests that a gene(s) associated with the replication machinery might have become altered during tumorigenesis . Based on mutation studies in Saccharomyces cerevisiae lacking Msh2 and DNA polymerase delta (DNA pol delta), we hypothesized that the acquisition of mutations in DNA pol delta could contribute to the hypermutator phenotype and tumorigenesis in Msh2(-/-) thymic tissue . Furthermore, previous reports have suggested that genes containing mononucleotide repeats are non-random mutational targets in the absence of MMR . Therefore, we sequenced all 26 exons of the DNA pol delta catalytic subunit, including the six exons containing mononucleotide repeats of >5 bp, from nine Msh2(-/-) thymic lymphomas and two wild-type controls . No DNA pol delta pathogenic mutations were found in the thymic lymphomas, although several DNA base differences compared with published DNA pol delta sequences were observed . We conclude, therefore, that inactivating mutations in DNA pol delta are not a contributing factor in the development of the hypermutator phenotype in MMR-deficient murine thymic lymphomas. Proc Natl Acad Sci U S A, 2001 Jan 16, 98(2), 519 - 24 Epub 2001 Jan 02. Interaction of Galpha 12 and Galpha 13 with the cytoplasmic domain of cadherin provides a mechanism for beta -catenin release; Meigs TE et al.; The G12 subfamily of heterotrimeric G proteins, comprised of the alpha-subunits Galpha12 and Galpha13, has been implicated as a signaling component in cellular processes ranging from cytoskeletal changes to cell growth and oncogenesis . In an attempt to elucidate specific roles of this subfamily in cell regulation, we sought to identify molecular targets of Galpha12 . Here we show a specific interaction between the G12 subfamily and the cytoplasmic tails of several members of the cadherin family of cell-surface adhesion proteins . Galpha12 or Galpha13 binding causes dissociation of the transcriptional activator beta-catenin from cadherins . Furthermore, in cells lacking the adenomatous polyposis coli protein required for beta-catenin degradation, expression of mutationally activated Galpha12 or Galpha13 causes an increase in beta-catenin-mediated transcriptional activation . These findings provide a potential molecular mechanism for the previously reported cellular transforming ability of the G12 subfamily and reveal a link between heterotrimeric G proteins and cellular processes controlling growth and differentiation. J Biol Chem, 2001 Mar 23, 276(12), 8848 - 55 Epub 2000 Dec 20. Mapping the DNA topoisomerase III binding domain of the Sgs1 DNA helicase; Fricke WM et al.; Several members of the RecQ family of DNA helicases are known to interact with DNA topoisomerase III (Top3) . Here we show that the Saccharomyces cerevisiae Sgs1 and Top3 proteins physically interact in cell extracts and bind directly in vitro . Sgs1 and Top3 proteins coimmunoprecipitate from cell extracts under stringent conditions, indicating that Sgs1 and Top3 are present in a stable complex . The domain of Sgs1 which interacts with Top3 was identified by expressing Sgs1 truncations in yeast . The results indicate that the NH(2)-terminal 158 amino acids of Sgs1 are sufficient for the high affinity interaction between Sgs1 and Top3 . In vitro assays using purified Top3 and NH(2)-terminal Sgs1 fragments demonstrate that at least part of the interaction is through direct protein-protein interactions with these 158 amino acids . Consistent with these physical data, we find that mutant phenotypes caused by a point mutation or small deletions in the Sgs1 NH(2) terminus can be suppressed by Top3 overexpression . We conclude that Sgs1 and Top3 form a tight complex in vivo and that the first 158 amino acids of Sgs1 are necessary and sufficient for this interaction . Thus, a primary role of the Sgs1 amino terminus is to mediate the Top3 interaction. J Biol Chem, 2001 Mar 23, 276(12), 8778 - 84 Epub 2000 Dec 20. Analysis of the binding of p53 to DNAs containing mismatched and bulged bases; Degtyareva N et al.; The tumor suppressor protein p53 modulates cellular response to DNA damage by a variety of mechanisms that may include direct recognition of some forms of primary DNA damage . Linear 49-base pair duplex DNAs were constructed containing all possible single-base mismatches as well as a 3-cytosine bulge . Filter binding and gel retardation assays revealed that the affinity of p53 for a number of these lesions was equal to or greater than that of the human mismatch repair complex, hMSH2-hMSH6, under the same binding conditions . However, other mismatches including G/T, which is bound strongly by hMSH2-hMSH6, were poorly recognized by p53 . The general order of affinity of p53 was greatest for a 3-cytosine bulge followed by A/G and C/C mismatches, then C/T and G/T mismatches, and finally all the other mismatches. J Biol Chem, 2001 Mar 23, 276(12), 9297 - 302 Epub 2000 Dec 19. The cytosolic O-acetylserine(thiol)lyase gene is regulated by heavy metals and can function in cadmium tolerance; Dominguez-Solis JR et al.; Regulation of the expression of the cytosolic O-acetylserine(thiol)lyase gene (Atcys-3A) from Arabidopsis thaliana under heavy metal stress conditions has been investigated . Northern blot analysis of Atcys-3A expression shows a 7-fold induction after 18 h of cadmium treatment . Addition of 50 microm CdCl(2) to the irrigation medium of mature Arabidopsis plants induces a rapid accumulation of the mRNA throughout the leaf lamina, the root and stem cortex, and stem vascular tissues when compared with untreated plants, as observed by in situ hybridization . High pressure liquid chromatography analysis of GSH content shows a transient increase after 18 h of metal treatment . Our results are compatible with a high cysteine biosynthesis rate under heavy metal stress required for the synthesis of GSH and phytochelatins, which are involved in the plant detoxification mechanism . Arabidopsis-transformed plants overexpressing the Atcys-3A gene by up to 9-fold show increased tolerance to cadmium when grown in medium containing 250 microm CdCl(2), suggesting that increased cysteine availability is responsible for cadmium tolerance . In agreement with these results, exogenous addition of cystine can, to some extent, also favor the growth of wild-type plants in cadmium-containing medium . Cadmium accumulates to higher levels in leaves of tolerant transformed lines than in wild-type plants. J Biol Chem, 2001 Apr 27, 276(17), 13517 - 23 Epub 2000 Dec 15. Functional conservation of phosphorylation-specific prolyl isomerases in plants; Yao JL et al.; The phosphorylation-specific peptidyl prolyl cis/trans isomerase (PPIase) Pin1 in humans and its homologues in yeast and animal species play an important role in cell cycle regulation . These PPIases consist of an NH(2)-terminal WW domain that binds to specific phosphoserine- or phosphothreonine-proline motifs present in a subset of phosphoproteins and a COOH-terminal PPIase domain that specifically isomerizes the phosphorylated serine/threonine-proline peptide bonds . Here, we describe the isolation of MdPin1, a Pin1 homologue from the plant species apple (Malus domestica) and show that it has the same phosphorylation-specific substrate specificity and can be inhibited by juglone in vitro, as is the case for Pin1 . A search in the plant expressed sequence tag data bases reveals that the Pin1-type PPIases are present in various plants, and there are multiple genes in one organism, such as soybean (Glycine max) and tomato (Lycopersicon esculentum) . Furthermore, all these plant Pin1-type PPIases, including AtPin1 in Arabidopsis thaliana, do not have a WW domain, but all contain a four-amino acid insertion next to the phospho-specific recognition site of the active site . Interestingly, like Pin1, both MdPin1 and AtPin1 are able to rescue the lethal mitotic phenotype of a temperature-sensitive mutation in the Pin1 homologue ESS1/PTF1 gene in Saccharomyces cerevisiae . However, deleting the extra four amino acid residues abolished the ability of AtPin1 to rescue the yeast mutation under non-overexpression conditions, indicating that these extra amino acids may be important for mediating the substrate interaction of plant enzymes . Finally, expression of MdPin1 is tightly associated with cell division both during apple fruit development in vivo and during cell cultures in vitro . These results have demonstrated that phosphorylation-specific PPIases are highly conserved functionally in yeast, animal, and plant species . Furthermore, the experiments suggest that although plant Pin1-type enzymes do not have a WW domain, they may fulfill the same functions as Pin1 and its homologues do in other organisms. J Biol Chem, 2001 Mar 23, 276(12), 8665 - 73 Epub 2000 Dec 15. The STAR/GSG family protein rSLM-2 regulates the selection of alternative splice sites; Stoss O et al.; We identified the rat Sam68-like mammalian protein (rSLM-2), a member of the STAR (signal transduction and activation of RNA) protein family as a novel splicing regulatory protein . Using the yeast two-hybrid system, coimmunoprecipitations, and pull-down assays, we demonstrate that rSLM-2 interacts with various proteins involved in the regulation of alternative splicing, among them the serine/arginine-rich protein SRp30c, the splicing-associated factor YT521-B and the scaffold attachment factor B . rSLM-2 can influence the splicing pattern of the CD44v5, human transformer-2beta and tau minigenes in cotransfection experiments . This effect can be reversed by rSLM-2-interacting proteins . Employing rSLM-2 deletion variants, gel mobility shift assays, and linker scan mutations of the CD44 minigene, we show that the rSLM-2-dependent inclusion of exon v5 of the CD44 pre-mRNA is dependent on a short purine-rich sequence . Because the related protein of rSLM-2, Sam68, is believed to play a role as an adapter protein during signal transduction, we postulate that rSLM-2 is a link between signal transduction pathways and pre-mRNA processing. Arch Toxicol, 2000 Nov, 74(9), 560 - 6 Genotoxic effect of substituted phenoxyacetic acids; Venkov P et al.; The potential toxic and mutagenic action of 2,4-dichlorophenoxyacetic acid has been studied in different test systems, and the obtained results range from increased chromosomal damage to no effect at all . We reexamined the effect of this herbicide by simultaneous using three tests based on yeast, transformed hematopoietic, and mouse bone marrow cells . The results obtained demonstrated that 2,4-dichlorophenoxyacetic acid has cytotoxic and mutagenic effects . The positive response of yeast and transformed hematopoietic cells was verified in kinetics and dose-response experiments . The analysis of metaphase chromosomes indicated a statistically proved induction of breaks, deletions, and exchanges after the intraperitoneal administration of 2,4-dichlorophenoxyacetic acid in mice . The study of phenoxyacetic acid and its differently chlorinated derivatives showed that cytotoxicity and mutagenicity are induced by chlorine atoms at position 2 and/or 4 in the benzene ring . The mutagenic effect was abolished by introduction of a third chlorine atom at position 5 . Thus 2,4,5-trichlorophenoxyacetic acid was found to have very weak, if any mutagenic effect; however, the herbicide preserved its toxic effect. Hybridoma, 2000 Oct, 19(5), 375 - 85 Generation of monoclonal antibodies to cryptic collagen sites by using subtractive immunization; Xu J et al.; The extracellular matrix (ECM) plays a fundamental role in the regulation of normal and pathological processes . The most abundantly expressed component found in the ECM is collagen . Triple helical collagen is known to be highly resistant to proteolytic cleavage except by members of the matrix metalloproteinase (MMP) family of enzymes . To date little is known concerning the biochemical consequences of collagen metabolism on human diseases . This is due in part to the lack of specific reagents that can distinguish between proteolyzed and triple helical forms of collagen . Here we used the technique of Subtractive Immunization (SI) to generate two unique monoclonal antibodies (MAbs HUIV26 and HUI77) that react with denatured and proteolyzed forms of collagen, but show little if any reaction with triple helical collagen . Importantly, HUIV26 and HUI77 react with cryptic sites within the ECM of human melanoma tumors, demonstrating their utility for immunohistochemical analysis in vivo . Thus, the generation of these novel MAbs not only identify specific cryptic epitopes within triple helical collagen, but also provide important new reagents for studying the roles of collagen remodeling in normal as well as pathological processes. Oncogene, 2000 Nov 30, 19(51), 5872 - 83 