S. cerevisiae

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 degradation between multicellular and unicellular eukaryotes . One such difference is the lack in plants and animals of mRNA decay intermediates like those generated in yeast when Xrn1p is blocked by poly(G) tracts that are inserted within mRNAs . We investigated the XRN-family in Arabidopsis thaliana and found it to have several novel features . First, the Arabidopsis genome contains three XRN-like genes (AtXRNs) that are structurally similar to Xrn2p/Rat1p, a characteristic unique to plants . Furthermore, our experimental results and sequence database searches indicate that Xrn1p orthologs may be absent from higher plants . Second, the lack of poly(G) mRNA decay intermediates in plants cannot be explained by the activity of the AtXRNs, because they are blocked by poly(G) tracts . Finally, complementation of yeast mutants and localization studies indicate that two of the AtXRNs likely function in the nucleus, whereas the third acts in the cytoplasm . Thus, the XRN-family in plants is more complex than in other eukaryotes, and, if an XRN-like enzyme plays a role in mRNA decay in plants, the likely participant is a cytoplasmic Xrn2p/Rat1p ortholog, rather than an Xrn1p ortholog.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14178 - 82
Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose; Tanner KG et al.; Conflicting reports have suggested that the silent information regulator 2 (SIR2) protein family employs NAD(+) to ADP-ribosylate histones {Tanny, J . C., Dowd, G . J., Huang, J., Hilz, H . & Moazed, D . (1999) Cell 99, 735-745; Frye, R . A . (1999) Biochem . Biophys . Res . Commun . 260, 273-279}, deacetylate histones {Landry, J., Sutton, A., Tafrov, S . T., Heller, R . C., Stebbins, J., Pillus, L . & Sternglanz, R . (2000) Proc . Natl . Acad . Sci . USA 97, 5807-5811; Smith, J . S., Brachmann, C . B., Celic, I., Kenna, M . A., Muhammad, S., Starai, V . J., Avalos, J . L., Escalante-Semerena, J . C., Grubmeyer, C., Wolberger, C . & Boeke, J . D . (2000) Proc . Natl . Acad . Sci . USA 97, 6658-6663}, or both {Imai, S., Armstrong, C . M., Kaeberlein, M . & Guarente, L . (2000) Nature (London) 403, 795-800} . Uncovering the true enzymatic function of SIR2 is critical to the basic understanding of its cellular function . Therefore, we set out to authenticate the reaction products and to determine the intrinsic catalytic mechanism . We provide direct evidence that the efficient histone/protein deacetylase reaction is tightly coupled to the formation of a previously unidentified acetyl-ADP-ribose product (1-O-acetyl-ADP ribose) . One molecule of NAD(+) and one molecule of acetyl-lysine are readily catalyzed to one molecule of deacetylated lysine, nicotinamide, and 1-O-acetyl-ADP-ribose . A unique reaction mechanism involving the attack of enzyme-bound acetate or the direct attack of acetyl-lysine on an oxocarbenium ADP-ribose intermediate is proposed . We suggest that the reported histone/protein ADP-ribosyltransferase activity is a low-efficiency side reaction that can be explained through the partial uncoupling of the intrinsic deacetylation and acetate transfer to ADP-ribose.

RNA, 2000 Nov, 6(11), 1625 - 34
Nascent 60S ribosomal subunits enter the free pool bound by Nmd3p; Ho JH et al.; Nmd3p from yeast is required for the export of the large (60S) ribosomal subunit from the nucleus (Ho et al., 2000) . Here, we show that Nmd3p forms a stable complex with free 60S subunits . Using an epitope-tagged Nmd3p, we show that free 60S subunits can be coimmunoprecipitated with Nmd3p . The interaction was specific for 60S subunits; 40S subunits were not coimmunoprecipitated . Using this coprecipitation technique and pulse-chase labeling of ribosomal subunit proteins we showed that Nmd3p bound nascent subunits, consistent with its role in export . However, under conditions in which ribosome biogenesis was inhibited (e.g., inhibition of transcription with thiolutin, inhibition of transcription of ribosomal protein and RNA genes in a sly1-1 mutant at nonpermissive temperature, and inhibition of translation in a conditional prt1 mutant), Nmd3p remained associated with 60S subunits . In addition, Nmd3delta120, a truncated protein that lacked a nuclear localization signal, retained 60S binding . These results suggest that Nmd3p recruits nascent 60S subunits into the pool of free 60S subunits and exchanges on 60S subunits as they recycle during translation.

RNA, 2000 Nov, 6(11), 1551 - 64
The human endogenous retrovirus K Rev response element coincides with a predicted RNA folding region; Yang J et al.; Human endogenous retrovirus K (HERV-K) is the name given to an approximately 30-million-year-old family of endogenous retroviruses present at >50 copies per haploid human genome . Previously, the HERV-K were shown to encode a nuclear RNA export factor, termed K-Rev, that is the functional equivalent of the H-Rev protein encoded by human immunodeficiency virus type 1 . HERV-K was also shown to contain a cis-acting target element, the HERV-K Rev response element (K-RRE), that allowed the nuclear export of linked RNA transcripts in the presence of either K-Rev or H-Rev . Here, we demonstrate that the functionally defined K-RRE coincides with a statistically highly significant unusual RNA folding region and present a potential RNA secondary structure for the approximately 416-nt K-RRE . Both in vitro and in vivo assays of sequence specific RNA binding were used to map two primary binding sites for K-Rev, and one primary binding site for H-Rev, within the K-RRE . Of note, all three binding sites map to discrete predicted RNA stem-loop subdomains within the larger K-RRE structure . Although almost the entire 416-nt K-RRE was required for the activation of nuclear RNA export in cells expressing K-Rev, mutational inactivation of the binding sites for K-Rev resulted in the selective loss of the K-RRE response to K-Rev but not to H-Rev . Together, these data strongly suggest that the K-RRE, like the H-RRE, coincides with an extensive RNA secondary structure and identify specific sites within the K-RRE that can recruit either K-Rev or H-Rev to HERV-K RNA transcripts.

Br J Cancer, 2000 Dec, 83(12), 1702 - 6
Expression of Ku70 correlates with survival in carcinoma of the cervix; Wilson CR et al.; Cervical carcinoma affects around 3400 women in the UK each year and advanced disease is routinely treated with radiation . As part of a programme to establish rapid and convenient methods of predicting tumour and patient responses to radiotherapy, we have examined the relationship between the pre-treatment expression of the Ku components of the DNA damage recognition complex DNA-PK and patient survival in cervical carcinoma . Using immunohistochemistry of formalin-fixed sections of tumour biopsies, antibodies to Ku70 and Ku80 stained identical regions of tumour and there was a high degree of correlation between the mean number of cells stained positive for the two components in 77 tumours (r = 0.82, P<0.001) . In 53 tumours there was a borderline significant correlation between measurements of tumour radiosensitivity (surviving fraction at 2 gray: SF2) and Ku70 expression (r = 0.26, P = 0.057) and no correlation for Ku80 (r = 0.18, P = 0.19) . However, all tumours with a low number of Ku70 or Ku80 positive cells were radiosensitive . Furthermore, using log-rank analysis there was significantly higher survival in the patients whose tumours had a low Ku70 expression (P = 0.046) . This difference was also reflected with Ku80, but did not reach statistical significance (P = 0.087) . The study suggests that lack of Ku protein leads to radiosensitivity in some tumours and that other factors are responsible for radiosensitive tumours with high Ku expression . It is likely that the most accurate prediction of treatment outcome will lie in assessing the expression of several proteins involved in the recognition and repair of DNA damage, one of which will be Ku .

J Biol Chem, 2001 Mar 2, 276(9), 6445 - 52 Epub 2000 Dec 04.
The GLFG regions of Nup116p and Nup100p serve as binding sites for both Kap95p and Mex67p at the nuclear pore complex; Strawn LA et al.; Our previous studies have focused on a family of Saccharomyces cerevisiae nuclear pore complex (NPC) proteins that contain domains composed of repetitive tetrapeptide glycine-leucine-phenylalanine-glycine (GLFG) motifs . We have previously shown that the GLFG regions of Nup116p and Nup100p directly bind the karyopherin transport factor Kap95p during nuclear protein import . In this report, we have further investigated potential roles for the GLFG region in mRNA export . The subcellular localizations of green fluorescent protein (GFP)-tagged mRNA transport factors were individually examined in yeast cells overexpressing the Nup116-GLFG region . The essential mRNA export factors Mex67-GFP, Mtr2-GFP, and Dbp5-GFP accumulated in the nucleus . In contrast, the localizations of Gle1-GFP and Gle2-GFP remained predominantly associated with the NPC, as in wild type cells . The localization of Kap95p was also not perturbed with GLFG overexpression . Coimmunoprecipitation experiments from yeast cell lysates resulted in the isolation of a Mex67p-Nup116p complex . Soluble binding assays with bacterially expressed recombinant proteins confirmed a direct interaction between Mex67p and the Nup116-GLFG or Nup100-GLFG regions . Mtr2p was not required for in vitro binding of Mex67p to the GLFG region . To map the Nup116-GLFG subregion(s) required for Kap95p and/or Mex67p association, yeast two-hybrid analysis was used . Of the 33 Nup116-GLFG repeats that compose the domain, a central subregion of nine GLFG repeats was sufficient for binding either Kap95p or Mex67p . Interestingly, the first 12 repeats from the full-length region only had a positive interaction with Mex67p, whereas the last 12 were only positive with Kap95p . Thus, the GLFG domain may have the capacity to bind both karyopherins and an mRNA export factor simultaneously . Taken together, our in vivo and in vitro results define an essential role for a direct Mex67p-GLFG interaction during mRNA export.

J Biol Chem, 2001 Feb 23, 276(8), 5753 - 9 Epub 2000 Dec 04.
Regulation of the TAK1 signaling pathway by protein phosphatase 2C; Hanada M et al.; Protein phosphatase 2C (PP2C) is implicated in the negative regulation of stress-activated protein kinase cascades in yeast and mammalian cells . In this study, we determined the role of PP2Cbeta-1, a major isoform of mammalian PP2C, in the TAK1 signaling pathway, a stress-activated protein kinase cascade that is activated by interleukin-1, transforming growth factor-beta, or stress . Ectopic expression of PP2Cbeta-1 inhibited the TAK1-mediated mitogen-activated protein kinase kinase 4-c-Jun amino-terminal kinase and mitogen-activated protein kinase kinase 6-p38 signaling pathways . In vitro, PP2Cbeta-1 dephosphorylated and inactivated TAK1 . Coimmunoprecipitation experiments indicated that PP2Cbeta-1 associates with the central region of TAK1 . A phosphatase-negative mutant of PP2Cbeta-1, PP2Cbeta-1 (R/G), acted as a dominant negative mutant, inhibiting dephosphorylation of TAK1 by wild-type PP2Cbeta-1 in vitro . In addition, ectopic expression of PP2Cbeta-1(R/G) enhanced interleukin-1-induced activation of an AP-1 reporter gene . Collectively, these results indicate that PP2Cbeta negatively regulates the TAK1 signaling pathway by direct dephosphorylation of TAK1.

Oncogene, 2000 Nov 9, 19(47), 5314 - 23
Reduction of Cdc25A contributes to cyclin E1-Cdk2 inhibition at senescence in human mammary epithelial cells; Sandhu C et al.; Replicative senescence may be an important tumor suppressive mechanism for human cells . We investigated the mechanism of cell cycle arrest at senescence in human mammary epithelial cells (HMECs) that have undergone a period of 'self-selection', and as a consequence exhibit diminished p16INK4A levels . As HMECs approached senescence, the proportion of cells with a 2N DNA content increased and that in S phase decreased progressively . Cyclin D1-cdk4, cyclin E-cdk2 and cyclin A-cdk2 activities were not abruptly inhibited, but rather diminished steadily with increasing population age . In contrast to observations in fibroblast, p21Cip1 was not increased at senescence in HMECs . There was no increase in p27Kip1 levels nor in KIP association with targets cdks . While p15INK4B and its binding to both cdk4 and cdk6 increased with increasing passage, some cyclin D1-bound cdk4 and cdk6 persisted in senescent cells, whose inhibition could not be attributed to p15INK4B . The inhibition of cyclin E-cdk2 in senescent HMECs was accompanied by increased inhibitory phosphorylation of cdk2, in association with a progressive loss of Cdc25A . Recombinant Cdc25A strongly reactivated cyclin E-cdk2 from senescent HMECs suggesting that reduction of Cdc25A contributes to cyclin E-cdk2 inhibition and G1 arrest at senescence . Although ectopic expression of Cdc25A failed to extend the lifespan of HMECs, the exogenous Cdc25A appeared to lack activity in these cells, since it neither shortened the G1-to-S phase interval nor activated cyclin E-cdk2 . In contrast, in the breast cancer-derived MCF-7 line, Cdc25A overexpression increased both cyclin E-cdk2 activity and the S phase fraction . Thus, mechanisms leading to HMEC immortalization may involve not only the re-induction of Cdc25A expression, but also activation of this phosphatase.

Curr Opin Chem Biol, 2000 Dec, 4(6), 632 - 8
Dissecting and manipulating the pathway for glycosylphos-phatidylinositol-anchor biosynthesis; Kinoshita T et al.; The pathway for glycosylphosphatidylinositol-anchor biosynthesis consists of at least 10 reaction steps . Many of the genes encoding the enzymes and regulators involved in this pathway have been recently cloned and their products characterised . These studies have revealed the common and also different characteristics of glycosylphosphatidyl-inositol biosynthesis enzymes in different organisms, leading to the development of species-specific inhibitors of the pathway.

Curr Biol, 2000 Nov 16, 10(22), 1463 - 6
The Drosophila RAD21 cohesin persists at the centromere region in mitosis; Warren WD et al.; 'Cohesin' is a highly conserved multiprotein complex thought to be the primary effector of sister-chromatid cohesion in all eukaryotes . Cohesin complexes in budding yeast hold sister chromatids together from S phase until anaphase, but in metazoans, cohesin proteins dissociate from chromosomes and redistribute into the whole cell volume during prophase, well before sister chromatids separate (reviewed in {1,2}) . Here we address this apparent anomaly by investigating the cell-cycle dynamics of DRAD21, the Drosophila orthologue of the Xenopus XRAD21 and Saccharomyces cerevisiae Scc1p/Mcd1p cohesins {3} . Analysis of DRAD21 in S2 Drosophila tissue culture cells and live embryos expressing a DRAD21-green fluorescent protein (GFP) fusion revealed the presence of four distinct subcellular pools of DRAD21: a cytoplasmic pool; a chromosome-associated pool which dissociates from chromatin as chromosomes condense in prophase; a short-lived centrosome-associated pool present during metaphase-anaphase; and a centromere-proximal pool which remains bound to condensed chromosomes, is found along the junction of sister chromatids between kinetochores, and persists until the metaphase-anaphase transition . We conclude that in Drosophila, and possibly all metazoans, a minor pool of cohesin remains bound to centromere-proximal chromatin after prophase and maintains sister-chromatid cohesion until the metaphase-anaphase transition.

Curr Biol, 2000 Nov 16, 10(22), 1427 - 37
A novel Dictyostelium RasGEF is required for normal endocytosis, cell motility and multicellular development; Wilkins A et al.; BACKGROUND: Dictyostelium possesses a surprisingly large number of Ras proteins and little is known about their activators, the guanine nucleotide exchange factors (GEFs) . It is also unclear, in Dictyostelium or in higher eukaryotes, whether Ras pathways are linear, with each Ras controlled by its own GEF, or networked, with multiple GEFs acting on multiple Ras proteins . RESULTS: We have identified the Dictyostelium gene that encodes RasGEFB, a protein with homology to known RasGEFs such as the Son-of-sevenless (Sos) protein . Dictyostelium cells in which the gene for RasGEFB was disrupted moved unusually rapidly, but lost the ability to perform macropinocytosis and therefore to grow in liquid medium . Crowns, the sites of macropinocytosis, were replaced by polarised lamellipodia . Mutant cells were also profoundly defective in early development, although they eventually formed tiny but normally proportioned fruiting bodies . This defect correlated with loss of discoidin Igamma mRNA, a starvation-induced gene, although other genes required for development were expressed normally or even precociously . RasGEFB was able to rescue a Saccharomyces CDC25 mutant, indicating that it is a genuine GEF for Ras proteins . CONCLUSIONS: RasGEFB appears to be the principal activator of the RasS protein, which regulates macropinocytosis and cell speed, but it also appears to regulate one or more other Ras proteins.

Mol Biol Cell, 2000 Dec, 11(12), 4403 - 11
The TRAPP complex is a nucleotide exchanger for Ypt1 and Ypt31/32; Jones S et al.; In yeast, the Ypt1 GTPase is required for ER-to-cis-Golgi and cis-to-medial-Golgi protein transport, while Ypt31/32 are a functional pair of GTPases essential for exit from the trans-Golgi . We have previously identified a Ypt1 guanine nucleotide exchange factor (GEF) activity and characterized it as a large membrane-associated protein complex that localizes to the Golgi and can be extracted from the membrane by salt, but not by detergent . TRAPP is a large protein complex that is required for ER-to-Golgi transport and that has properties similar to those of Ypt1 GEF . Here we show that TRAPP has Ypt1 GEF activity . GST-tagged Bet3p or Bet5p, two of the TRAPP subunits, were expressed in yeast cells and were precipitated by glutathione-agarose (GA) beads . The resulting precipitates can stimulate both GDP release and GTP uptake by Ypt1p . The majority of the Ypt1 GEF activity associated with the GST-Bet3p precipitate has an apparent molecular weight of > 670 kDa, indicating that the GEF activity resides in the TRAPP complex . Surprisingly, TRAPP can also stimulate nucleotide exchange on the Ypt31/32 GTPases, but not on Sec4p, a Ypt-family GTPase required for the last step of the exocytic pathway . Like the previously characterized Ypt1 GEF, the TRAPP Ypt1-GEF activity can be inhibited by the nucleotide-free Ypt1-D124N mutant protein . This mutant protein also inhibits the Ypt32 GEF activity of TRAPP . Coprecipitation and overexpression studies suggest that TRAPP can act as a GEF for Ypt1 and Ypt31/32 in vivo . These data suggest the exciting possibility that a GEF complex common to Ypt1 and Ypt31/32 might coordinate the function of these GTPases in entry into and exit from the Golgi.

Mol Biol Cell, 2000 Dec, 11(12), 4259 - 75
Exocyst is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins; Lipschutz JH et al.; Epithelial cyst and tubule formation are critical processes that involve transient, highly choreographed changes in cell polarity . Factors controlling these changes in polarity are largely unknown . One candidate factor is the highly conserved eight-member protein complex called the exocyst . We show that during tubulogenesis in an in vitro model system the exocyst relocalized along growing tubules consistent with changes in cell polarity . In yeast, the exocyst subunit Sec10p is a crucial component linking polarized exocytic vesicles with the rest of the exocyst complex and, ultimately, the plasma membrane . When the exocyst subunit human Sec10 was exogenously expressed in epithelial Madin-Darby canine kidney cells, there was a selective increase in the synthesis and delivery of apical and basolateral secretory proteins and a basolateral plasma membrane protein, but not an apical plasma membrane protein . Overexpression of human Sec10 resulted in more efficient and rapid cyst formation and increased tubule formation upon stimulation with hepatocyte growth factor . We conclude that the exocyst plays a central role in the development of epithelial cysts and tubules.

Mol Biol Cell, 2000 Dec, 11(12), 4051 - 65
A snc1 endocytosis mutant: phenotypic analysis and suppression by overproduction of dihydrosphingosine phosphate lyase; Grote E et al.; The v-SNARE proteins Snc1p and Snc2p are required for fusion of secretory vesicles with the plasma membrane in yeast . Mutation of a methionine-based sorting signal in the cytoplasmic domain of either Sncp inhibits Sncp endocytosis and prevents recycling of Sncp to the Golgi after exocytosis . snc1-M43A mutant yeast have reduced growth and secretion rates and accumulate post-Golgi secretory vesicles and fragmented vacuoles . However, cells continue to grow and secrete for several hours after de novo Snc2-M42A synthesis is repressed . DPL1, the structural gene for dihydrosphingosine phosphate lyase, was selected as a high copy number snc1-M43A suppressor . Because DPL1 also partially suppresses the growth and secretion phenotypes of a snc deletion, we propose that enhanced degradation of dihydrosphingosine-1-phosphate allows an alternative protein to replace Sncp as the secretory vesicle v-SNARE.