The isoform-specific stretch of hSos1 defines a new Grb2-binding domain; Zarich N et al.; hSos1 isoform II, defined by the presence of a 15 amino acid stretch in its carboxy-terminal region, exhibits higher Grb2 affinity than hSos1 isoform I . In this study, we investigated the cause for this difference and observed that, in addition to the four currently accepted Grb2-binding motifs, a number of additional, putative SH3-minimal binding sites (SH3-MBS) could be identified . The isoform II-specific 15 amino acid stretch contained one of them . Indeed, we demonstrated by site-directed mutagenesis that these SH3-MBS were responsible for the Grb2 interaction, and we found that C-terminal fragments of the two hSos1 isoforms (lacking the four cannonical Grb2-binding motifs, but containing the SH3-minimal binding sites) were able to bind Grb2, with the isoform II fragment showing higher Grb2 affinity than the corresponding isoform I fragment . Furthermore, we provide evidence that C-terminal truncated mutants of either hSos1 isoform, containing only the SH3-minimal binding sites, were able to originate in vivo stable complexes with Grb2 . Although, Grb2-binding remains higher in both full-length isoforms, compared to the C-terminal truncated mutants, these mutants were also able to activate Ras, supporting a potential role of this C-terminal region as negative modulator of Sos1 activity . These findings document the existence of a new, functional, SH3-minimal binding site located in the specific stretch of hSos1 isoform II which may be responsible for the increased Grb2 affinity of this isoform in comparison to isoform I, and for the physiological properties differences between both isoforms . Moreover, these SH3-minimal binding sites may be sufficient to attain stable and functional hSosl-Grb2 complexes. Curr Genet, 2000 Nov, 38(4), 188 - 90 Mis-targeting of multiple gene disruption constructs containing hisG; Davidson JF et al.; Gene targeting by homologous recombination occurs in Saccharomyces cerevisiae efficiently when there are as few as 30 base pairs of sequence homology at both ends of the targeting construct . Multiple gene disruptions within a single cell are possible using the hisG cassette, which allows recovery of the marker but leaves a single hisG sequence imbedded in the disrupted gene(s) . We use an integration hisG construct, which has limited homology to the target at one end, to show that a single genomic copy of hisG decreases the percentage of integration at the target locus from 44% to 4.5% and two genomic hisG copies decrease it to less than 1% . Enlarging the homology at the disruption construct abolishes this effect . Thus competition between endogenous hisG sequences and successive hisG cassette transformations occurs if there is limited homology at one end of the targeting construct . Therefore, methods using limited homology, such as PCR-mediated gene targeting, are inefficient when significant internal homology exists. Science, 2000 Dec 22, 290(5500), 2306 - 9 Genome-wide location and function of DNA binding proteins; Ren B et al.; Understanding how DNA binding proteins control global gene expression and chromosomal maintenance requires knowledge of the chromosomal locations at which these proteins function in vivo . We developed a microarray method that reveals the genome-wide location of DNA-bound proteins and used this method to monitor binding of gene-specific transcription activators in yeast . A combination of location and expression profiles was used to identify genes whose expression is directly controlled by Gal4 and Ste12 as cells respond to changes in carbon source and mating pheromone, respectively . The results identify pathways that are coordinately regulated by each of the two activators and reveal previously unknown functions for Gal4 and Ste12 . Genome-wide location analysis will facilitate investigation of gene regulatory networks, gene function, and genome maintenance. J Mol Biol, 2001 Jan 12, 305(2), 219 - 30 Isolation of mutations that disrupt cooperative DNA binding by the Drosophila bicoid protein; Burz DS et al.; Cooperative DNA binding is thought to contribute to the ability of the Drosophila melanogaster protein, Bicoid, to stimulate transcription of target genes in precise sub-domains within the embryo . As a first step toward testing this idea, we devised a genetic screen to isolate mutations in Bicoid that specifically disrupt cooperative interactions, but do not disrupt DNA recognition or transcription activation . The screen was carried out in Saccharomyces cerevisiae and 12 cooperativity mutants were identified . The mutations map across most of the Bicoid protein, with some located within the DNA-binding domain (homeodomain) . Four homeodomain mutants were characterized in yeast and shown to activate a single-site reporter gene to levels comparable to that of wild-type, indicating that DNA binding per se is not affected . However, these mutants failed to show cooperative coupling between high and low-affinity sites, and showed reduced activation of a reporter gene carrying a natural Drosophila enhancer . Homology modeling indicated that none of the four mutations is in residues that contact DNA . Instead, these residues are likely to interact with other DNA-bound Bicoid monomers or other parts of the Bicoid protein . In vitro, the isolated homeodomains did not show strong cooperativity defects, supporting the idea that other regions of Bicoid are also important for cooperativity . This study describes the first systematic screen to identify cooperativity mutations in a eukaryotic DNA-binding protein . J Biol Chem, 2001 Apr 20, 276(16), 13034 - 8 Epub 2000 Dec 08. A mutational epitope for cytochrome C binding to the apoptosis protease activation factor-1; Yu T et al.; Cytochrome c (Cc) binding to apoptosis protease activation factor-1 (Apaf-1) is a critical activation step in the execution phase of apoptosis . Here we report studies that help define the Cc:Apaf-1 binding surface . It is shown that a large number of Cc residues, including residues 7, 25, 39, 62-65, and 72, are involved in the Cc:Apaf-1 interaction . Mutation of residue 72 eliminated Cc activity whereas mutations of residues 7, 25, 39, and 62-65 showed reduced activity in an additive fashion . The implications of this binding model for both recognition and modulation of protein-protein interactions are briefly discussed. Genes Dev, 2000 Dec 15, 14(24), 3204 - 14 The bHLH regulator pMesogenin1 is required for maturation and segmentation of paraxial mesoderm; Yoon JK et al.; Paraxial mesoderm in vertebrates gives rise to all trunk and limb skeletal muscles, the trunk skeleton, and portions of the trunk dermis and vasculature . We show here that germline deletion of mouse pMesogenin1, a bHLH class gene specifically expressed in developmentally immature unsegmented paraxial mesoderm, causes complete failure of somite formation and segmentation of the body trunk and tail . At the molecular level, the phenotype features dramatic loss of expression within the presomitic mesoderm of Notch/Delta pathway components and oscillating somitic clock genes that are thought to control segmentation and somitogenesis . Subsequent patterning and specification steps for paraxial mesoderm also fail, leading to a complete absence of all trunk paraxial mesoderm derivatives, which include skeletal muscle, vertebrae, and ribs . We infer that pMesogenin1 is an essential upstream regulator of trunk paraxial mesoderm development and segmentation. Biochemistry, 2000 Dec 26, 39(51), 15895 - 900 Identification of Asp218 and Asp326 as the principal Mg2+ binding ligands of the homing endonuclease PI-SceI; Schottler S et al.; The monomeric homing endonuclease PI-SceI harbors two catalytic centers which cooperate in the cleavage of the two strands of its extended recognition sequence . Structural and biochemical data suggest that catalytic center I contains Asp218, Asp229, and Lys403, while catalytic center II contains Asp326, Thr341, and Lys301 . The analogy with I-CreI, for which the cocrystal structure with the DNA substrate has been determined, suggests that Asp218 and Asp229 in catalytic center I and Asp326 and Thr341 in catalytic center II serve as ligands for Mg(2+), the essential divalent metal ion cofactor which can be replaced by Mn(2+) in vitro . We have carried out a mutational analysis of these presumptive Mg(2+) ligands . The variants carrying an alanine or asparagine substitution bind DNA, but (with the exception of the D229N variant) are inactive in DNA cleavage in the presence of Mg(2+), demonstrating that these residues are important for cleavage . Our finding that the PI-SceI variants carrying single cysteine substitutions at these positions are inactive in the presence of the oxophilic Mg(2+) but active in the presence of the thiophilic Mn(2+) suggests that the amino acid residues at these positions are involved in cofactor binding . From the fact that in the presence of Mn(2+) the D218C and D326C variants are even more active than the wild-type enzyme, it is concluded that Asp218 and Asp326 are the principal Mg(2+) ligands of PI-SceI . On the basis of these findings and the available structural information, a model for the composition of the two Mg(2+) binding sites of PI-SceI is proposed. Plant J, 2000 Dec, 24(5), 679 - 91 A stress-induced calcium-dependent protein kinase from Mesembryanthemum crystallinum phosphorylates a two-component pseudo-response regulator; Patharkar OR et al.; McCDPK1 is a salinity- and drought-induced calcium-dependent protein kinase (CDPK) isolated from the common ice plant, Mesembryanthemum crystallinum . A yeast two-hybrid experiment was performed, using full-length McCDPK1 and truncated forms of McCDPK1 as baits, to identify interacting proteins . A catalytically impaired bait isolated a cDNA clone encoding a novel protein, CDPK substrate protein 1 (CSP1) . CSP1 interacted with McCDPK1 in a substrate-like fashion in both yeast two-hybrid assays and wheat germ interaction assays . Furthermore, McCDPK1 was capable of phosphorylating CSP1 in vitro in a calcium-dependent manner . Our results demonstrate that the use of catalytically impaired and unregulated CDPKs with the yeast two-hybrid system can accelerate the discovery of CDPK substrates . The deduced CSP1 amino acid sequence indicated that it is a novel member of a class of pseudo-response regulator-like proteins that have a highly conserved helix-loop-helix DNA binding domain and a C-terminal activation domain . McCDPK1 and CSP1 co-localized to nuclei of NaCl-stressed ice plants . Csp1 transcript accumulation was not regulated by NaCl or dehydration stress . Our results strongly suggest that McCDPK1 may regulate the function of CSP1 by reversible phosphorylation. Plant J, 2000 Dec, 24(5), 645 - 54 Tonoplast subcellular localization of maize cytochrome b5 reductases; Bagnaresi P et al.; Plant cytochrome b5 reductases (b5R) are assumed to be part of an ER-associated redox chain that oxidizes NADH to provide electrons via cytochrome b5 (cyt b5) to ER-associated fatty acyl desaturase and related hydroxylases, as in mammalian cells . Here we report on cDNA cloning of a novel maize b5R, NFR II, strongly related to a previously cloned cDNA, NFR I (Bagnaresi et al., 1999, Biochem . J . 