Genetics, 2000 Dec, 156(4), 1717 - 25
Specific genetic interference with behavioral rhythms in Drosophila by expression of inverted repeats; Martinek S et al.; We describe a new experimental technique that allows for a tissue-specific reduction of gene activity in the Drosophila nervous system . On the basis of the observation that certain gene functions can be ubiquitously blocked by injecting double-stranded RNA into Drosophila embryos, we employed a method to interfere with an individual gene function permanently in a predetermined cell type . This was achieved by the formation of an inverted-repeat RNA sequence in the tissue of interest under control of the GAL4/UAS binary expression system . As an example, we show that inverted-repeat-mediated interference with the period gene produces a hypomorphic period phenotype . A selective decrease of period RNA appears to be a component of the cellular response.

Genetics, 2000 Dec, 156(4), 1559 - 71
The Aspergillus nidulans xprF gene encodes a hexokinase-like protein involved in the regulation of extracellular proteases; Katz ME et al.; The extracellular proteases of Aspergillus nidulans are produced in response to limitation of carbon, nitrogen, or sulfur, even in the absence of exogenous protein . Mutations in the A . nidulans xprF and xprG genes have been shown to result in elevated levels of extracellular protease in response to carbon limitation . The xprF gene was isolated and sequence analysis indicates that it encodes a 615-amino-acid protein, which represents a new type of fungal hexokinase or hexokinase-like protein . In addition to their catalytic role, hexokinases are thought to be involved in triggering carbon catabolite repression . Sequence analysis of the xprF1 and xprF2 alleles showed that both alleles contain nonsense mutations . No loss of glucose or fructose phosphorylating activity was detected in xprF1 or xprF2 mutants . There are two possible explanations for this observation: (1) the xprF gene may encode a minor hexokinase or (2) the xprF gene may encode a protein with no hexose phosphorylating activity . Genetic evidence suggests that the xprF and xprG genes are involved in the same regulatory pathway . Support for this hypothesis was provided by the identification of a new class of xprG(-) mutation that suppresses the xprF1 mutation and results in a protease-deficient phenotype.

J Biol Chem, 2001 Mar 30, 276(13), 10185 - 90 Epub 2000 Nov 21.
The structure of the C4C4 ring finger of human NOT4 reveals features distinct from those of C3HC4 RING fingers; Hanzawa H et al.; The NOT4 protein is a component of the CCR4.NOT complex, a global regulator of RNA polymerase II transcription . Human NOT4 (hNOT4) contains a RING finger motif of the C(4)C(4) type . We expressed and purified the N-terminal region of hNOT4 (residues 1-78) encompassing the RING finger motif and determined the solution structure by heteronuclear NMR . NMR experiments using a (113)Cd-substituted hNOT4 RING finger showed that two metal ions are bound through cysteine residues in a cross-brace manner . The three-dimensional structure of the hNOT4 RING finger was refined with root mean square deviation values of 0.58 +/- 0.13 A for the backbone atoms and 1.08 +/- 0.12 A for heavy atoms . The hNOT4 RING finger consists of an alpha-helix and three long loops that are stabilized by zinc coordination . The overall folding of the hNOT4 RING finger is similar to that of the C(3)HC(4) RING fingers . The relative orientation of the two zinc-chelating loops and the alpha-helix is well conserved . However, for the other regions, the secondary structural elements are distinct.

Nat Struct Biol, 2000 Dec, 7(12), 1156 - 64
Crystal structures of ribosome anti-association factor IF6; Groft CM et al.; Ribosome anti-association factor eIF6 (originally named according to translation initiation terminology as eukaryotic initiation factor 6) binds to the large ribosomal subunit, thereby preventing inappropriate interactions with the small subunit during initiation of protein synthesis . We have determined the X-ray structures of two IF6 homologs, Methanococcus jannaschii archaeal aIF6 and Sacchromyces cerevisiae eIF6, revealing a phylogenetically conserved 25 kDa protein consisting of five quasi identical alpha/beta subdomains arrayed about a five-fold axis of pseudosymmetry . Yeast eIF6 prevents ribosomal subunit association . Comparative protein structure modeling with other known archaeal and eukaryotic homologs demonstrated the presence of two conserved surface regions, one or both of which may bind the large ribosomal subunit.

EMBO J, 2000 Dec 1, 19(23), 6582 - 91
RNA helicase dynamics in pre-mRNA splicing; Schwer B et al.; The DExH-box NTPase/helicase Prp22p plays two important roles in pre-mRNA splicing . It promotes the second transesterification reaction and then catalyzes the ATP-dependent release of mature mRNA from the spliceosome . Evidence that helicase activity is important emerged from the analysis of Prp22p motif III (SAT) mutations that uncouple the NTPase and helicase activities . We find that S635A and T637A hydrolyse ATP, but are defective in unwinding duplex RNA and releasing mRNA from the spliceosome . The S635A mutation is lethal in vivo at </=30 degrees C and results in slow growth at 34-37 degrees C . Further insights into helicase action during splicing were gleaned by isolating and characterizing intragenic suppressors of prp22-S635A . Biochemical analysis of the S27 suppressor protein showed that a second mutation of Val539 to Ile in motif Ia revived the helicase activity of the S635A mutant together with the ability to catalyze mRNA release . These findings underscore the tight correlation of RNA unwinding and spliceosome disassembly and demonstrate how suppressor analysis can be used to dissect the subtle internal domain dynamics of helicase action.

EMBO J, 2000 Dec 1, 19(23), 6498 - 507
Spt4 modulates Rad26 requirement in transcription-coupled nucleotide excision repair; Jansen LE et al.; The nucleotide excision repair machinery can be targeted preferentially to lesions in transcribed sequences . This mode of DNA repair is referred to as transcription-coupled repair (TCR) . In yeast, the Rad26 protein, which is the counterpart of the human Cockayne syndrome B protein, is implicated specifically in TCR . In a yeast strain genetically deprived of global genome repair, a deletion of RAD26 renders cells UV sensitive and displays a defect in TCR . Using a genome-wide mutagenesis approach, we found that deletion of the SPT4 gene suppresses the rad26 defect . We show that suppression by the absence of Spt4 is specific for a rad26 defect and is caused by reactivation of TCR in a Rad26-independent manner . Spt4 is involved in the regulation of transcription elongation . The absence of this regulation leads to transcription that is intrinsically competent for TCR . Our findings suggest that Rad26 acts as an elongation factor rendering transcription TCR competent and that its requirement can be modulated by Spt4.

EMBO J, 2000 Dec 1, 19(23), 6475 - 88
Nud1p links astral microtubule organization and the control of exit from mitosis; Gruneberg U et al.; The budding yeast spindle pole body (SPB) not only organizes the astral and nuclear microtubules but is also associated with a number of cell-cycle regulators that control mitotic exit . Here, we describe that the core SPB component Nud1p is a key protein that functions in both processes . The astral microtubule organizing function of Nud1p is mediated by its interaction with the gamma-tubulin complex binding protein Spc72p . This function of Nud1p is distinct from its role in cell-cycle control: Nud1p binds the spindle checkpoint control proteins Bfa1p and Bub2p to the SPB, and is part of the mitotic exit network (MEN) in which it functions upstream of CDC15 but downstream of LTE1 . In conditional lethal nud1-2 cells, the MEN component Tem1p, a GTPase, is mislocalized, whereas the kinase Cdc15p is still associated with the SPB . Thus, in nud1-2 cells the failure of Tem1p to interact with Cdc15p at the SPB probably prevents mitotic exit.

EMBO J, 2000 Dec 1, 19(23), 6440 - 52
LHS1 and SIL1 provide a lumenal function that is essential for protein translocation into the endoplasmic reticulum; Tyson JR et al.; Lhs1p is an Hsp70-related chaperone localized in the endoplasmic reticulum (ER) lumen . Deltalhs1 mutant cells are viable but are constitutively induced for the unfolded protein response (UPR) . Here, we demonstrate a severe growth defect in Deltaire1Deltalhs1 double mutant cells in which the UPR can no longer be induced . In addition, we have identified a UPR- regulated gene, SIL1, whose overexpression is sufficient to suppress the Deltaire1Deltalhs1 growth defect . SIL1 encodes an ER-localized protein that interacts directly with the ATPase domain of Kar2p (BiP), suggesting some role in modulating the activity of this vital chaperone . SIL1 is a non-essential gene but the Deltalhs1Deltasil1 double mutation is lethal and correlates with a complete block of protein translocation into the ER . We conclude that the IRE1-dependent induction of SIL1 is a vital adaptation in Deltalhs1 cells, and that the activities associated with the Lhs1 and Sil1 proteins constitute an essential function required for protein translocation into the ER . The Sil1 protein appears widespread amongst eukaryotes, with homologues in Yarrowia lipolytica (Sls1p), Drosophila and mammals.

EMBO J, 2000 Dec 1, 19(23), 6392 - 400
The role of the TIM8-13 complex in the import of Tim23 into mitochondria; Paschen SA et al.; Tim8 and Tim13 are non-essential, conserved proteins of the mitochondrial intermembrane space, which are organized in a hetero-oligomeric complex . They are structurally related to Tim9 and Tim10, essential components of the import machinery for mitochondrial carrier proteins . Here we show that the TIM8-13 complex interacts with translocation intermediates of Tim23, which are partially translocated across the outer membrane but not with fully imported or assembled Tim23 . The TIM8-13 complex binds to the N-terminal or intermediate domain of Tim23 . It traps the incoming precursor in the intermembrane space thereby preventing retrograde translocation . The TIM8-13 complex is strictly required for import of Tim23 under conditions when a low membrane potential exists in the mitochondria . The human homologue of Tim8 is encoded by the DDP1 (deafness/dystonia peptide 1) gene, which is associated with the Mohr-Tranebjaerg syndrome (MTS), a progressive neurodegenerative disorder leading to deafness . It is demonstrated that import of human Tim23 is dependent on a high membrane potential . A mechanism to explain the pathology of MTS is discussed.

Biochemistry, 2000 Dec 5, 39(48), 14720 - 7
Heterodimer formation between superoxide dismutase and its copper chaperone; Lamb AL et al.; Copper, zinc superoxide dismutase (SOD1) is activated in vivo by the copper chaperone for superoxide dismutase (CCS) . The molecular mechanisms by which CCS recognizes and docks with SOD1 for metal ion insertion are not well understood . Two models for the oligomerization state during copper transfer have been proposed: a heterodimer comprising one monomer of CCS and one monomer of SOD1 and a dimer of dimers involving interactions between the two homodimers . We have investigated protein-protein complex formation between copper-loaded and apo yeast CCS (yCCS) and yeast SOD1 for both wild-type SOD1 (wtSOD1) and a mutant SOD1 in which copper ligand His 48 has been replaced with phenylalanine (H48F-SOD1) . According to gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and chemical cross-linking experiments, yCCS and this mutant SOD1 form a complex with the correct molecular mass for a heterodimer . No higher order oligomers were detected . Heterodimer formation is facilitated by the presence of zinc but does not depend on copper loading of yCCS . The complex formed with H48F-SOD1 is more stable than that formed with wtSOD1, suggesting that the latter is a more transient species . Notably, heterodimer formation between copper-loaded yCCS and wtSOD1 is accompanied by SOD1 activation only in the presence of zinc . These findings, taken together with structural, biochemical, and genetic studies, strongly suggest that in vivo copper loading of yeast SOD1 occurs via a heterodimeric intermediate.

Int J Med Microbiol, 2000 Oct, 290(6), 549 - 58
Molecular characterization and influence on fungal development of ALP2, a novel serine proteinase from Aspergillus fumigatus; Reichard U et al.; A novel subtilisin-related serine proteinase (ALP2) {EC 3.4.21.48} with a broad range of activity between pH 4.5 and 11.0 was released from a cell wall fraction of Aspergillus fumigatus by an alkaline pH shift . The enzyme which was not detected in the culture supernatant was partially purified by phenylbutylamine agarose chromatography . The N-terminal sequence revealed that ALP2 is the same protein identified as the major allergen of A . fumigatus in patients suffering from extrinsic bronchial asthma (Shen et al . 1999, Int . Arch . Allergy Immunol . 119, 259-264) . Based on this N-terminal sequence and on a conserved region of fungal subtilisins, a specific PCR probe was generated and the ALP2 genomic and cDNA were isolated from corresponding phage libraries . ALP2 shares a 49% identity with the vacuolar proteinase B (PrB) of Saccharomyces cerevisiae . In addition there is a 78% identity with PEPC, a serine proteinase which has been described in Aspergillus niger . Targeted disruption of the ALP2-encoding gene resulted in a slightly decreased speed of vegetative growth and in a more than 80% reduction of sporulation in the alp2-negative mutants, correlated with an approximately 50% reduction of the median diameter of conidiophore vesicles . The requirement of ALP2 for regular sporulation, in addition to its role in allergic asthma, raises further interest in cellular proteinases in respect to morphogenesis and pathogenesis in A . fumigatus.

Nature, 2000 Nov 23, 408(6811), 488 - 92
A ubiquitin-like system mediates protein lipidation; Ichimura Y et al.; Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole . Apg8/Aut7 is an essential factor for autophagy in yeast . We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus . Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane . Here we report a new mode of protein lipidation . Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine . This lipidation is mediated by a ubiquitination-like system . Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref . 9) . Apg7 activates two different ubiquitin-like proteins, Apg12 (ref . 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref . 11) and Apg3, respectively . These reactions are necessary for the formation of Apg8-phosphatidylethanolamine . This lipidation has an essential role in membrane dynamics during autophagy.

J Antibiot (Tokyo), 2000 Sep, 53(9), 903 - 11
Phellinsin A, a novel chitin synthases inhibitor produced by Phellinus sp . PL3; Hwang EI et al.; Phellinsin A, a novel chitin synthases inhibitor was isolated from the cultured broth of fungus PL3, which was identified as Phellinus sp . PL3 . Phellinsin A was purified by solvent partition, silica gel, ODS column chromatographies, and preparative HPLC, consecutively . The structure of phellinsin A was assigned as a phenolic compound on the basis of various spectroscopic analyses including UV, IR, Mass, and NMR . Its molecular weight and formula were found to be 358 and C18H14O8, respectively . Phellinsin A selectively inhibited chitin synthase I and II of Saccharomyces cerevisiae with an IC50 value of 76 and 28 microg/ml, respectively, in our cell free assay system . This compound showed antifungal activity against Colletotrichum lagenarium, Pyricularia oryzae, Rhizoctonia solani, Aspergillus fumigatus, and Trichophyton mentagrophytes.

Nature, 2000 Nov 16, 408(6810), 381 - 6
Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex; Schulman BA et al.; F-box proteins are members of a large family that regulates the cell cycle, the immune response, signalling cascades and developmental programmes by targeting proteins, such as cyclins, cyclin-dependent kinase inhibitors, IkappaBalpha and beta-catenin, for ubiquitination (reviewed in refs 1-3) . F-box proteins are the substrate-recognition components of SCF (Skp1-Cullin-F-box protein) ubiquitin-protein ligases . They bind the SCF constant catalytic core by means of the F-box motif interacting with Skp1, and they bind substrates through their variable protein-protein interaction domains . The large number of F-box proteins is thought to allow ubiquitination of numerous, diverse substrates . Most organisms have several Skp1 family members, but the function of these Skp1 homologues and the rules of recognition between different F-box and Skp1 proteins remain unknown . Here we describe the crystal structure of the human F-box protein Skp2 bound to Skp1 . Skp1 recruits the F-box protein through a bipartite interface involving both the F-box and the substrate-recognition domain . The structure raises the possibility that different Skp1 family members evolved to function with different subsets of F-box proteins, and suggests that the F-box protein may not only recruit substrate, but may also position it optimally for the ubiquitination reaction.

Gene Expr, 2000, 9(1-2), 37 - 61
Role of histone acetylation in the assembly and modulation of chromatin structures; Annunziato AT et al.; The acetylation of the core histone N-terminal "tail" domains is now recognized as a highly conserved mechanism for regulating chromatin functional states . The following article examines possible roles of acetylation in two critically important cellular processes: replication-coupled nucleosome assembly, and reversible transitions in chromatin higher order structure . After a description of the acetylation of newly synthesized histones, and of the likely acetyltransferases involved, an overview of histone octamer assembly is presented . Our current understanding of the factors thought to assemble chromatin in vivo is then described . Genetic and biochemical investigations of the function the histone tails, and their acetylation, in nucleosome assembly are detailed, followed by an analysis of the importance of histone deacetylation in the maturation of newly replicated chromatin . In the final section the involvement of the histone tail domains in chromatin higher order structures is addressed, along with the role of histone acetylation in chromatin folding . Suggestions for future research are offered in the concluding remarks.

Radiat Res, 2000 Dec, 154(6), 650 - 8
Correlation between the clonogenic initial slope and the response of polykaryon-forming units: the behavior of strains defective in XRCC5 and ATM and the heritability of small variations in radioresponse; Manti L et al.; The polykaryon-forming unit (PFU) assay measures the survival of multiple cycles of DNA synthesis after exposure to ionizing radiation, and it is known that there is a strong correlation between the slope of the PFU dose-response curve and the clonogenic initial slope . This suggests that DNA lesions expressed in clonogens are also important in PFU . Cells having a mutation in XRCC5 (also known as Ku80; strain xrs-6) and ATM (strain AT5BIVA) were hypersensitive in the PFU assay and in clonogens, while a strain of xrs-6 cells transfected with hamster wild-type XRCC5 cDNA displayed wild-type resistance in both assays . These data suggest that the DNA double-strand break (DSB) is an important lesion in PFU, although the relative radioresistance of PFU compared to clonogens indicates differential DSB toxicity . We propose that this results from the absence of cytokinesis-related loss of DNA fragments . Small variations in the radioresponse of PFU were observed between CHO K1 cell substrains, such that the xrs parental substrain RR-CHOK1 (carrying wild-type XRCC5) was more sensitive than an independent K1 substrain (E-CHOK1) . Somatic hybridization showed that this variation is heritable and that the resistant E phenotype is dominant . In RR-CHOK1 cells there was a biphasic PFU radioresponse, which suggests that there may be transient expression at a locus selectively affecting PFU sensitivity.

Radiat Res, 2000 Dec, 154(6), 609 - 15
cDNA expression array analysis of DNA repair genes in human glioma cells that lack or express DNA-PK; Galloway AM et al.; M059J cells provide the only example of DNA-PKcs (now known as PRKDC) deficiency in a human cell line . M059K cells, derived from the same tumor specimen, express PRKDC protein and activity and, together with M059J, provide a useful model in which to study the role of DNA-PK in cellular responses to DNA-damaging agents . Because these cells are of tumor origin, we used Atlas human cancer cDNA expression arrays to investigate possible differential expression of other DNA repair genes in control and irradiated samples . cDNA array results indicated differential expression of 14 genes . Northern blotting confirmed relatively greater expression of replication factor C 37-kDa subunit mRNA in M059J cells compared to M059K cells and reduced expression of DNA ligase IV compared to ligase III in both cell lines independent of irradiation . These results suggest that other DNA repair proteins are altered in these cell lines and that repair mechanisms predicted from the study of normal tissues may be fundamentally altered in human cancer cells.

Biochem Biophys Res Commun, 2000 Nov 30, 278(3), 685 - 90
Role of NAD(+) in the deacetylase activity of the SIR2-like proteins; Landry J et al.; In this report we describe the role of NAD(+) in the deacetylation reaction catalyzed by the SIR2 family of enzymes . We first show that the products of the reaction detected by HPLC analysis are ADP-ribose, nicotinamide, and a deacetylated peptide substrate . These products are in a 1:1:1 molar ratio, indicating that deacetylation involves the hydrolysis of one NAD(+) to ADP-ribose and nicotinamide for each acetyl group removed . Three results suggest that deacetylation requires an enzyme-ADP-ribose intermediate . First, the enzyme can promote an NAD(+) if nicotinamide exchange reaction that depends on an acetylated substrate . Second, a non-hydrolyzable NAD(+) analog is a competitive inhibitor of the enzyme, and, third, nicotinamide shows product inhibition of deacetylase activity .