338, 499-505) . Maize b5R isoforms are produced by a small multi-gene family . The NFR cDNAs were shown to encode active b5Rs by heterologous expression in yeast . Both reductases, in addition to Fe3+-chelates, efficiently reduced Cu2+-chelates . Using a polyclonal antibody able to recognize both NFR I and NFR II isoforms, no ER or mitochondrial localization could be detected in maize roots . Unexpectedly, maize b5Rs were found to be targeted to the tonoplast . Using the most specific assay to measure NFR activity, we confirmed that the highest NFR specific activity is associated with tonoplast-enriched maize root fractions . Tonoplast targeting is not consistent with a role in desaturase reactions or with the other functions ascribed to date to plant b5R . This indicates that alternative ER-associated electron donors for desaturases need to be sought, and that plant b5Rs may have previously unexpected functions. J Immunol, 2001 Jan 1, 166(1), 197 - 206 Negative-feedback regulation of CD28 costimulation by a novel mitogen-activated protein kinase phosphatase, MKP6; Marti F et al.; TCR and CD28 costimulatory receptor-cooperative induction of T cell IL-2 secretion is dependent upon activation of mitogen-activated protein (MAP) kinases . Using yeast-hybrid technology, we cloned a novel CD28 cytoplasmic tail (CD28 CYT) interacting protein, MAP kinase phosphatase-6 (MKP6), which we demonstrate inactivates MAP kinases . Several lines of evidence indicate that MKP6 plays an important functional role in CD28 costimulatory signaling . First, in human peripheral blood T cells (PBT), expression of MKP6 is strongly up-regulated by CD28 costimulation . Second, transfer of dominant-negative MKP6 to PBT with the use of retroviruses primes PBT for the secretion of substantially larger quantities of IL-2, specifically in response to CD28 costimulation . A similar enhancement of IL-2 secretion is observed neither in response to TCR plus CD2 costimulatory receptor engagement nor in response to other mitogenic stimuli such as phorbol ester and ionomycin . Furthermore, this hypersensitivity to CD28 costimulation is associated with CD28-mediated hyperactivation of MAP kinases . Third, a retroviral transduced chimeric receptor with a CD28 CYT that is specifically unable to bind MKP6 costimulates considerably larger quantities of IL-2 from PBT than a similar transduced chimeric receptor that contains a wild-type CD28 CYT . Taken together, these results suggest that MKP6 functions as a novel negative-feedback regulator of CD28 costimulatory signaling that controls the activation of MAP kinases. Curr Opin Microbiol, 2000 Dec, 3(6), 573 - 81 Pheromone response, mating and cell biology; Elion EA; Saccharomyces cerevisiae responds to mating pheromones by activating a receptor-G-protein-coupled mitogen-activated protein kinase (MAPK) cascade that is also used by other signaling pathways . The activation of the MAPK cascade may involve conformational changes through prebound receptor and heterotrimeric G-protein . G beta may then recruit Cdc42-bound MAPKKKK Ste20 to MAPKKK Ste11 through direct interactions with Ste20 and the Ste5 scaffold . Ste20 activates Ste11 by derepressing an autoinhibitory domain . An underlying nuclear shuttling machinery may be required for proper recruitment of Ste5 to G beta . Subsequent polarized growth is mediated by a similar mechanism involving Far1, which binds G beta in addition to Cdc24 and Bem1 . Far1 and Cdc24 also undergo nuclear shuttling and the nuclear pool of Far1 may temporally regulate access of Cdc24 to the cell cortex. Free Radic Biol Med, 2000 Dec, 29(11), 1166 - 76 Enhanced sensitivity and long-term G2 arrest in hydrogen peroxide-treated Ku80-null cells are unrelated to DNA repair defects; Arrington ED et al.; While the Ku complex, comprised of Ku70 and Ku80, is primarily involved in the repair of DNA double-strand breaks, it is also believed to participate in additional cellular processes . Here, treatment of embryo fibroblasts (MEFs) derived from either wild-type or Ku80-null (Ku80(-/-)) mice with various stress agents revealed that hydrogen peroxide (H(2)O(2)) was markedly more cytotoxic for Ku80(-/-) MEFs and led to their long-term accumulation in the G2 phase . This differential response was not due to differences in DNA repair, since H(2)O(2)-triggered DNA damage was repaired with comparable efficiency in both Wt and Ku80(-/-) MEFs, but was associated with differences in the expression of important cell cycle regulatory genes . Our results support the notion that Ku80-mediated cytoprotection and G2-progression are not only dependent on the cell's DNA repair but also may reflect Ku80's influence on additional cellular processes such as gene expression. J Cell Biol, 2000 Dec 11, 151(6), 1337 - 44 The cortical protein Num1p is essential for dynein-dependent interactions of microtubules with the cortex; Heil-Chapdelaine RA et al.; In budding yeast, the mitotic spindle moves into the neck between the mother and bud via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud . How dynein and microtubules interact with the cortex is unknown . We found that cells lacking Num1p failed to exhibit dynein-dependent microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation . Num1p localized to the bud cortex, and that localization was independent of microtubules, dynein, or dynactin . These data are consistent with Num1p being an essential element of the cortical attachment mechanism for dynein-dependent sliding of microtubules in the bud. Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14494 - 9 Developmentally dynamic histone acetylation pattern of a tissue-specific chromatin domain; Forsberg EC et al.; We have defined the histone acetylation pattern of the endogenous murine beta-globin domain, which contains the erythroidspecific beta-globin genes . The beta-globin locus control region (LCR) and transcriptionally active promoters were enriched in acetylated histones in fetal liver relative to fetal brain, whereas the inactive promoters were hypoacetylated . In contrast, the LCR and both active and inactive promoters were hyperacetylated in yolk sac . Hypersensitive site two of the LCR was also hyperacetylated in murine embryonic stem cells, whereas beta-globin promoters were hypoacetylated . Thus, the acetylation pattern varied at different developmental stages . Histone deacetylase inhibition selectively increased acetylation at a hypoacetylated promoter in fetal liver, suggesting that active deacetylation contributes to silencing of promoters . We propose that dynamic histone acetylation and deacetylation play an important role in the developmental control of beta-globin gene expression. Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14200 - 5 A stable complex of a novel transcription factor IIB- related factor, human TFIIIB50, and associated proteins mediate selective transcription by RNA polymerase III of genes with upstream promoter elements; Teichmann M et al.; Transcription factor IIIB (TFIIIB) is directly involved in transcription initiation by RNA polymerase III in eukaryotes . Yeast contain a single TFIIIB activity that is comprised of the TATA-binding protein (TBP), TFIIB-related factor 1 (BRF1), and TFIIIB", whereas two distinct TFIIIB activities, TFIIIB-alpha and TFIIIB-beta, have been described in human cells . Human TFIIIB-beta is required for transcription of genes with internal promoter elements, and contains TBP, a TFIIIB" homologue (TFIIIB150), and a BRF1 homologue (TFIIIB90), whereas TFIIIB-alpha is required for transcription of genes with promoter elements upstream of the initiation site . Here we describe the identification, cloning, and characterization of TFIIIB50, a novel homologue of TFIIB and TFIIIB90 . TFIIIB50 and tightly associated factors, along with TBP and TFIIIB150, reconstitute human TFIIIB-alpha activity . Thus, higher eukaryotes, in contrast to the yeast Saccharomyces cerevisiae, have evolved two distinct TFIIB-related factors that mediate promoter selectivity by RNA polymerase III. Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14127 - 32 ATP-dependent structural change of the eukaryotic clamp-loader protein, replication factor C; Shiomi Y et al.; The eukaryotic DNA sliding clamp that keeps DNA polymerase engaged at a replication fork, called proliferating cell nuclear antigen (PCNA), is loaded onto the 3' ends of primer DNA through its interaction with a heteropentameric protein complex called replication factor C (RFC) . The ATPase activity of RFC is necessary for formation of a functional PCNA clamp . In the present study, the sensitivity of RFC to partial proteolysis is used to show that addition of ATP, ATPgammaS, or ADP induces different structural changes in RFC . Direct observation by electron microscopy reveals that RFC has a closed two-finger structure called the U form in the absence of ATP . This is converted into a more open C form on addition of ATP . In contrast, the structural changes induced by ATPgammaS or ADP are limited . These results suggest that RFC adapts on opened configuration intermediately after ATP hydrolysis . We further observe that PCNA is held between the two fingers of RFC and propose that the RFC structure change we observe during ATP hydrolysis causes the attached PCNA to form its active ring-like clamp on DNA. FEBS Lett, 2000 Dec 15, 486(3), 285 - 90 Kinesin subfamily UNC104 contains a FHA domain: boundaries and physicochemical characterization; Westerholm-Parvinen A et al.; By sequence analysis we show that the U104 domain found in the UNC104 subfamily of kinesins is a forkhead homology-associated domain (FHA) . A combination of limited proteolysis, mass spectroscopy, and physicochemical analysis define this domain as a genuine autonomously folding domain . Our data show that the FHA domain is shorter than previously reported since the C-terminal alpha-helix is not part of its minimum core . Key amino acids postulated to recognize phosphorylated residues are conserved . These data suggest that the kinesin FHA domains are functional domains involved in protein-protein interactions regulated by phosphorylation. Brain Res, 2000 Dec 15, 886(1-2), 73 - 81 A comparison of the expression and properties of Apaf-1 and Apaf-1L; Walke DW et al.; Apaf-1 is a mammalian homolog of CED-4 that regulates cell death by participating in a ternary complex with cytochrome c, and procaspase-9 . In the case of CED-4, two splice variants exist . The smaller (CED-4S) is proapoptotic while the larger (CED-4L) contains a short in-frame insert and is anti-apoptotic . We cloned a murine variant of apaf-1, termed apaf-1L, which contains an eleven amino acid insert similar to a recently described human apaf-1L clone . apaf-1 and apaf-1L have similar distributions in adult and fetal tissues, although apaf-1L transcripts are more abundant . Apaf-1L, undergoes homomerization and heteromerization with Apaf-1 in yeast . Apaf-1L also binds to caspase-9 and a dominant-negative isoform of caspase-9 . Unlike CED-4, neither Apaf-1 variant was lethal in yeast . However, both Apaf-1 and Apaf-1L elicit cell death when cotransfected with caspase-9 into 293 EBNA cells . Although Apaf-1L was more potent than Apaf-1, their biological properties were qualitatively similar. Biochim Biophys Acta, 2000 Dec 11, 1499(1-2), 154 - 163 A genetic interaction between a ubiquitin-like protein and ubiquitin-mediated proteolysis in Dictyostelium discoideum(1); Pukatzki S et al.; A ubiquitination factor, NosA, is essential for cellular differentiation in Dictyostelium discoideum . In the absence of nosA, development is blocked, resulting in a developmental arrest at the tight-aggregate stage, when cells differentiate into two precursor cell types, prespore and prestalk cells . Development is restored when a second gene, encoding the ubiquitin-like protein SonA, is inactivated in nosA-mutant cells . SonA has homology over its entire length to Dsk2 from Saccharomyces cerevisiae, a ubiquitin-like protein that is involved in the assembly of the spindle pole body . Dsk2 and SonA are both stable proteins that do not seem to be subjected to degradation via the ubiquitin pathway . SonA does not become ubiquitinated and the intracellular levels of SonA are not affected by the absence of NosA . The high degree of suppression suggests that SonA rescues most or all of the defects caused by the absence of nosA . We propose that NosA and SonA act in concert to control the activity of a developmental regulator that must be deactivated for cells to cross a developmental boundary. Virology, 2000 Dec 20, 278(2), 501 - 13 Ser(2194) is a highly conserved major phosphorylation site of the hepatitis C virus nonstructural protein NS5A; Katze MG et al.; Phosphorylation of the nonstructural NS5A protein is highly conserved among hepatitis C virus (HCV) genotypes . However, the precise site or sites of phosphorylation of NS5A have not been determined, and the functional significance of phosphorylation remains unknown . Here, we showed by two-dimensional phosphopeptide mapping that a protein kinase or kinases present in yeast, insect, and mammalian cells phosphorylated a highly purified HCV genotype 1b NS5A from insect cells on identical serine residues . We identified a major phosphopeptide (corresponding to amino acids 2193-2212 of the HCV 1b polyprotein) by using negative-ion electrospray ionization-microcapillary high performance liquid chromatography-mass spectrometry . The elution time of the phosphopeptide determined by negative-ion electrospray ionization-mass spectrometry corresponded with the elution time of the majority of (32)P-label that was incorporated into the phosphopeptide by an in vitro kinase reaction . Subsequent analysis of the peak fraction by automated positive-ion electrospray ionization-tandem mass spectrometry revealed that Ser(2194) was the major phosphorylated residue on the phosphopeptide GpSPPSLASSSASQLSAPSLK . Substitution for Ser(2194) with Ala resulted in the concomitant disappearance of major in vivo phosphorylated peptides . Ser(2194) and surrounding amino acids are highly conserved in all HCV genotypes, suggesting NS5A phosphorylation at Ser(2194) may be an important mechanism for modulating NS5A biological functions . Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 732 - 7 p29, a novel GCIP-interacting protein, localizes in the nucleus; Chang MS et al.; GCIP, a newly identified cyclin D-interacting protein, was found to reduce the phosphorylation of retinoblastoma protein and inhibit E2F1-mediated transcriptional activity . To explore more GCIP interacting proteins, the yeast two-hybrid screening using GCIP as a bait protein was performed . One novel gene, p29, was demonstrated to associate with GCIP in the yeast two-hybrid method and in vitro GST pull-down assay . Multiple tissue Northern blot analysis showed that p29 was abundantly expressed in the heart, skeletal muscle, and kidney relative to other tissues . The transient expression of HA-tagged p29 in HeLa cells localized in the nucleus . Taken together, we have isolated a novel protein, p29, which may be involved in the functional regulation of GCIP . Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 341 - 7 The P(174)L mutation in the human hSCO1 gene affects the assembly of cytochrome c oxidase; Paret C et al.; Mutations of the yeast SCO1 gene result in impaired COX assembly . Recently, heterozygous mutations in the human homologue hSCO1 have been reported in infants suffering from neonatal ketoacidotic coma and isolated COX deficiency (Valnot et al., 2000) . One of the hSCO1 alleles harboured a frame shift mutation resulting in a premature stop codon, the other a missense mutation leading to a substitution of proline(174) by leucine . This position is next to the essential CXXXC motif, which is conserved in all Sco1p homologues . We used chimeric proteins with the amino-terminal portion derived from yeast Sco1p and carboxy-terminal portion including the CXXXC motif from the human hSco1p to provide experimental evidence for the pathogenic nature of the P(174)L mutation . These chimeras are able to complement yeast sco1 null mutants . Introduction of the P(174)L mutation affects the function of these chimeric proteins severely, as shown by impaired COX assembly and loss of COX activity . J Biol Chem, 2001 Mar 16, 276(11), 8005 - 13 Epub 2000 Dec 15. Activation of the cyclin-dependent kinase CTDK-I requires the heterodimerization of two unstable subunits; Hautbergue G et al.; RNA polymerase II CTD kinases are key elements in the control of mRNA synthesis . They constitute a family of cyclin-dependent kinases activated by C-type cyclins . Unlike most cyclin-dependent kinase complexes, which are composed of a catalytic and a regulatory subunit, the yeast CTD kinase I complex contains three specific subunits: a kinase subunit (Ctk1), a cyclin subunit (Ctk2), and a third subunit (Ctk3) of unknown function that does not exhibit any similarity to known proteins . Like the Ctk2 cyclin that is regulated at the level of protein turnover, Ctk3 is an unstable protein processed through a ubiquitin-proteasome pathway . Interestingly, Ctk2 and Ctk3 physical interaction is required to protect both subunits from degradation, pointing to a new mechanism for cyclin turnover regulation . We also show that Ctk2 and Ctk3 can each interact independently with the kinase . However, despite the formation of CDK/cyclin complexes in vitro, the Ctk2 cyclin is unable to activate its CDK: both Ctk2 and Ctk3 are required for Ctk1 CTD kinase activation . The different specific features governing CTDK-I regulation probably reflect requirement for the transcriptional response to multiple growth conditions. J Biol Chem, 2001 Mar 23, 276(12), 9246 - 52 Epub 2000 Dec 04. Structure of a multifunctional protein . Mammalian phosphatidylinositol transfer protein complexed with phosphatidylcholine; Yoder MD et al.; Eukaryotic phosphatidylinositol transfer protein is a ubiquitous multifunctional protein that transports phospholipids between membrane surfaces and participates in cellular phospholipid metabolism during signal transduction and vesicular trafficking . The three-dimensional structure of the alpha-isoform of rat phosphatidylinositol transfer protein complexed with one molecule of phosphatidylcholine, one of its physiological ligands, has been determined to 2.2 A resolution by x-ray diffraction techniques . A single beta-sheet and several long alpha-helices define an enclosed internal cavity in which a single molecule of the phospholipid is accommodated with its polar head group in the center of the protein and fatty acyl chains projected toward the surface . Other structural features suggest mechanisms by which cytosolic phosphatidylinositol transfer protein interacts with membranes for lipid exchange and associates with a variety of lipid and protein kinases. EMBO J, 2000 Dec 15, 19(24), 6891 - 9 A new pathway of translational regulation mediated by eukaryotic initiation factor 3; Guo J et al.; We report a new pathway of translation regulation that may operate in interferon-treated or virus-infected mammalian cells . This pathway is activated by P56, a protein whose synthesis is strongly induced by interferons or double-stranded RNA . Using a yeast two-hybrid screen, we identified the P48 subunit of the mammalian translation initiation factor eIF-3 as a protein that interacts with P56 . The P56-P48 interaction was confirmed in human cells by co-immunoprecipitation assays and confocal microscopy . Gel filtration assays revealed that P56 binds to the large eIF-3 complex that contains P48 . Purified recombinant P56 inhibited in vitro translation of reporter mRNAs in a dose-dependent fashion, and that inhibition was reversed by the addition of purified eIF-3 . In vivo, expression of transfected P56 or induction of the endogenous P56 by interferon caused an inhibition of overall cellular protein synthesis and the synthesis of a transfected reporter protein . As expected, a P56 mutant that does not interact with P48 and eIF-3 failed to inhibit protein synthesis in vitro and in vivo. EMBO J, 2000 Dec 15, 19(24), 6845 - 52 Srb7p is a physical and physiological target of Tup1p; Gromoller A et al.; The holoenzyme of transcription integrates the positive and negative signals from the promoters of eukaryotic genes . We demonstrate that the essential holoenzyme component Srb7p is a physiologically relevant target of the global repressor Tup1p in Saccharomyces cerevisiae . Tup1p binds Srb7p in vivo and in vitro, and all genes tested that are repressed by Tup1p are derepressed when wild-type Srb7p is replaced by a mutant derivative of Srb7p that is no longer capable of interacting with Tup1p . Therefore, Srb7p is the first holoenzyme component essential for repression by Tup1p for which a physical interaction with Tup1p has been demonstrated . Furthermore, we find that Srb7p also binds Med6p and that this interaction is necessary for full transcriptional activation by different activators . Our finding that Med6p and Tup1p compete for the interaction with Srb7p suggests a model for Tup1p-mediated repression. EMBO J, 2000 Dec 15, 19(24), 6759 - 69 A novel ability of Smad3 to regulate proteasomal degradation of a Cas family member HEF1; Liu X et al.; Smad3 is a key signal transducer of transforming growth factor-ss (TGF-ss) and activin, and is known to be a DNA-binding transcriptional regulator . Here we report a novel property of Smad3 in regulating the proteasomal degradation of the human enhancer of filamentation 1 (HEF1), which is a member of the Cas family of cytoplasmic docking proteins . Our studies revealed that Smad3 interacts with HEF1 and triggers the proteasomal degradation of HEF1 in overexpression systems . In addition, TGF-ss stimulation induces rapid proteasomal degradation of endogenous HEF1 in different TGF-ss-responsive cell lines . Interestingly, the degradation of HEF1 protein in epithelial cells is followed closely by an increase in HEF1 mRNA, resulting in a time-dependent increase in HEF1 protein level in TGF-ss-treated cells . Furthermore, we observed that an elevated HEF1 protein level inhibits TGF-ss-induced Smad3-mediated gene responses . These data provide the first evidence for a novel cytoplasmic activity of Smad3 in regulating proteasomal degradation of HEF1 and also suggest a role for HEF1 in a negative feedback mechanism of the TGF-ss signaling pathway. EMBO J, 2000 Dec 15, 19(24), 6713 - 20 Sequential action of two GTPases to promote vacuole docking and fusion; Eitzen G et al.; Homotypic vacuole fusion occurs by sequential priming, docking and fusion reactions . Priming frees the HOPS complex (Vps 11, 16, 18, 33, 39 and 41) to activate Ypt7p for docking . Here we explore the roles of the GDP and GTP states of Ypt7p using Gdi1p (which extracts Ypt7:GDP), Gyp7p (a GTPase-activating protein for Ypt7p:GTP), GTPgammaS or GppNHp (non-hydrolyzable nucleotides), and mutant forms of Ypt7p that favor either GTP or GDP states . GDP-bound Ypt7p on isolated vacuoles can be extracted by Gdi1p, although only the GTP-bound state allows docking . Ypt7p is converted to the GTP-bound state after priming and stably associates with HOPS . Gyp7p can cause Ypt7p to hydrolyze bound GTP to GDP, driving HOPS release and accelerating Gdi1p-mediated release of Ypt7p . Ypt7p extraction does not inhibit the Ca(2+)-triggered cascade that leads to fusion . However, in the absence of Ypt7p, fusion is still sensitive to GTPgammaS and GppNHp, indicating that there is a second specific GTPase that regulates the calcium flux and hence fusion . Thus, two GTPases sequentially govern vacuole docking and fusion. Science, 2000 Dec 15, 290(5499), 2105 - 10 Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes; Riechmann JL et al.; The completion of the Arabidopsis thaliana genome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms . Arabidopsis dedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific to plants . Arabidopsis transcription factors that belong to families common to all eukaryotes do not share significant similarity with those of the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to each lineage . The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription. Mol Endocrinol, 2000 Dec, 14(12), 2001 - 9 Temporal formation of distinct thyroid hormone receptor coactivator complexes in HeLa cells; Sharma D et al.; Thyroid hormone receptors (TRs) regulate transcription by recruiting distinct coregulatory complexes to target gene promoters . Coactivators implicated in ligand-dependent activation by TR include p300, the CREB-binding protein (CBP), members of the p160/SRC family, and the multisubunit TR-associated protein (TRAP) complex . Using a stable TR-expressing HeLa cell line, we show that interaction of TR with members of the p160/SRC family, CBP, and the p300/CBP-associated factor (PCAF) occurs rapidly (approximately 10 min) following addition of thyroid hormone (T3) . In close agreement with these observations, we find that TR is associated with potent histone acetyltransferase activity rapidly following T3-treatment . By contrast, we observe that formation of TR-TRAP complexes occurs significantly later (approximately 3 h) post T3 treatment . An examination of the kinetics of T3-induced gene expression in HeLa cells reveals bimodal or delayed activation on specific T3-responsive promoters . Taken together, our data are consistent with the hypothesis that T3-dependent activation at specific target promoters may involve the regulated action of multiple TR-coactivator complexes. Mol Endocrinol, 2000 Dec, 14(12), 1907 - 17 The PDX-1 activation domain provides specific functions necessary for transcriptional stimulation in pancreatic beta-cells; Peshavaria M et al.; PDX-1 is a homeodomain transcription factor whose targeted disruption results in a failure of the pancreas to develop . Mutations in the human pdx-1 gene are linked to an early onset form of non-insulin-dependent diabetes mellitus . PDX-1 binds to and transactivates the promoters of several physiologically relevant genes within the beta-cell, including insulin, glucose transporter 2, glucokinase, and islet amyloid polypeptide . This study focuses on the mechanisms by which PDX-1 activates insulin gene transcription . To evaluate the role of PDX-1 in transcription of the insulin gene, a chloramphenicol acetyltransferase reporter construct was designed with a single yeast GAL4-DNA binding site in place of the A3/PDX-1 binding element in the rat insulin II enhancer . In the presence of GAL4:PDX chimeras containing N-terminal transactivation domain sequences, this GAL4-substituted insulin construct was active in PDX-1-expressing beta-cells and not non-beta-cells . PDX-1 activation was mediated through three highly conserved segments of the transactivation domain . In addition, when cotransfected together with the GAL4-substituted insulin enhancer reporter gene in glucose-responsive