Biochem Biophys Res Commun, 2000 Nov 30, 278(3), 659 - 64
Nuclear translocation of TSC-22 (TGF-beta-stimulated clone-22) concomitant with apoptosis: TSC-22 as a putative transcriptional regulator; Hino S et al.; We examined the alteration of the subcellular localization of TSC-22 (TGF-beta-stimulated clone-22) after induction of apoptosis and the transcription-regulatory activity of TSC-22 . In the living cells, TSC-22-green fluorescent protein (GFP) fusion protein was clearly localized to the cytoplasm, however, in the apoptotic cells, the TSC-22-GFP fusion protein was translocated from the cytoplasm to the nucleus . TSC-22 fused to GAL4-DNA binding domain (GAL4BD) did not show the transcriptional activity on the reporter genes in yeast and in HSG (salivary gland cancer cells) and Hela . However, in CHO cells, TSC-22-GAL4BD fusion protein strongly activated the reporter gene . The transcriptional activity of the leucine zipper structure of TSC-22 is greater than that of the full-length TSC-22 . These findings suggest that after receiving the apoptotic stimuli, TSC-22 translocates from the cytoplasm to the nucleus and shows the transcription-regulatory activity .

Nucleic Acids Res . 2000 Dec 1;28(23):E102.
Design and calibration of a semi-synthetic DNA phasing assay; Hardwidge PR et al.; Electrophoretic assays of intrinsic DNA shape and shape changes induced by ligand binding are extremely useful because of their convenience and simplicity . The development of calibrations and empirical quantitative relationships permits highly accurate measurement of DNA shape using electrophoresis . Many conventional analyses employ the unidirectional ligation of short DNA duplexes . However, many oligonucleotides (typically more than 20) must often be synthesized for a single experiment . Additionally, the length of the DNA duplex can become limiting, preventing the analysis of certain DNA sequences . We now describe a semi-synthetic electrophoretic phasing method that offers several advantages, including a reduced number of required synthetic oligonucleotides, the ability to analyze longer DNA duplexes and a simplified approach for data analysis . We characterize semi-synthetic DNA probes in electrophoretic phasing assays by ligation of synthetic duplexes containing A(5) tracts between two longer restriction fragments . Upon electrophoresis, the gel mobility is strongly correlated with the predicted DNA curvature provided by the reference A(5) tracts . Having obtained this calibration, we show that the semi-synthetic phasing assay can be readily and economically applied to analyze DNA curvature induced by DNA charge modifications and DNA bending due to peptide binding.

Nucleic Acids Res, 2000 Dec 1, 28(23), 4783 - 9
Essential regions of the tRNA primer required for HIV-1 infectivity; Yu Q et al.; Human immunodeficiency virus (HIV), like all retroviruses, requires a cellular tRNA as a primer for initiation of reverse transcription . In a previous study, we demonstrated that an HIV-1 with a primer binding site complementary to yeast tRNA(Phe) (psHIV-Phe) was not infectious unless yeast tRNA(Phe) was supplied in trans . This unique in vivo complementation system has now been used to define the elements of the tRNA required for HIV-1 replication . Mutant tRNA(Phe) with deletions in TPsiC stem-loop, anticodon stem-loop or D stem-loop of the tRNA were generated and assessed for the capacity to rescue psHIV-Phe . Mutant tRNA(Phe) with disrupted TPsiC stem-loop did not rescue psHIV-Phe . In contrast, a mutant tRNA(Phe) without the D stem-loop was fully functional for the rescue . The tRNA anticodon stem-loop region was found to be important for efficient complementation . The results of our studies demonstrate for the first time the importance of specific structural and sequence elements of the tRNA primer for HIV-1 reverse transcription and define new targets for interruption of HIV-1 replication.

Nucleic Acids Res, 2000 Dec 1, 28(23), 4778 - 82
A Ku80 fragment with dominant negative activity imparts a radiosensitive phenotype to CHO-K1 cells; Marangoni E et al.; DNA non-homologous end joining, the major mechanism for the repair of DNA double-strands breaks (DSB) in mammalian cells requires the DNA-dependent protein kinase (DNA-PK), a complex composed of a large catalytic subunit of 460 kDa (DNA-PKcs) and the heterodimer Ku70-Ku80 that binds to double-stranded DNA ends . Mutations in any of the three subunits of DNA-PK lead to extreme radiosensitivity and DSB repair deficiency . Here we show that the 283 C-terminal amino acids of Ku80 introduced into the Chinese hamster ovary cell line CHO-K1 have a dominant negative effect . Expression of Ku(449-732) in CHO cells was verified by northern blot analysis and resulted in decreased Ku-dependent DNA end-binding activity, a diminished capacity to repair DSBs as determined by pulsed field gel electrophoresis and decreased radioresistance determined by clonogenic survival . The stable modifications observed at the molecular and cellular level suggest that this fragment of Ku80 confers a dominant negative effect providing an important mechanism to sensitise radioresistant cells.

Nucleic Acids Res, 2000 Dec 1, 28(23), 4665 - 73
Signaling through regulated transcription factor interaction: mapping of a regulatory interaction domain in the Myb-related Bas1p; Pinson B et al.; Gene activation in eukaryotes is inherently combinatorial depending on cooperation between different transcription factors . An example where this cooperation seems to be directly exploited for regulation is the Bas1p/Bas2p couple in yeast . Bas1p is a Myb-related transcription factor that acts together with the homeodomain-related Bas2p (Pho2p) to regulate purine and histidine biosynthesis genes in response to extracellular purine limitation . We show that fusion of the two factors abolished adenine repression, suggesting that what is regulated by adenine is the Bas1p-Bas2p interaction . Analysis of Bas1p deletions revealed a critical domain (Bas1p interaction and regulatory domain, BIRD) acting in two-hybrid assays as an adenine-dependent Bas1p-Bas2p interaction domain . BIRD had a dual function, as an internal repressor of a centrally located Bas1p transactivation domain on the ADE1 promoter and as a Bas2p-dependent activator on the HIS4 promoter . This promoter-dependent behavior reflected a differential binding to the two promoters in vivo . On ADE1 Bas1p bound the promoter efficiently by itself, but required adenine limitation and Bas2p interaction through BIRD for derepression . On HIS4 efficient promoter binding and derepression required both factors and adenine limitation . We propose a promoter-dependent model for adenine regulation in yeast based on controlled Bas1p-Bas2p interactions through BIRD and exploited differentially by the two promoters.

Nucleic Acids Res, 2000 Dec 1, 28(23), 4634 - 41
Cisplatin-DNA adducts inhibit translocation of the Ku subunits of DNA-PK; Turchi JJ et al.; We have determined the effect of cisplatin-DNA damage on the ability of the DNA-dependent protein kinase (DNA-PK) to interact with duplex DNA molecules in vitro . The Ku DNA binding subunits of DNA-PK display a reduced ability to translocate on duplex DNA containing cisplatin-DNA adducts compared to control, undamaged duplex DNA . The decreased rates of translocation resulted in a decrease in the association of the p460 catalytic subunit of DNA-PK (DNA-PKcs) with the Ku-DNA complex . In addition to a decrease in DNA-PKcs association, the DNA-PKcs that is bound with Ku at a DNA end containing cisplatin-DNA adducts has a reduced catalytic rate compared to heterotrimeric DNA-PK assembled on undamaged DNA . The position of the cisplatin-DNA lesion from the terminus also effects kinase activation, with maximal inhibition occurring when the lesion is closer to the terminus . These results are consistent with a model for DNA-PK activation where the Ku dimer translocates away from the DNA terminus and facilitates the association of DNA-PKcs which interacts with both Ku and DNA resulting in kinase activation . The presence of cisplatin adducts decreases the ability to translocate away from the terminus and results in the formation of inactive kinase complexes at the DNA terminus . The results are discussed with respect to the ability of cisplatin to sensitize cells to DNA damage induced by ionizing radiation and the ability to repair DNA double-strand breaks.

FEBS Lett, 2000 Nov 24, 485(2-3), 189 - 94
Function of the cytosolic N-terminus of sucrose transporter AtSUT2 in substrate affinity; Schulze W et al.; AtSUT2 was found to be a low-affinity sucrose transporter (K(M)=11.7 mM at pH 4) . Chimeric proteins between AtSUT2 and the high-affinity StSUT1 were constructed in which the extended N-terminus and central loop of AtSUT2 were exchanged with those domains of StSUT1 and vice versa . Chimeras containing the N-terminus of AtSUT2 showed significantly lower affinity for sucrose compared to chimeras containing the N-terminus of StSUT1 . The results indicate a significant function of the N-terminus but not the central cytoplasmic loop in determining substrate affinity . Expression of AtSUT2 in major veins of source leaves and in flowers is compatible with a role as a second low-affinity sucrose transporter or as a sucrose sensor.

Mol Cell Biol, 2000 Dec, 20(24), 9262 - 70
Role of HSP90 in salt stress tolerance via stabilization and regulation of calcineurin; Imai J et al.; The role of HSP90 in stress tolerance was investigated in Saccharomyces cerevisiae . Cells showing 20-fold overexpression of Hsc82, an HSP90 homologue in yeast, were hypersensitive to high-NaCl or H-LiCl stresses . Hsc82-overexpressing cells appeared similar to calcineurin-defective cells in salt sensitivity and showed reduced levels of calcineurin-dependent gene expression . Co-overexpression of Cna2, the catalytic subunit of calcineurin, suppressed the hypersensitivity . Cna2 and Hsc82 coimmunoprecipitated from control cells grown under normal conditions but not from stressed cells . In contrast, coimmunoprecipitation was detected with Hsc82-overexpressing cells even after exposure to stresses . Cna2 immune complexes from stressed control cells showed a significant level of calcineurin activity, whereas those from stressed Hsc82-overexpressing cells did not . Treatment of extracts from Hsc82-overexpressing cells with Ca(2+)-calmodulin increased the calcineurin activity associated with Cna2 immune complexes . Geldanamycin, an inhibitor of HSP90 abolished the coimmunoprecipitation but did not activate calcineurin . When the expression level of Hsc82 decreased to below 30% of the normal level, cells also became hypersensitive to salt stress . In these cells, the amount of Cna2 was reduced, likely as a result of degradation . The present results showed that Hsc82 binds to and stabilizes Cna2 and that dissociation of Cna2 from Hsc82 is necessary for its activation.

J Biol Chem, 2001 Mar 2, 276(9), 6112 - 8 Epub 2000 Nov 28.
Mitochondrial Hsp70 Ssc1: role in protein folding; Liu Q et al.; Ssc1, the major Hsp70 of the mitochondrial matrix, is involved in the translocation of proteins from the cytosol into the matrix and their subsequent folding . To better understand the physiological mechanism of action of this Hsp70, we have undertaken a biochemical analysis of Ssc1 and two mutant proteins, Ssc1--2 and Ssc1--201 . ssc1--2 is a temperature-sensitive mutant defective in both translocation and folding; ssc1--201 contains a second mutation in this ssc1 gene that suppresses the temperature-sensitive growth defect of ssc1--2, correcting the translocation but not the folding defect . We found that although Ssc1 was competent to facilitate the refolding of denatured luciferase in vitro, both Ssc1--2 and Ssc1--201 showed significant defects, consistent with the data obtained with isolated mitochondria . Purified Ssc1--2 had a lowered affinity for a peptide substrate compared with wild-type Ssc1 but only in the ADP-bound state . This peptide binding defect was reversed in the suppressor protein Ssc1--201 . However, a defect in the ability of Hsp40 to stimulate the ATPase activity of Ssc1--2 was not corrected in Ssc1--201 . Thus, the inability of these two mutant proteins to efficiently facilitate luciferase refolding correlates with their defect in stimulation of ATPase activity by Hsp40s, indicating that this interaction is critical for protein folding in mitochondria.

J Biol Chem, 2001 Mar 2, 276(9), 6105 - 11 Epub 2000 Nov 28.
RINT-1, a novel Rad50-interacting protein, participates in radiation-induced G(2)/M checkpoint control; Xiao J et al.; Rad50, an structural maintenance of chromosomes (SMC) protein family member, participates in a variety of cellular processes, including DNA double-strand break repair, cell cycle checkpoint activation, telomere maintenance, and meiosis . Disruption of Rad50 in mice leads to lethality during early embryogenesis, indicating its essential function in normal proliferating cells . In addition to its ability to form a complex with the DNA double-strand break repair proteins Mre11 and NBS1, Rad50 may interact with other cellular proteins to execute its full range of biological activities . A novel 87-kDa protein named RINT-1 was identified using the C-terminal region of human Rad50 as the bait in a yeast two-hybrid screen . Human RINT-1 shares sequence homology with a novel protein identified in Drosophila melanogaster, including a coiled-coil domain within its N-terminal 150 amino acids, a conserved central domain of about 350 amino acids, and a C-terminal region of 90 amino acids exhibiting 35--38% identity . The conserved central and C-terminal regions of RINT-1 are required for its interaction with Rad50 . While Rad50 and RINT-1 are both expressed throughout the cell cycle, RINT-1 specifically binds to Rad50 only during late S and G(2)/M phases, suggesting that RINT-1 may be involved in cell cycle regulation . Consistent with this possibility, MCF-7 cells expressing an N-terminally truncated RINT-1 protein displayed a defective radiation-induced G(2)/M checkpoint . These results suggest that RINT-1 may play a role in the regulation of cell cycle control after DNA damage.

J Biol Chem, 2001 Mar 2, 276(9), 6463 - 7 Epub 2000 Nov 28.
Regulation of APG14 expression by the GATA-type transcription factor Gln3p; Chan TF et al.; Gln3p is a nitrogen catabolite repression-sensitive GATA-type transcription factor . Its nuclear accumulation was recently shown to be under the control of TOR signaling . Gln3p normally resides in the cytoplasm . When cells are starved from nitrogen nutrients or treated with rapamycin, however, Gln3p becomes translocated into the nucleus, thereby activating the expression of genes involved in nitrogen utilization and transport . To identify other genes under the control of Gln3p, we searched for the Gln3p-binding GATAA motifs within 500 base pairs of the promoter sequences upstream of the yeast open reading frames in the Saccharomyces Genome Database . APG14, a gene essential for autophagy, was found to have the most GATAA motifs . We show that nitrogen starvation or rapamycin treatment rapidly causes a more than 20-fold induction of APG14 . The expression of APG14 is dependent on Gln3p; deletion of Gln3p severely reduced its induction by rapamycin, whereas depletion of Ure2p caused its constitutive expression . However, overexpression of APG14 led to only a slight increase in autophagy in nitrogen-rich medium . Therefore, these results define a signaling cascade leading to the expression of APG14 in response to the availability of nitrogen nutrients and suggest that the regulated expression of APG14 contributes to but is not sufficient for the control of autophagy.

J Biol Chem, 2001 Feb 9, 276(6), 4509 - 21 Epub 2000 Nov 28.
The PEA3 Ets transcription factor comprises multiple domains that regulate transactivation and DNA binding; Bojovic BB et al.; PEA3, a member of the Ets family of transcription factors, is a nuclear phosphoprotein capable of activating transcription . Mouse PEA3 comprises 480 amino acids and bears an approximately 85-amino acid ETS domain near its carboxyl terminus . Whereas analyses of bacterially expressed PEA3 revealed that the ETS domain is required for sequence-specific DNA binding, little is known of the functional domains in the protein required for its activity in mammalian cells . To this end, we defined the location of the PEA3 functional domains in COS cells . PEA3 bears a strong activation domain near its amino terminus, which is flanked by two regions that independently negatively regulate its activity . PEA3 expressed in COS cells was incapable of binding to DNA in vitro . However, DNA binding activity could be unmasked by incubation with a PEA3-specific antibody . Analyses of the DNA binding activity of PEA3 deletion mutants revealed that two regions flanking the ETS domain independently inhibited DNA binding; deletion of both regions was required to detect DNA binding in the absence of a PEA3-specific antibody . Under these conditions, the ETS domain was sufficient for sequence-specific DNA binding . These findings suggest that the activity of PEA3 is exquisitely controlled at multiple functional levels.

Proteins, 2001 Jan 1, 42(1), 23 - 37
Fold recognition and accurate query-template alignment by a combination of PSI-BLAST and threading; Shan Y et al.; A homology-based structure prediction method ideally gives both a correct fold assignment and an accurate query-template alignment . In this article we show that the combination of two existing methods, PSI-BLAST and threading, leads to significant enhancement in the success rate of fold recognition . The combined approach, termed COBLATH, also yields much higher alignment accuracy than found in previous studies . It consists of two-way searches both by PSI-BLAST and by threading . In the PSI-BLAST portion, a query is used to search for hits in a library of potential templates and, conversely, each potential template is used to search for hits in a library of queries . In the threading portion, the scoring function is the sum of a sequence profile and a 6x6 substitution matrix between predicted query and known template secondary structure and solvent exposure . "Two-way" in threading means that the query's sequence profile is used to match the sequences of all potential templates and the sequence profiles of all potential templates are used to match the query's sequence . When tested on a set of 533 nonhomologous proteins, COBLATH was able to assign folds for 390 (73%) . Among these 390 queries, 265 (68%) had root-mean-square deviations (RMSDs) of less than 8 A between predicted and actual structures . Such high success rate and accuracy make COBLATH an ideal tool for structural genomics.

Cell Physiol Biochem, 2000, 10(4), 203 - 8
h-sgk serine-threonine protein kinase as transcriptional target of p38/MAP kinase pathway in HepG2 human hepatoma cells; Waldegger S et al.; The human serum and glucocorticoid dependent serine/threonine kinase h-sgk has previously been discovered as cell volume regulated gene . The present study has been performed to elucidate the involvement of p38-kinase in the transcriptional control of h-sgk by osmotic cell shrinkage . The p38-kinase has previously been cloned as the mammalian homologue of HOG1 kinase, which constitutes a part of the osmosensor in the yeast Saccharomyces cerevisiae . Phosphorylated (active) p38-kinase has been estimated with Western blotting, transcription of hsgk using Northern blotting . Both, increase of extracellular NaCl concentration by 50 mmol/l and addition of 10 micromol/l anisomycin increase phosphorylation of the p38-kinase within 5 to 10 minutes . h-sgk transcription is upregulated by addition of 50 mmol/l NaCl and by anisomycin (10 micromol/l), effects completely inhibited by the specific p38-kinase inhibitor, SB 203580 (10 micromol/l) . In conclusion, the stimulation of h-sgk transcription by osmotic cell shrinkage is mediated by p38-kinase .

Biochemistry (Mosc), 2000 Oct, 65(10), 1129 - 34
Affinity labeling of RNA-polymerase II in the transcriptionally active complex by a phosphorylating analog of the initiation substrate; Savinkova LK et al.; Affinity modification of RNA-polymerase II by a phosphorylating analog of the initiation substrate carrying a zwitterionic 5;-terminal phosphate group with a 4-N,N-dimethylaminopyridine residue (DMAP-pA) was studied during specific transcription initiation controlled by the late adenoviral promotor . Super-selective affinity labeling and standard conditions of affinity modification resulted in labeling a polypeptide with molecular weight corresponding to that of the third subunit of the enzyme, RPB3 (45 kD) . The initiation substrate (ATP) protects RNA-polymerase II from modification . The third subunit may be involved in the formation of the substrate-binding site of the enzyme.

DNA Seq, 2000, 11(3-4), 315 - 9
A recQ family DNA helicase gene from Aspergillus nidulans; Appleyard MV et al.; We have identified an Aspergillus nidulans gene encoding a RecQ family helicase which we have therefore named recQ . The A . nidulans recQ protein is most closely related in sequence to human recQ helicase 5 . Like the latter polypeptide, A . nidulans recQ consists of little more than the conserved helicase domain, lacking the long amino- and carboxy-terminal extensions seen in other recQ family members such as BLM and WRN and in the sole RecQ family helicase of the yeast Saccharomyces cerevisiae (Sgs1p) . By analogy with other eukaryotic RecQ helicases, A . nidulans recQ helicase is likely to play an important role in the maintenance of genomic integrity.

DNA Seq, 2000, 11(3-4), 309 - 14
Cloning and molecular characterization of a gene encoding a Cryptosporidium parvum putative 20S proteasome beta1-type subunit; Chung PA et al.; A DNA sequence composed of 1281 nucleotides (nt) consisting of a single open reading frame (ORF) encoding a putative 20S proteasome beta1-type subunit was isolated from clones derived from genomic libraries constructed from the KSU-1 isolate of Cryptosporidium parvum . Southern blot analysis suggested that the sequenced DNA exists in the C . parvum genome as a single copy; transcription was verified through reverse transcription-polymerase chain reaction (RT-PCR) performed on total RNA isolated from C . parvum sporozoites . The predicted protein consists of 210 amino acids (aa), contains characteristic amino acids common to all proteasomal subunits, and shares stronger similarity to the beta1-type subunit of yeast than to other types of beta-subunits.