MIN-6 beta-cells, glucose-induced activation is observed with GAL4:PDX-1 but not with fusions of the heterologous activation domains from herpes virus VP16 or adenovirus-5 E1A proteins . Using A3 element-substituted GAL4 insulin enhancer reporter constructs containing mutations in two additional key control elements, E1 and C1, we also show that full activation requires cooperative interactions between other enhancer-bound factors, particularly the E1 element activators . In contrast, the activity of the VP16 activation factor was not dependent on the activators of either the E1 or C1 sites . These results suggest that the PDX-1 transactivation domain is specifically required for appropriate regulation of insulin enhancer function in beta-cells. J Inherit Metab Dis, 2000 Nov, 23(7), 713 - 29 Studies of the V94M-substituted human UDPgalactose-4-epimerase enzyme associated with generalized epimerase-deficiency galactosaemia; Wohlers TM et al.; Impairment of the human enzyme UDPgalactose 4-epimerase (hGALE) results in epimerase-deficiency galactosaemia, an inborn error of metabolism with variable biochemical presentation and clinical outcomes reported to range from benign to severe . Molecular studies of the hGALE loci from patients with epimerase deficiency reveal significant allelic heterogeneity, raising the possibility that variable genotypes may constitute at least one factor contributing to the biochemical and clinical heterogeneity observed . Previously we have identified a single substitution mutation, V94M, present in the homozygous state in all patients genotyped with the severe, generalized form of epimerase-deficiency galactosaemia . We report here further studies of the V94M-hGALE enzyme, overexpressed and purified from a null-background yeast expression system . Our results demonstrate that the mutant protein is impaired relative to the wild-type enzyme predominantly at the level of Vmax rather than of Km . Studies using UDP-N-acetylgalactosamine as a competitor of UDPgalactose further demonstrate that the Km values for these two substrates vary by less than a factor of 3 for both the wild-type and mutant proteins . Finally, we have explored the impact of the V94M substitution on susceptibility of yeast expressing human GALE to galactose toxicity, including changes in the levels of galactose 1-phosphate (gal-1-P) accumulated in these cells at different times following exposure to galactose . We have observed an inverse correlation between the level of GALE activity expressed in a given culture and the degree of galactose toxicity observed . We have further observed an inverse correlation between the level of GALE activity expressed in a culture and the concentration of gal-1-P accumulated in the cells . These data support the hypothesis that elevated levels of gal-1-P may underlie the observed toxicity . They further raise the intriguing possibility that yeast may provide a valuable model not only for assessing the impact of given patient mutations on hGALE function, but also for exploring the metabolic imbalance resulting from impaired activity of GALE in living cells. J Biol Chem, 2001 Mar 9, 276(10), 7122 - 8 Epub 2000 Dec 14. Constitutive signaling by Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor desensitizes calcium mobilization by other receptors; Lupu-Meiri M et al.; We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells . In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca(2+)-activated chloride current responses to TRH and acetylcholine . Similar inhibitions of cytoplasmic free Ca(2+) responses to TRH were observed in human embryonic kidney HEK 293 EM cells and in mouse pituitary AtT20 cells . Further study of oocytes showed that this inhibition was partially reversed by interferon-gamma-inducible protein 10 (IP-10), an inverse agonist of KSHV-GPCR . The basal rate of (45)Ca(2+) efflux in oocytes expressing KSHV-GPCRs was 4.4 times greater than in control oocytes, and IP-10 rapidly inhibited increased (45)Ca(2+) efflux . In the absence of IP-10, growth-related oncogene alpha caused a further 2-fold increase in (45)Ca(2+) efflux . In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-gamma-inducible protein 10 . Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs . These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools. Plant Physiol, 2000 Dec, 124(4), 1844 - 53 Interaction specificity of Arabidopsis calcineurin B-like calcium sensors and their target kinases; Kim KN et al.; Calcium is a critical component in a number of plant signal transduction pathways . A new family of calcium sensors called calcineurin B-like proteins (AtCBLs) have been recently identified from Arabidopsis . These calcium sensors have been shown to interact with a family of protein kinases (CIPKs) . Here we report that each individual member of AtCBL family specifically interacts with a subset of CIPKs and present structural basis for the interaction and for the specificity underlying these interactions . Although the C-terminal region of CIPKs is responsible for interaction with AtCBLs, the N-terminal region of CIPKs is also involved in determining the specificity of such interaction . We have also shown that all three EF-hand motifs in AtCBL members are required for the interaction with CIPKs . Several AtCBL members failed to interact with any of the CIPKs presented in this study, suggesting that these AtCBL members either have other CIPKs as targets or they target distinct proteins other than CIPKs . These results may provide structural basis for the functional specificity of CBL family of calcium sensors and their targets. Plant Physiol, 2000 Dec, 124(4), 1658 - 67 An enhancer trap line associated with a D-class cyclin gene in Arabidopsis; Swaminathan K et al.; In yeast and animals, cyclins have been demonstrated to be important regulators of cell cycle progression . In recent years, a large number of A-, B-, and D-class cyclins have been isolated from a variety of plant species . One class of cyclins, the D-class cyclins, is important for progression through G1 phase of the cell cycle . In Arabidopsis, four D-class cyclins have been isolated and characterized (CYCLIN-D1;1, CYCLIN-D2;1, CYCLIN-D3;1, and CYCLIN-D4;1) . In this report we describe the characterization of a fifth D-class cyclin gene, CYCLIN-D3;2 (CYCD3;2), from Arabidopsis . An enhancer trap line, line 5580, contains a T-DNA insertion in CYCD3;2 . Enhancer trap line 5580 exhibits expression in young vegetative and floral primordia . In line 5580, T-DNA is inserted in the first exon of the CYCD3;2 gene; in homozygous 5580 plants CYCD3;2 RNA is not detectable . Even though CYCD3;2 gene function is eliminated, homozygous 5580 plants do not exhibit an obvious growth or developmental phenotype . Via in situ hybridization we demonstrate that CYCD3;2 RNA is expressed in developing vegetative and floral primordia . In addition, CYCD3;2 is also capable of rescuing a yeast strain that is deficient in G1 cyclin activity. Int J Oncol, 2001 Jan, 18(1), 97 - 103 The error-prone DNA polymerase zeta catalytic subunit (Rev3) gene is ubiquitously expressed in normal and malignant human tissues; Kawamura K et al.; Mutagenesis induced by UV light and chemical agents in yeast is largely dependent on the function of Rev3, the catalytic subunit of DNA polymerase zeta that carries out translesion DNA synthesis . Human and mouse homologues of the yeast Rev3 gene have recently been identified, and inhibition of Rev3 expression in cultured human fibroblasts by Rev3 anti-sense was shown to reduce UV-induced mutagenesis, indicating that Rev3 also plays a crucial role in mutagenesis in mammalian cells . A common variant transcript with an insertion of 128-bp between nucleotides +139 and +140 is found in both human and mouse Rev3 cDNAs, but its biological significance has not been defined . We show here that the insertion variant is not translatable either under in vitro or in vivo conditions . We also found that the translational efficiency of Rev3 gene is enhanced by the 5' untranslated region that contains a putative stem-loop structure postulated to inhibit the translation . Since the human Rev3 gene is localized to chromosome 6q21, a region previously shown to contain genes involved in tumor suppression and cellular senescence, we examined its expression in various normal and malignant tissues . Rev3 and its insertion variant transcripts were ubiquitously detected in all 27 normal human tissues studied, with an additional variant species found in tissues with relatively high levels of Rev3 expression . Levels of Rev3 transcripts were similar in lung, gastric, colon and renal tumors compared to normal tissue counterparts . The data indicate that Rev3 expression is ubiquitous and is not dysregulated in malignancies. Biochem J, 2001 Jan 1, 353(Pt 1), 13 - 22 Msx3 protein recruits histone deacetylase to down-regulate the Msx1 promoter; Mehra-Chaudhary R et al.; Msx1 promoter is known to be repressed by Msx1 protein {Shetty, Takahashi, Matsui, Iyengar and Raghow (1999) Biochem . J . 339, 751-758} . We show that in the transiently transfected C(2)C(12) myoblasts, co-expression of Msx3 also causes potent repression of Msx1 promoter that can be relieved by exogenous expression of cAMP-response-element-binding protein-binding protein (CBP) and p300 in a dose-dependent manner . Co-immunoprecipitation and Western blot analyses revealed that Msx3 interacts with CBP and p300 and this interaction significantly decreases the histone acetyltransferase (HAT) activity of both proteins . We also discovered that Msx3-mediated repression of Msx1 promoter is synergized by the exogenous co-expression of histone deacetylase 1 (HDAC1) . Furthermore, the repression of Msx1 promoter by Msx3 could be relieved by treating transfected cells with trichostatin A, an inhibitor of HDAC(s) . Finally, we show that Msx3 and HDAC1 can be co-immunoprecipitated in a complex that does not contain CBP and that Msx3 and HDAC1 proteins are co-localized in the nucleus . Taken together, our results strongly suggest that two distinct multiprotein complexes are present within the nuclei of C(2)C(12) cells: one containing Msx3 and HDAC(s) and another containing Msx3 and CBP and/or p300 . On the basis of these results, we propose a dual mechanism of repression by Msx3 protein that involves the squelching of the HAT activity of co-activators, CBP and p300, and recruitment of HDAC(s). Plant J, 2000 Nov, 24(4), 511 - 22 Na+/myo-inositol symporters and Na+/H+-antiport in Mesembryanthemum crystallinum; Chauhan S et al.; Mitr1 and Mitr2 from Mesembryanthemum crystallinum (common ice plant) are members of a family of genes homologous to H+{or Na+}/myo-inositol symporters (ITRs), not previously studied in plants . MITR1 complemented an Itr1-deficient yeast strain . Mitr1 is strongly expressed in roots, moderately in stems, and weakly in leaves . Its transcripts increased in all organs, most dramatically in roots, under salinity stress . Mitr2 constitutes a rare transcript, slightly upregulated by salt stress in leaves only . Mitr1 transcripts are present in all cells in the root tip, but become restricted to phloem-associated cells in mature roots . Peptide antibodies against the two proteins indicated the presence of MITR1 in all organs and of MITR2 in leaves . Both are located in the tonoplast . MITR1 acts in removing sodium from root vacuoles, correlated with findings of low root sodium, while leaf vacuoles accumulate sodium in the ice plant . Up-regulation in leaves and stems is also found for Na+/H+-antiporter (Nhx-type) transcripts . Under comparable stress conditions, Nhx-and Itr-like transcripts in Arabidopsis were regulated differently . In the ice plant, co-ordinate induction of Na+/H+-antiporters and Na+/myo-inositol symporters transfers sodium from vacuoles in root cells into the leaf mesophyll as a halophytic strategy that lowers the osmotic potential . The tissue-specific differential expression of Itr- and Nhx-type transcripts suggests that the vacuolar sodium/inositol symporters function to reduce sodium amounts in cells of the root and vascular tissue, while sodium/proton antiporters in leaf tissues function to partition sodium into vacuoles for storage. Plant J, 2000 Nov, 24(4), 503 - 9 Plant sucrose-H+ symporters mediate the transport of vitamin H; Ludwig A et al.; A cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake-deficient yeast mutant . Vitamin H transport by this protein was sensitive to the SH-group inhibitor p-chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP), suggesting an energy-dependent biotin-H+ symport mechanism . The transport activity could contribute to the so-far uncharacterized plant sucrose-H+ symporter AtSUC5 which mediates the energy-dependent transport of biotin and sucrose, and restores growth of the biotin transport-deficient yeast mutant on medium with low biotin concentrations . Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin-H+ symporters) . This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters . The physiological relevance of this finding is discussed. Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14400 - 5 Activation of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14-3-3 to histone deacetylase 5; McKinsey TA et al.; Skeletal muscle differentiation is controlled by interactions between myocyte enhancer factor-2 (MEF2) and myogenic basic helix-loop-helix transcription factors . Association of MEF2 with histone deacetylases (HDAC) -4 and -5 results in repression of MEF2 target genes and inhibition of myogenesis . Calcium/calmodulin-dependent protein kinase (CaMK) signaling promotes myogenesis by disrupting MEF2-HDAC complexes and stimulating HDAC nuclear export . To further define the mechanisms that confer CaMK responsiveness to HDAC4 and -5, we performed yeast two-hybrid screens to identify HDAC-interacting factors . These screens revealed interactions between HDAC4 and members of the 14-3-3 family of proteins, which function as signal-dependent intracellular chaperones . HDAC4 binds constitutively to 14-3-3 in yeast and mammalian cells, whereas HDAC5 binding to 14-3-3 is largely dependent on CaMK signaling . CaMK phosphorylates serines -259 and -498 in HDAC5, which subsequently serve as docking sites for 14-3-3 . Our studies suggest that 14-3-3 binding to HDAC5 is required for CaMK-dependent disruption of MEF2-HDAC complexes and nuclear export of HDAC5, and implicate 14-3-3 as a signal-dependent regulator of muscle cell differentiation. Neuroscience, 2000, 101(3), 767 - 77 Nerve injury-associated kinase: a sterile 20-like protein kinase up-regulated in dorsal root ganglia in a rat model of neuropathic pain; Rausch O et al.; Partial injury of the rat sciatic nerve elicits a variety of characteristic chemical, electrophysical and anatomical changes in primary sensory neurons and constitutes a physiologically relevant model of neuropathic pain . To elucidate molecular mechanisms that underlie the physiology of neuropathic pain, we have used messenger RNA differential display to identify genes that exhibit increased ipsilateral expression in L4/5 dorsal root ganglia, following unilateral partial ligation of the rat sciatic nerve . One set of partial complementary DNA clones identified in this screen was found to encode a protein kinase, nerve injury-associated kinase . Cloning of the full-length human nerve injury-associated kinase complementary DNA, together with recombinant expression analysis, reveal nerve injury-associated kinase to be a functional member of a subgroup of sterile 20-like protein kinases characterised by the presence of a putative carboxy terminal autoregulatory domain . Induction of nerve injury-associated kinase expression in dorsal root ganglia in the rat neuropathic pain model was confirmed by quantitative reverse transcription-polymerase chain reaction, and RNA in situ hybridization analysis revealed enhanced levels of nerve injury-associated kinase within neurons.Together, our data implicate nerve injury-associated kinase as a novel upstream component of an intracellular signalling cascade that is up-regulated in dorsal root ganglia neurons in response to sciatic nerve injury. Mol Cell Biol, 2001 Jan, 21(1), 281 - 8 DNA damage-dependent nuclear dynamics of the Mre11 complex; Mirzoeva OK et al.; The Mre11 complex has been implicated in diverse aspects of the cellular response to DNA damage . We used in situ fractionation of human fibroblasts to carry out cytologic analysis of Mre11 complex proteins in the double-strand break (DSB) response . In situ fractionation removes most nucleoplasmic protein, permitting immunofluorescent localization of proteins that become more avidly bound to nuclear structures after induction of DNA damage . We found that a fraction of the Mre11 complex was bound to promyelocyte leukemia protein bodies in undamaged cells . Within 10 min after gamma irradiation, nuclear retention of the Mre11 complex in small granular foci was observed and persisted until 2 h postirradiation . In light of the previous demonstration that the Mre11 complex associated with ionizing radiation (IR)-induced DSBs, we infer that the protein retained under these conditions was associated with DNA damage . We also observed increased retention of Rad51 following IR treatment, although IR induced Rad51 foci were distinct from Mre11 foci . The ATM kinase, which phosphorylates Nbs1 during activation of the S-phase checkpoint, was not required for the Mre11 complex to associate with DNA damage . These data suggest that the functions of the Mre11 complex in the DSB response are implicitly dependent upon its ability to detect DNA damage. Mol Cell Biol, 2001 Jan, 21(1), 271 - 80 Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1; Philip B et al.; Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae . They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis . These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade . We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1 . At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain . This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1 . We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein . In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele . Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins . We demonstrate that Mid2 is not mannosylated in a pmt2 mutant and that this modification is important for signaling by Mid2. Mol Cell Biol, 2001 Jan, 21(1), 51 - 60 Ptc1, a type 2C Ser/Thr phosphatase, inactivates the HOG pathway by dephosphorylating the mitogen-activated protein kinase Hog1; Warmka J et al.; The HOG (high-osmolarity glycerol) mitogen-activated protein kinase (MAPK) pathway regulates the osmotic stress response in the yeast Saccharomyces cerevisiae . Three type 2C Ser/Thr phosphatases (PTCs), Ptc1, Ptc2, and Ptc3, have been isolated as negative regulators of this pathway . Previously, multicopy expression of PTC1 and PTC3 was shown to suppress lethality of the sln1Delta strain due to hyperactivation of the HOG pathway . In this work, we show that PTC2 also suppresses sln1Delta lethality . Furthermore, the phosphatase activity of these PTCs was needed for suppression, as mutation of a conserved Asp residue, likely to coordinate a metal ion, inactivated PTCs . Further analysis of Ptc1 function in vivo showed that it inactivates the MAPK, Hog1, but not the MEK, Pbs2 . In the wild type, Hog1 kinase activity increased transiently, approximately 12-fold in response to osmotic stress, while overexpression of PTC1 limited activation to approximately 3-fold . In contrast, overexpression of PTC1 did not inhibit phosphorylation of Hog1 Tyr in the phosphorylation lip, suggesting that Ptc1 does not act on Pbs2 . Deletion of PTC1 also strongly affected Hog1, leading to high basal Hog1 activity and sustained Hog1 activity in response to osmotic stress, the latter being consistent with a role for Ptc1 in adaptation . In vitro, Ptc1 but not the metal binding site mutant, Ptc1D58N, inactivated Hog1 by dephosphorylating the phosphothreonine but not the phosphotyrosine residue in the phosphorylation lip . Consistent with its role as a negative regulator of Hog1, which accumulates in the nucleus upon activation, Ptc1 was found in both the nucleus and the cytoplasm . Thus, one function of Ptc1 is to inactivate Hog1. Mol Cell Biol, 2001 Jan, 21(1), 39 - 50 Analysis of the steroid receptor coactivator 1 (SRC1)-CREB binding protein interaction interface and its importance for the function of SRC1; Sheppard HM et al.; The transcriptional activity of nuclear receptors is mediated by coactivator proteins, including steroid receptor coactivator 1 (SRC1) and its homologues and the general coactivators CREB binding protein (CBP) and p300 . SRC1 contains an activation domain (AD1) which functions via recruitment of CBP and and p300 . In this study, we have used yeast two-hybrid and in vitro interaction-peptide inhibition experiments to map the AD1 domain of SRC1 to a 35-residue sequence potentially containing two alpha-helices . We also define a 72-amino-acid sequence in CBP necessary for SRC1 binding, designated the SRC1 interaction domain (SID) . We show that in contrast to SRC1, direct binding of CBP to the estrogen receptor is weak, suggesting that SRC1 functions primarily as an adaptor to recruit CBP and p300 . In support of this, we show that the ability of SRC1 to enhance ligand-dependent nuclear receptor activity in transiently transfected cells is dependent upon the integrity of the AD1 region . In contrast, the putative histone acetyltransferase domain, the Per-Arnt-Sim basic helix-loop-helix domain, the glutamine-rich domain, and AD2 can each be removed without loss of ligand-induced activity . Remarkably, a construct corresponding to residues 631 to 970, which contains only the LXXLL motifs and the AD1 region of SRC1, retained strong coactivator activity in our assays. J Biol Chem, 2001 Mar 9, 276(10), 7176 - 86 Epub 2000 Dec 11. Assembly of scaffold-mediated complexes containing Cdc42p, the exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regulated phosphorylation of Cdc24p; Bose I et al.; In budding yeast cells, the cytoskeletal polarization and depolarization events that shape the bud are triggered at specific times during the cell cycle by the cyclin-dependent kinase Cdc28p . Polarity establishment also requires the small GTPase Cdc42p and its exchange factor, Cdc24p, but the mechanism whereby Cdc28p induces Cdc42p-dependent polarization is unknown . Here we show that Cdc24p becomes phosphorylated in a cell cycle-dependent manner, triggered by Cdc28p . However, the role of Cdc28p is indirect, and the phosphorylation appears to be catalyzed by the p21-activated kinase family member Cla4p and also depends on Cdc42p and the scaffold protein Bem1p . Expression of GTP-Cdc42p, the product of Cdc24p-mediated GDP/GTP exchange, stimulated Cdc24p phosphorylation independent of cell cycle cues, raising the possibility that the phosphorylation is part of a feedback regulatory pathway . Bem1p binds directly to Cdc24p, to Cla4p, and to GTP-bound Cdc42p and can mediate complex formation between these proteins in vitro . We suggest that Bem1p acts to concentrate polarity establishment proteins at a discrete site, facilitating polarization and promoting Cdc24p phosphorylation at specific times during the cell cycle. J Biol Chem, 2001 Mar 9, 276(10), 7654 - 60 Epub 2000 Dec 11. Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase; Nakanishi Y et al.; Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase) uses PP(i) as an energy donor and requires free Mg(2+) for enzyme activity and stability . To determine the catalytic domain, we analyzed charged residues (Asp(253), Lys(261), Glu(263), Asp(279), Asp(283), Asp(287), Asp(723), Asp(727), and Asp(731)) in the putative PP(i)-binding site and two conserved acidic regions of mung bean V-PPase by site-directed mutagenesis and heterologous expression in yeast . Amino acid substitution of the residues with alanine and conservative residues resulted in a marked decrease in PP(i) hydrolysis activity and a complete loss of H(+) transport activity . The conformational change of V-PPase induced by the binding of the substrate was reflected in the susceptibility to trypsin . Wild-type V-PPase was completely digested by trypsin but not in the presence of Mg-PP(i), while two V-PPase mutants, K261A and E263A, became sensitive to trypsin even in the presence of the substrate . These results suggest that the second acidic region is also implicated in the substrate hydrolysis and that at least two residues, Lys(261) and Glu(263), are essential for the substrate-binding function . From the observation that the conservative mutants K261R and E263D showed partial activity of PP(i) hydrolysis but no proton pump activity, we estimated that two residues, Lys(261) and Glu(263), might be related to the energy conversion from PP(i) hydrolysis to H(+) transport . The importance of two residues, Asp(253) and Glu(263), in the Mg(2+)-binding function was also suggested from the trypsin susceptibility in the presence of Mg(2+) . Furthermore, it was found that the two acidic regions include essential common motifs shared among the P-type ATPases. J Biol Chem, 2001 Mar 9, 276(10), 7541 - 8 Epub 2000 Dec 11. Characterization of CAF4 and CAF16 reveals a functional connection between the CCR4-NOT complex and a subset of SRB proteins of the RNA polymerase II holoenzyme; Liu HY et al.; The