Mol Cell, 2000 Oct, 6(4), 769 - 80
A histone variant, Htz1p, and a Sir1p-like protein, Esc2p, mediate silencing at HMR; Dhillon N et al.; Silencing at HMR requires silencers, and one of the roles of the silencer is to recruit Sir proteins . This work focuses on the function of Sir1p once it is recruited to the silencer . We have generated mutants of Sir1p that are recruited to the silencer but are unable to silence, and we have utilized these mutants to identify four proteins, Sir3p, Sir4p, Esc2p, and Htz1p, that when overexpressed, restored silencing . The isolation of Sir3p and Sir4p validated this screen . Molecular analysis suggested that Esc2p contributed to silencing in a manner similar to Sir1p and probably helped recruit or stabilize the other Sir proteins, while Htz1p present at HMR assembled a specialized chromatin structure necessary for silencing.

Science, 2000 Nov 24, 290(5496), 1571 - 4
Biochemical basis of oxidative protein folding in the endoplasmic reticulum; Tu BP et al.; The endoplasmic reticulum (ER) supports disulfide bond formation by a poorly understood mechanism requiring protein disulfide isomerase (PDI) and ERO1 . In yeast, Ero1p-mediated oxidative folding was shown to depend on cellular flavin adenine dinucleotide (FAD) levels but not on ubiquinone or heme, and Ero1p was shown to be a FAD-binding protein . We reconstituted efficient oxidative folding in vitro using FAD, PDI, and Ero1p . Disulfide formation proceeded by direct delivery of oxidizing equivalents from Ero1p to folding substrates via PDI . This kinetic shuttling of oxidizing equivalents could allow the ER to support rapid disulfide formation while maintaining the ability to reduce and rearrange incorrect disulfide bonds.

J Virol, 2000 Dec, 74(24), 11522 - 30
Correct integration of model substrates by Ty1 integrase; Moore SP et al.; The retrovirus-like mobile genetic element of Saccharomyces cerevisiae, Ty1, transposes to new genomic locations via the element-encoded integrase (IN) . Here we report that purified recombinant IN catalyzed correct integration of a linear DNA into a supercoiled target plasmid . Ty1 virus-like particles (VLPs) integrated donor DNA more efficiently than IN . VLP and IN-mediated insertions occurred at random sites in the target . Mg(2+) was preferred over Mn(2+) for correct integration, and neither cation enhanced nonspecific nuclease activity of IN . Products consistent with correct integration events were also obtained by Southern analysis . Recombinant IN and VLPs utilized many, but not all, linear donor fragments containing non-Ty1 ends, including a U3 mutation which has been shown to be defective for transposition in vivo . Together, our results suggest that IN is sufficient for Ty1 integration in vitro and IN interacts with exogenous donors less stringently than with endogenous elements.

Genes Dev, 2000 Nov 15, 14(22), 2807 - 12
Ku acts in a unique way at the mammalian telomere to prevent end joining; Hsu HL et al.; Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusion events and to distinguish the chromosome ends from double-strand breaks . We report that TRF1 and Ku form a complex at the telomere . The Ku and TRF1 complex is a specific high-affinity interaction, as demonstrated by several in vitro methods, and exists in human cells as determined by coimmunoprecipitation experiments . Ku does not bind telomeric DNA directly but localizes to telomeric repeats via its interaction with TRF1 . Primary mouse embryonic fibroblasts that are deficient for Ku80 accumulated a large percentage of telomere fusions, establishing that Ku plays a critical role in telomere capping in mammalian cells . We propose that Ku localizes to internal regions of the telomere via a high-affinity interaction with TRF1 . Therefore, Ku acts in a unique way at the telomere to prevent end joining.

Nature, 2000 Nov 9, 408(6809), 255 - 62
Genetic pathways that regulate ageing in model organisms; Guarente L et al.; Searches for genes involved in the ageing process have been made in genetically tractable model organisms such as yeast, the nematode Caenorhabditis elegans, Drosophila melanogaster fruitflies and mice . These genetic studies have established that ageing is indeed regulated by specific genes, and have allowed an analysis of the pathways involved, linking physiology, signal transduction and gene regulation . Intriguing similarities in the phenotypes of many of these mutants indicate that the mutations may also perturb regulatory systems that control ageing in higher organisms.

Nature, 2000 Nov 9, 408(6809), 225 - 9
A transcription reinitiation intermediate that is stabilized by activator; Yudkovsky N et al.; High levels of gene transcription by RNA polymerase II depend on high rates of transcription initiation and reinitiation . Initiation requires recruitment of the complete transcription machinery to a promoter, a process facilitated by activators and chromatin remodelling factors . Reinitiation probably occurs through a different pathway . After initiation, a subset of the transcription machinery remains at the promoter, forming a platform for assembly of a second transcription complex . Here we describe the isolation of a reinitiation intermediate that includes transcription factors TFIID, TFIIA, TFIIH, TFIIE and Mediator . This intermediate can act as a scaffold for formation of a functional reinitiation complex . Formation of this scaffold is dependent on ATP and TFIIH . The scaffold is stabilized in the presence of the activator Gal4-VP16, but not Gal4-AH, suggesting a new role for some activators and Mediator in promoting high levels of transcription.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13027 - 32
A nucleolar protein related to ribosomal protein L7 is required for an early step in large ribosomal subunit biogenesis; Dunbar DA et al.; The Saccharomyces cerevisiae Rlp7 protein has extensive identity and similarity to the large ribosomal subunit L7 proteins and shares an RNA-binding domain with them . Rlp7p is not a ribosomal protein; however, it is encoded by an essential gene and therefore must perform a function essential for cell growth . In this report, we show that Rlp7p is a nucleolar protein that plays a critical role in processing of precursors to the large ribosomal subunit RNAs . Pulse-chase labeling experiments with Rlp7p-depleted cells reveal that neither 5.8S(S), 5.8S(L), nor 25S is produced, indicating that both the major and minor processing pathways are affected . Analysis of processing intermediates by primer extension indicates that Rlp7p-depleted cells accumulate the 27SA(3) precursor RNA, which is normally the major substrate (85%) used to produce the 5.8S and 25S rRNAs, and the ratio of 27SB(L) to 27SB(S) precursors changes from approximately 1:8 to 8:1 (depleted cells) . Because 27SA(3) is the direct precursor to 27SB(S), we conclude that Rlp7p is specifically required for the 5' to 3' exonucleolytic trimming of the 27SA(3) into the 27SB(S) precursor . As it is essential for processing in both the major and minor pathways, we propose that Rlp7p may act as a specificity factor that binds precursor rRNAs and tethers the enzymes that carry out the early 5' to 3' exonucleolytic reactions that generate the mature rRNAs . Rlp7p may also be required for the endonucleolytic cleavage in internal transcribed spacer 2 that separates the 5.8S rRNA from the 25S rRNA.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13021 - 6
Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy; Talaga DS et al.; We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4 . The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus . Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer . End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea) . The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass . As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface . The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations . Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale . A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics . The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states . Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

Biochemistry, 2000 Nov 21, 39(46), 14103 - 12
Biochemical and structural analysis of the interaction between the UBA(2) domain of the DNA repair protein HHR23A and HIV-1 Vpr; Withers-Ward ES et al.; The DNA repair protein HHR23A is a highly conserved protein that functions in nucleotide excision repair . HHR23A contains two ubiquitin associated domains (UBA) that are conserved in a number of proteins with diverse functions involved in ubiquitination, UV excision repair, and signaling pathways via protein kinases . The cellular binding partners of UBA domains remain unclear; however, we previously found that the HHR23A UBA(2) domain interacts specifically with the HIV-1 Vpr protein . Analysis of the low resolution solution structure of HHR23A UBA(2) revealed a hydrophobic loop region of the UBA(2) domain that we predicted was the interface for protein/protein interactions . Here we present results of in vitro binding studies that demonstrate the requirement of this hydrophobic loop region for interaction with human immunodeficiency virus (HIV-1) Vpr . A single point mutation of the Pro at residue 333 to a Glu totally abolishes the binding of HIV-1 Vpr to UBA(2) . High resolution NMR structures of the binding deficient UBA(2) mutant P333E as well as of the wild-type UBA(2) domain were determined to compare the effect of this mutation on the structure . Small but significant differences are observed only locally at the site of the mutation . The biochemical and structural analysis confirms the function of the HHR23A UBA(2) GFP-loop as the protein/protein interacting domain.

J Biol Chem, 2001 Feb 16, 276(7), 5403 - 11 Epub 2000 Nov 21.
The N-terminal domain of rat liver carnitine palmitoyltransferase 1 contains an internal mitochondrial import signal and residues essential for folding of its C-terminal catalytic domain; Cohen I et al.; We have previously shown that the first 147 N-terminal residues of the rat liver carnitine palmitoyltransferase 1 (CPT1), encompassing its two transmembrane (TM) segments, specify both mitochondrial targeting and anchorage at the outer mitochondrial membrane (OMM) . In the present study, we have identified the precise import sequence in this polytopic OMM protein . In vitro import studies with fusion and deletion CPT1 proteins demonstrated that none of its TM segments behave as a signal anchor sequence . Analysis of the regions flanking the TM segments revealed that residues 123-147, located immediately downstream of TM2, function as a noncleavable, matrix-targeting signal . They specify mitochondrial targeting, whereas the hydrophobic TM segment(s) acts as a stop-transfer sequence that stops and anchors the translocating CPT1 into the OMM . Heterologous expression in Saccharomyces cerevisiae of several deleted CPT1 proteins not only confirms the validity of the "stop-transfer" import model but also indicates that residues 1-82 of CPT1 contain a putative microsomal targeting signal whose cellular significance awaits further investigation . Finally, we identified a highly folded core within the C-terminal domain of CPT1 that is hidden in the entire protein by its cytosolic N-terminal residues . Functional analysis of the deleted CPT1 proteins indicates that this folded C-terminal core, which may belong to the catalytic domain of CPT1, requires TM2 for its correct folding achievement and is in close proximity to residues 1-47.