CCR4-NOT transcriptional regulatory complex affects transcription both positively and negatively and consists of the following two complexes: a core 1 x 10(6) dalton (1 MDa) complex consisting of CCR4, CAF1, and the five NOT proteins and a larger, less defined 1.9-MDa complex . We report here the identification of two new factors that associate with the CCR4-NOT proteins as follows: CAF4, a WD40-containing protein, and CAF16, a putative ABC ATPase . Whereas neither CAF4 nor CAF16 was part of the core CCR4-NOT complex, both CAF16 and CAF4 appeared to be present in the 1.9-MDa complex . CAF4 also displayed physical interactions with multiple CCR4-NOT components and with DBF2, a likely component of the 1.9-MDa complex . In addition, both CAF4 and CAF16 were found to interact in a CCR4-dependent manner with SRB9, a component of the SRB complex that is part of the yeast RNA polymerase II holoenzyme . The three related SRB proteins, SRB9, SRB10, and SRB11, were found to interact with and to coimmunoprecipitate DBF2, CAF4, CCR4, NOT2, and NOT1 . Defects in SRB9 and SRB10 also affected processes at the ADH2 locus known to be controlled by components of the CCR4-NOT complex; an srb9 mutation was shown to reduce ADH2 derepression and either an srb9 or srb10 allele suppressed spt10-enhanced expression of ADH2 . In addition, srb9 and srb10 alleles increased ADR1(c)-dependent ADH2 expression; not4 and not5 deletions are the only other known defects that elicit this phenotype . These results suggest a close physical and functional association between components of the CCR4-NOT complexes and the SRB9, -10, and -11 components of the holoenzyme. J Cell Sci, 2001 Jan, 114(Pt 1), 89 - 99 Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits; Bertinato J et al.; The Ku antigen is a heteromeric (Ku70/Ku80), mostly nuclear protein . Ku participates in multiple nuclear processes from DNA repair to V(D)J recombination to telomere maintenance to transcriptional regulation and serves as a DNA binding subunit and allosteric regulator of DNA-dependent protein kinase . While some evidence suggests that subcellular localization of Ku may be subject to regulation, how Ku gains access to the nucleus is poorly understood . In this work, using a combination of indirect immunofluorescence and direct fluorescence, we have demonstrated that transfer of the Ku heterodimer to the nucleus is determined by basic nuclear localization signals in each of the Ku subunits that function independently . A bipartite basic nuclear localization signal between amino acids 539-556 of Ku70 was observed to be required for nuclear import of full-length Ku70 monomer, while a short Ku80 motif of four amino acids from 565-568 containing three lysines was required for the nuclear import of full-length Ku80 . Ku heterodimers containing only one nuclear localization signal accumulated in the nucleus as efficiently as wild-type Ku, while site directed mutagenesis inactivating the basic motifs in each subunit, resulted in a Ku heterodimer that was completely localized to the cytoplasm . Lastly, our results indicate that mutations in Ku previously proposed to abrogate Ku70/Ku80 heterodimerization, markedly reduced the accumulation of Ku70 without affecting heterodimer formation in mammalian cells. J Biol Chem, 2001 Mar 16, 276(11), 8321 - 7 Epub 2000 Dec 08. Human Ku70 interacts with heterochromatin protein 1alpha; Song K et al.; Ku is involved in the metabolism of DNA ends, DNA repair, and the maintenance of telomeres . It consists of a heterodimer of 70- and 80-kDa subunits . Recently we have demonstrated that Ku70 interacted with TRF2, a mammalian telomere-binding protein . Using the same yeast two-hybrid screening system, we now show that Ku70 also interacts with heterochromatin protein 1alpha (HP1alpha), a protein known to be associated with telomeres as well as heterochromatin . HP1 is a suppressor of the position effect variegation in Drosophila and acts as a transcriptional suppressor in mammalian cells . The interaction with Ku70 in the two-hybrid system was confirmed by a glutathione S-transferase pull-down study using bacterial recombinant proteins in vitro . The interaction was also reproduced in vivo in HeLa cells, where endogenous Ku70 coimmunoprecipitated with HP1alpha . This interaction was more effective in acidic pH and weakened considerably as the pH of the reaction buffer was elevated up to 7.5 . Ku80 did not interact with HP1alpha directly . The interaction domains of Ku70 and HP1alpha included the Leu-Ser repeat (amino acids 200-385) and the chromo shadow domain, respectively . Ku70 was largely colocalized with transfected HP1alpha but not with a C-terminal deletion mutant, HP1alpha(Delta)C . In contrast to HP1alpha, Ku70 did not repress transcriptional activity of the reporter gene when tethered to DNA after transfection to mammalian cells . The implication of this interaction is discussed. Virology, 2000 Dec 5, 278(1), 276 - 88 CD4-Dependent and CD4-independent utilization of coreceptors by human immunodeficiency viruses type 2 and simian immunodeficiency viruses; Liu HY et al.; More than 10 G protein-coupled receptors (GPCRs) have been reported to act as coreceptors for entry of human and simian immunodeficiency viruses (HIV and SIV) . We investigated the utilization of six GPCRs as coreceptors by T-cell-line-adapted HIV-2 strains (CBL-20, CBL-21, CBL-23, GH-1, ROD, and SBL6669) and SIV strains (SIVagmTYO-1, SIVmac251, and SIVmndGB-1) . NP-2/CD4 cells were transduced with CCR3, CCR5, CCR8, CXCR4, GPR1, or APJ, and examined for susceptibilities to cell-free HIV/SIV . HIV-2 strains were grouped into two types by their coreceptor usage . The first group, CBL-20 and CBL-21, used CXCR4 exclusively; the other four strains used a few or all of the six coreceptors . These strains could further infect CD4-negative NP-2/CXCR4 or NP-2/CCR5 cells in the presence (all strains) or absence (SBL6669 and ROD strains) of soluble CD4 . SIVagm and SIVmnd infected NP-2/CD4/GPR1 cells . The coreceptors CCR3, CCR8, GPR1, and APJ did not mediate the CD4-independent infection . Although HIV-2ROD and SIVmnd infected both NP-2/CD4/CXCR4 and NP-2/CD4/CCR5 cells, only CXCR4 and CCR5, respectively, were used in CD4-independent infection . Binding of virions to CD4-negative cells occurred at 4 degrees C . These findings suggest that there may be a correlation between the promiscuous use of coreceptors by HIV-2/SIV strains and their ability to infect CD4-negative cells . Biochem Biophys Res Commun, 2000 Dec 9, 279(1), 95 - 102 Distinct regions of the chicken p46 polypeptide are required for its in vitro interaction with histones H2B and H4 and histone acetyltransferase-1; Ahmad A et al.; We cloned cDNA encoding the chicken p46 polypeptide, chp46, homologous to the p48 subunit of chicken chromatin assembly factor-1, chCAF-1p48 . It comprises 424 amino acids including a putative initiation Met, is a member of the WD protein family, with seven WD repeat motifs, and exhibits 90.3% identity to chCAF-1p48 and 94.3% identity to the human and mouse p46 polypeptides (hup46 and mop46) . The in vitro immunoprecipitation experiment established that chp46 interacts with histones H2B and H4 and chicken histone acetyltransferase-1, chHAT-1, whereas hup46 interacts with histones H2A and H4 and chHAT-1 and chCAF-1p48 with histone H4 and chHAT-1 . The in vitro immunoprecipitation experiment, involving truncated mutants of chp46, revealed not only that two regions comprising amino acids 33-179 and 375-404 are necessary for its binding to H2B, but also that two regions comprising amino acids 1-32 and 405-424 are necessary for its binding to H4 . Furthermore, the GST pulldown affinity assay, involving truncated mutants of chp46, revealed that a region comprising amino acids 359-404 (in fact, 375-404) binds to chHAT-1 in vitro . Taken together, these results indicate not only that chp46 should participate differentially in a number of DNA-utilizing processes through interactions of its distinct regions with chHAT-1 and histones H2B and H4, but also that the proper propeller structure of chp46 is not necessary for its interaction with chHAT-1 . Anal Biochem, 2000 Dec 15, 287(2), 319 - 28 Application of a fluorescent histone acetyltransferase assay to probe the substrate specificity of the human p300/CBP-associated factor; Trievel RC et al.; Histone N-acetyltransferases (HATs) are a group of enzymes which acetylate specific lysine residues in the N-terminal tails of nucleosomal histones to promote transcriptional activation . Recent structural and enzymatic work on the GCN5/PCAF HAT family has elucidated the structure of their catalytic domain and mechanism of histone acetylation . However, the substrate specificity of these enzymes has not been quantitatively investigated . Utilizing a novel microplate fluorescent HAT assay which detects the enzymatic production of coenzyme A (CoA), we have compared the activities of the HAT domains of human PCAF and its GCN5 homologue from yeast and Tetrahymena and found that they have similar kinetic parameters . PCAF was further assayed with a series of different length histone H3 peptide substrates, which revealed that the determinants for substrate recognition lie within a 19-residue sequence . Finally, we evaluated the acetylation of three putative PCAF substrates, histones H3 and H4 and the transcription factor p53, and have determined that histone H3 is significantly preferred over the histone H4 and p53 substrates . Taken together, the fluorescent acetyltransferase assay presented here should be widely applicable to other HAT enzymes, and the results obtained with PCAF demonstrate a strong substrate preference for the N-terminal residues of histone H3 . Biochim Biophys Acta, 2000 Dec 15, 1529(1-3), 188 - 202 Cholesterol modification of proteins; Mann RK et al.; The demonstration over 30 years ago that inhibitors of cholesterol biosynthesis disrupt animal development suggested an intriguing connection between fundamental cellular metabolic processes and the more global processes of embryonic tissue patterning . Adding a new dimension to this relationship is the more recent finding that the Hedgehog family of tissue patterning factors are covalently modified by cholesterol . Here we review the mechanism of the Hedgehog autoprocessing reaction that results in this modification, and compare this reaction to that undergone by other autoprocessing proteins . We also discuss the biological consequences of cholesterol modification, in particular the use of cholesterol as a molecular handle in the spatial deployment of the protein signal in developing tissues . Finally, the developmental consequences of chemical and genetic disruption of cholesterol homeostasis are summarized, along with the potential importance of cholesterol-rich lipid rafts in production of and response to the Hh signal. FEBS Lett, 2000 Dec 1, 486(1), 14 - 8 Preferential interaction of loach DNA polymerase delta with DNA duplexes containing single-stranded gaps; Sharova NP et al.; We studied the interaction of DNA polymerase delta (pol delta) purified from the eggs of the teleost fish Misgurnus fossilis (loach) with DNA duplexes containing single-stranded gaps of 1-13 nucleotides (nt) . In the absence of processivity factors (PCNA, RF-C, and ATP), pol delta elongated primers on single-stranded DNA templates in a distributive manner . However, the enzyme was capable of processive synthesis by filling gaps of 5-9 nt in DNA duplexes . These data suggest that, upon filling a small gap, pol delta interacts with the 5'-terminus downstream of the gap as well as with the 3'-terminus of the primer . Interaction of pol delta with the proximal 5'-terminus restricting the gap was confirmed by electrophoretic mobility shift assay . Analysis of the enzyme binding to DNA duplexes containing gaps of various sizes showed a much higher affinity of pol delta for duplexes with gaps of about 10 nt than for DNA substrates with primers annealed to single-stranded templates . The most efficient pol delta binding was observed in experiments with DNA substrates containing unpaired 3'-tails in primers . The data obtained suggest that DNA molecules with small gaps and single-stranded tails may serve as substrates for direct action of pol delta in the course of DNA repair. Bioinformatics, 2000 Sep, 16(9), 750 - 9 Ballast: blast post-processing based on locally conserved segments; Plewniak F et al.; MOTIVATION: Blast programs are very efficient in finding relatively strong similarities but some very distantly related sequences are given a very high Expect value and are ranked very low in Blast results . We have developed Ballast, a program to predict local maximum segments (LMSs-i.e . sequence segments conserved relatively to their flanking regions) from a single Blast database search and to highlight these divergent homologues . The TBlastN database searches can also be processed with the help of information from a joint BlastP search . RESULTS: We have applied the Ballast algorithm to BlastP searches performed with sequences belonging to well described dispersed families (aminoacyl-tRNA synthetases; helicases) against the SwissProt 38 database . We show that Ballast is able to build an appropriate conservation profile and that LMSs are predicted that are consistent with the signatures and motifs described in the literature . Furthermore, by comparing the Blast, PsiBlast and Ballast results obtained on a well defined database of structurally related sequences, we show that the LMSs provide a scoring scheme that can concentrate on top ranking distant homologues better than Blast . Using the graphical user interface available on the Web, specific LMSs may be selected to detect divergent homologues sharing the corresponding properties with the query sequence without requiring any additional database search. J Electron Microsc (Tokyo), 2000, 49(3), 415 - 21 Atomic force microscopy with carbon nanotube probe resolves the subunit organization of protein complexes; Hohmura KI et al.; Among many scanning probe microscopies, atomic force microscopy (AFM) is a useful technique to analyse the structure of biological materials because of its applicability to non-conductors in physiological conditions with high resolution . However, the resolution has been limited to an inherent property of the technique; tip effect associated with a large radius of the scanning probe . To overcome this problem, we developed a carbon nanotube probe by attaching a carbon nanotube to a conventional scanning probe under a well-controlled process . Because of the constant and small radius of the tip (2.5-10 nm) and the high aspect ratio (1:100) of the carbon nanotube, the lateral resolution has been much improved judging from the apparent widths of DNA and nucleosomes . The carbon nanotube probes also possessed a higher durability than the conventional probes . We further evaluated the quality of carbon nanotube probes by three parameters to find out the best condition for AFM imaging: the angle to the tip axis; the length; and the tight fixation to the conventional tip . These carbon nanotube probes, with high vertical resolution, enabled us to clearly visualize the subunit organization of multi-subunit proteins and to propose structural models for proliferating cell nuclear antigen and replication factor C . This success in the application of carbon nanotube probes provides the current AFM technology with an additional power for the analyses of the detailed structure of biological materials and the relationship between the structure and function of proteins. Cell, 2000 Nov 10, 103(4), 655 - 65 Solution structure of the interacting domains of the Mad-Sin3 complex: implications for recruitment of a chromatin-modifying complex; Brubaker K et al.; Gene-specific targeting of the Sin3 corepressor complex by DNA-bound repressors is an important mechanism of gene silencing in eukaryotes . The Sin3 corepressor specifically associates with a diverse group of transcriptional repressors, including members of the Mad family, that play crucial roles in development . The NMR structure of the complex formed by the PAH2 domain of mammalian Sin3A with the transrepression domain (SID) of human Mad1 reveals that both domains undergo mutual folding transitions upon complex formation generating an unusual left-handed four-helix bundle structure and an amphipathic alpha helix, respectively . The SID helix is wedged within a deep hydrophobic pocket defined by two PAH2 helices . Structure-function analyses of the Mad-Sin3 complex provide a basis for understanding the underlying mechanism(s) that lead to gene silencing. Biophys J, 2000 Dec, 79(6), 3105 - 17 Heme structure and orientation in single monolayers of cytochrome c on polar and nonpolar soft surfaces; Edwards AM et al.; Polarized x-ray absorption fine structure (XAFS) spectroscopy has been performed in fluorescence mode under total external reflection conditions on frozen hydrated single monolayers of yeast cytochrome c (YCC) . The protein molecules were vectorially oriented within the monolayer by tethering their naturally occurring and unique surface cysteine residues to the sulfhydryl-endgroups at the surface of a mixed organic self-assembled monolayer, itself covalently attached to an ultrapure silicon wafer . The sulfhydryl-endgroups were isolated by dilution with either methyl- or hydroxyl-endgroups, producing macroscopically nonpolar or uncharged-polar soft surfaces, respectively . Independent information on the heme-plane orientation relative to the monolayer plane was obtained experimentally via optical linear dichroism . The polarized XAFS data have been analyzed both qualitatively and by a global mapping approach limited to systematically altering the various iron-ligand distances within a model for the local atomic environment of the heme prosthetic group, and comparing the theoretically generated XAFS spectra with those obtained experimentally . A similar analysis of unpolarized XAFS data from a frozen solution of YCC was performed using either the heme environment from the NMR solution or the x-ray crystallographic data for YCC as the model structure . All resulting iron-ligand distances were then used in molecular dynamics (MD) computer simulations of YCC in these three systems to investigate the possible effects of anisotropic ligand motions on the fits of the calculated to the experimental XAFS spectra. Am J Pathol, 2000 Dec, 157(6), 1917 - 25 Reduced heparan sulfate accumulation in enterocytes contributes to protein-losing enteropathy in a congenital disorder of glycosylation; Westphal V et al.; Intestinal biopsy in a boy with gastroenteritis-induced protein-losing enteropathy (PLE) showed loss of heparan sulfate (HS) and syndecan-1 core protein from the basolateral surface of the enterocytes, which improved after PLE subsided . Isoelectric focusing analysis of serum transferrin indicated a congenital disorder of glycosylation (CDG) and subsequent analysis showed three point mutations in the ALG6 gene encoding an alpha1,3-glucosyltransferase needed for the addition of the first glucose to the dolichol-linked oligosaccharide . The maternal mutation, C998T, causing an A333V substitution, has been shown to cause CDG-Ic, whereas the two paternal mutations, T391C (Y131H) and C924A (S308R) have not previously been reported . The mutations were tested for their ability to rescue faulty N:-linked glycosylation of carboxypeptidase Y in an ALG6-deficient Saccharomyces cerevisiae strain . Normal human ALG6 rescues glycosylation and A333V partially rescues, whereas the combined paternal mutations (Y131H and S308R) are ineffective . Underglycosylation resulting from each of these mutations is much more severe in rapidly dividing yeast . Similarly, incomplete protein glycosylation in the patient is most severe in rapidly dividing enterocytes during gastroenteritis-induced stress . Incomplete N:-linked glycosylation of an HS core protein and/or other biosynthetic enzymes may explain the selective localized loss of HS and PLE. Biochemistry, 2000 Dec 12, 39(49), 15190 - 8 N-Terminal domain of annexin 2 regulates Ca(2+)-dependent membrane aggregation by the core domain: a site directed mutagenesis study; Ayala-Sanmartin J et al.; Annexin 2 binds and aggregates biological membranes in a Ca(2+)-dependent manner . This protein exists as a monomer (p36) or as a heterotetramer (p90) in which two p36 chains are associated with a dimer of p11, a member of the S100 protein family . Protein kinase C phosphorylates the protein at the level of the N-terminal tail on serines 11 and 25, thereby modifying its oligomeric structure and its properties of membrane aggregation . To analyze these effects, the properties of a series of mutants in which serines 11 and 25 were replaced by alanine and/or glutamic acid were investigated . The affinity for p11 light chain was decreased in the S11E mutants . Glutamic acid residues in positions 11 or 25 did not change membrane binding, either in the tetrameric or in the monomeric form . On the other hand, these mutations affected the aggregation properties of the two forms . For the tetramer, the aggregation efficiency was decreased but not the Ca(2+) sensitivity, whereas the latter was affected in the case of the monomer . The effects were stronger in the S11E mutants, and they were cumulative in the double mutant . They suggest a different conformation of the N-terminal domain in the mutants (and in the phosphorylated protein), a hypothesis which is supported by proteolysis experiments . This conformational change would affect aggregation by the monomer through a dimerization step. Mol Cell, 2000 Nov, 6(5), 1089 - 98 The apoptosis-promoting factor TIA-1 is a regulator of alternative pre-mRNA splicing; Forch P et al.; We report here that the apoptosis-promoting protein TIA-1 regulates alternative pre-mRNA splicing of the Drosophila melanogaster gene male-specific-lethal 2 and of the human apoptotic gene Fas . TIA-1 associates selectively with pre-mRNAs that contain 5' splice sites followed by U-rich sequences . TIA-1 binding to the U-rich stretches facilitates 5' splice site recognition by U1 snRNP . This activity is critical for activation of the weak 5' splice site of msl-2 and for modulating the choice of splice site partner in Fas . Structural and functional similarities with the Saccharomyces cerevisiae splicing factor Nam8 suggest striking evolutionary conservation of a mechanism of pre-mRNA splicing regulation that controls biological processes as diverse as meiosis in yeast, dosage compensation in fruit flies, or programmed cell death in humans. Mol Cell, 2000 Nov, 6(5), 1049 - 58 ATP-driven chromatin remodeling activity and histone acetyltransferases act sequentially during transactivation by RAR/RXR In vitro; Dilworth FJ et al.; Using a "crude" chromatin-based transcription system that mimics transactivation by RAR/RXR heterodimers in vivo, we could not demonstrate that chromatin remodeling was required to relieve nucleosomal repression . Using "purified" chromatin templates, we show here that, irrespective of the presence of histone H1, both ATP-driven chromatin remodeling activities and histone acetyltransferase (HAT) activities of coactivators recruited by liganded receptors are required to achieve transactivation . DNA footprinting, ChIP analysis, and order of addition experiments indicate that coactivator HAT activities and two ATP-driven remodeling activities are sequentially involved at distinct steps preceding initiation of transcription . Thus, both ATP-driven chromatin remodeling and HAT activities act in a temporally ordered and interdependent manner to alleviate the repressive effects of nucleosomal histones on transcription by RARalpha/RXRalpha heterodimers. J Biol Chem, 2001 Mar 2, 276(9), 6861 - 6 Epub 2000 Dec 05. Inefficient bypass of an abasic site by DNA polymerase eta; Haracska L et al.; DNA polymerase eta (Pol eta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately, and inactivation of Pol eta in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum . Also, Pol eta bypasses the 8-oxoguanine lesion efficiently by predominantly inserting a C opposite this lesion, and it bypasses the O(6)-methylguanine lesion by inserting a C or a T . To further assess the range of DNA lesions tolerated by Pol eta, here we examine the bypass of an abasic site, a prototypical noninstructional lesion . Steady-state kinetic analyses show that both yeast and human Pol eta are very inefficient in both inserting a nucleotide opposite an abasic site and in extending from the nucleotide inserted . Hence, Pol eta bypasses this lesion extremely poorly . These results suggest that Pol eta requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation. Proc Natl Acad Sci U S A, 2000 Dec 5, 97(25), 13985 - 90 Novel features of the XRN-family in Arabidopsis: evidence that AtXRN4, one of several orthologs of nuclear Xrn2p/Rat1p, functions in the cytoplasm; Kastenmayer JP et al.; The 5'-3' exoribonucleases Xrn1p and Xrn2p/Rat1p function in the degradation and processing of several classes of RNA in Saccharomyces cerevisiae . Xrn1p is the main enzyme catalyzing cytoplasmic mRNA degradation in multiple decay pathways, whereas Xrn2p/Rat1p functions in the processing of rRNAs and small nucleolar RNAs (snoRNAs) in the nucleus . Much less is known about the XRN-like proteins of multicellular eukaryotes; however, differences in their activities could explain differences in mRNA deg