J Biol Chem, 2001 Feb 16, 276(7), 5052 - 8 Epub 2000 Nov 21.
Regulator of G protein signaling 8 (RGS8) requires its NH2 terminus for subcellular localization and acute desensitization of G protein-gated K+ channels; Saitoh O et al.; Functional roles of the NH(2)-terminal region of RGS (regulators of G protein signaling) 8 in G protein signaling were studied . The deletion of the NH(2)-terminal region of RGS8 (DeltaNRGS8) resulted in a partial loss of the inhibitory function in pheromone response of yeasts, although Galpha binding was not affected . To examine roles in subcellular distribution, we coexpressed two fusion proteins of RGS8-RFP and DeltaNRGS8-GFP in DDT1MF2 cells . RGS8-RFP was highly concentrated in nuclei of unstimulated cells . Coexpression of constitutively active Galpha(o) resulted in translocation of RGS8 protein to the plasma membrane . In contrast, DeltaNRGS8-GFP was distributed diffusely through the cytoplasm in the presence or absence of active Galpha(o) . When coexpressed with G protein-gated inwardly rectifying K(+) channels, DeltaNRGS8 accelerated both turning on and off similar to RGS8 . Acute desensitization of G protein-gated inwardly rectifying K(+) current observed in the presence of RGS8, however, was not induced by DeltaNRGS8 . Thus, we, for the first time, showed that the NH(2) terminus of RGS8 contributes to the subcellular localization and to the desensitization of the G protein-coupled response.

J Immunol, 2000 Dec 1, 165(11), 6347 - 55
Ku86 variant expression and function in multiple myeloma cells is associated with increased sensitivity to DNA damage; Tai YT et al.; Ku is a heterodimer of Ku70 and Ku86 that binds to double-stranded DNA breaks (DSBs), activates the catalytic subunit (DNA-PKcs) when DNA is bound, and is essential in DSB repair and V(D)J recombination . Given that abnormalities in Ig gene rearrangement and DNA damage repair are hallmarks of multiple myeloma (MM) cells, we have characterized Ku expression and function in human MM cells . Tumor cells (CD38(+)CD45RA(-)) from 12 of 14 (86%) patients preferentially express a 69-kDa variant of Ku86 (Ku86v) . Immunoblotting of whole cell extracts (WCE) from MM patients shows reactivity with Abs targeting Ku86 N terminus (S10B1) but no reactivity with Abs targeting Ku86 C terminus (111), suggesting that Ku86v has a truncated C terminus . EMSA confirmed a truncated C terminus in Ku86v and further demonstrated that Ku86v in MM cells had decreased Ku-DNA end binding activity . Ku86 forms complexes with DNA-PKcs and activates kinase activity, but Ku86v neither binds DNA-PKcs nor activates kinase activity . Furthermore, MM cells with Ku86v have increased sensitivity to irradiation, mitomycin C, and bleomycin compared with patient MM cells or normal bone marrow donor cells with Ku86 . Therefore, this study suggests that Ku86v in MM cells may account for decreased DNA repair and increased sensitivity to radiation and chemotherapeutic agents, whereas Ku86 in MM cells confers resistance to DNA damaging agents . Coupled with a recent report that Ku86 activity correlates with resistance to radiation and chemotherapy, these results have implications for the potential role of Ku86 as a novel therapeutic target.

J Cell Biol, 2000 Nov 27, 151(5), 1057 - 66
Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit; Ho JH et al.; In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran . However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified . We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit . Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits . Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p . 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES . To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B . These results identify Crm1p as the receptor for Nmd3p export . Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.

J Cell Biol, 2000 Nov 27, 151(5), 1047 - 56
Chromosomal addresses of the cohesin component Mcd1p; Laloraya S et al.; We identified the chromosomal addresses of a cohesin subunit, Mcd1p, in vivo by chromatin immunoprecipitation coupled with high resolution PCR-based chromosomal walking . The mapping of new Mcd1p-binding sites (cohesin-associated regions {CARs}) in single-copy sequences of several chromosomes establish their spacing ( approximately 9 kb), their sequestration to intergenic regions, and their association with AT-rich sequences as general genomic properties of CARs . We show that cohesins are not excluded from telomere proximal regions, and the enrichment of cohesins at the centromere at mitosis reflects de novo loading . The average size of a CAR is 0.8-1.0 kb . They lie at the boundaries of transcriptionally silenced regions, suggesting they play a direct role in defining the silent chromatin domain . Finally, we identify CARs in tandem (rDNA) and interspersed repetitive DNA (Ty2 and subtelomeric repeats) . Each 9-kb rDNA repeat has a single CAR proximal to the 5S gene . Thus, the periodicity of CARs in single-copy regions and the rDNA repeats is conserved . The presence and spacing of CARs in repetitive DNA has important implications for genomic stability and chromosome packaging/condensation.

Biochem J, 2000 Dec 1, 352 Pt 2, 409 - 18
The peroxisomal targeting sequence type 1 receptor, Pex5p, and the peroxisomal import efficiency of alanine:glyoxylate aminotransferase; Knott TG et al.; Unlike most organellar proteins, some peroxisomal proteins are often found in significant amounts in the cytosol . Such apparent import inefficiency is very marked in guinea pig (Cavia porcellus) hepatocytes in which the cytosolic levels of two peroxisomal proteins, catalase and alanine:glyoxylate aminotransferase (AGT), are much higher than those found in human (Homo sapiens) hepatocytes, for example . In an attempt to provide an explanation for this phenomenon, we have cloned the guinea pig CpPEX5 gene, which encodes the peroxisomal targeting sequence type 1 (PTS1) import receptor Pex5p, and functionally compared it with its human homologue, HsPex5p . Our results showed the following: (1) CpPEX5, like its human homologue, encodes two splice variants differing by the presence or absence of an internal region of 37 amino acids; (2) both variants were expressed in all guinea pig tissues studied; (3) both variants were equally able to complement peroxisomal import of PTS1 proteins in microinjected Deltapex5 human fibroblasts; (4) CpPex5p was as efficient as HsPex5p in mediating the peroxisomal import of proteins possessing the consensus PTS1, Ser-Lys-Leu, but much less efficient in mediating the import of proteins possessing non-consensus PTS1s (i.e . Lys-Lys-Leu of human AGT and Ala-Asn-Leu of human catalase); (5) reporter proteins with the consensus PTS1, Ser-Lys-Leu, inhibited the peroxisomal import of endogenous catalase, whereas AGT with the non-consensus Lys-Lys-Leu did not; (6) high concentrations of HsPex5p, but not CpPex5p, markedly inhibited the import of AGT, but not catalase or proteins ending in Ser-Lys-Leu; and (7) in the yeast two-hybrid system, AGT-Ser-Lys-Leu interacted with the tetratricopeptide repeat domain of HsPex5p, but AGT-Lys-Lys-Leu did not . In addition, AGT-Ser-Lys-Leu was targeted to peroxisomes in Saccharomyces cerevisiae, whereas AGT-Lys-Lys-Leu was not . These data suggest that the inefficient peroxisomal import of AGT and catalase in guinea pig cells is due to the inefficiency with which CpPex5p mediates the peroxisomal import of proteins containing non-consensus PTS1s . They also suggest that the non-consensus PTS1 of human AGT might interact with HsPex5p very differently compared with the consensus PTS1, Ser-Lys-Leu.

Pediatr Dermatol, 2000 Sep-Oct, 17(5), 381 - 3
A juvenile case of overlap syndrome of systemic lupus erythematosus and polymyositis, later accompanied by systemic sclerosis with the development of anti-Scl 70 and anti-Ku antibodies; Nitta Y et al.; We describe a 16-year-old girl with an overlap syndrome consisting of systemic lupus erythematosus (SLE) from the age of 7 and polymyositis (PM) from the age of 10, later accompanied by systemic sclerosis (SSc) from the age of 15 . She was diagnosed as having SLE with exudative malar erythema, photosensitivity, and discoid rashes with positive antinuclear antibody (ANA) and anti-DNA antibody titers . The diagnosis of PM was also made in accordance with findings of a high titer of muscle enzymes and a muscle biopsy specimen demonstrating marked degeneration of the muscle fibers and perivascular infiltration of mononuclear cells . She developed Raynaud's phenomenon and pitting ulcers on her fingers with positive anti-Scl 70 and anti-Ku antibodies, leading to a diagnosis of SSc . The patient was treated with prednisolone . To our knowledge this is the youngest case of SLE-PM overlap syndrome later accompanied by SSc.

J Biol Inorg Chem, 2000 Oct, 5(5), 584 - 92
Changing the heme ligation in flavocytochrome b2: substitution of histidine-66 by cysteine; Mowat CG et al.; Substitution by cysteine of one of the heme iron axial ligands (His66) of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase from Saccharomyces cerevisiae) has resulted in an enzyme (H66C-b2) which remains a competent L-lactate dehydrogenase (kcat 272+/-6 s(-1), L-lactate KM 0.60+/-0.06 mM, 25 degrees C, I 0.10, Tris-HCl, pH 7.5) but which has no cytochrome c reductase activity . As a result of the mutation, the reduction potential of the heme was found to be -265+5 mV, over 240 mV more negative than that of the wild-type enzyme, and therefore unable to be reduced by L-lactate . Surface-enhanced resonance Raman spectroscopy indicates similarities between the heme of H66C-b2 and those of cytochromes P450, with a nu4 band at 1,345 cm(-1) which is indicative of cysteine heme-iron ligation . In addition, EPR spectroscopy yields g-values at 2.33, 2.22 and 1.94, typical of low-spin ferric cytochromes P450, optical spectra show features between 600 and 900 nm which are characteristic of sulfur coordination of the heme iron, and MCD spectroscopy shows a blue-shifted NIR CT band relative to the wild-type, implying that the H66C-b2 heme is P450-like . Interestingly, EPR evidence also suggests that the second histidine heme-iron ligand (His43) is displaced in the mutant enzyme.

Biotechniques, 2000 Nov, 29(5), 1084 - 90, 1092
High-accuracy DNA sequence variation screening by DHPLC; Spiegelman JI et al.; Genetic maps based on biallelic single-nucleotide polymorphisms amenable to microarray-based genotyping have significantly accelerated the mapping of mono- and multigenic traits in model organisms such as Saccharomyces cerevisiae and Arabidopsis thaliana . This advance needs to be matched by highly accurate, inexpensive and robust methodology for fine-structure mapping of the candidate region(s) and the eventual identification of the causative mutation(s) . To establish the usefulness of denaturing high-performance liquid chromatography (DHPLC) for those purposes, we have amplified 476 fragments from two A . thaliana ecotypes with an average length of 563 bp covering various candidate regions on chromosomes 1, 2 and 4 . Parallel analysis by DHPLC and dye terminator sequencing showed that DHPLC detected 165 out of 166 polymorphic fragments with only four false positives, amounting to a sensitivity, specificity and accuracy of 99.4%, 98.7% and 99%, respectively . It proved beneficial to analyze the fragments not only at the highest but also at the lower temperatures recommended by the algorithm freely available at inverted question markinsertion.stanford.edu/melt.html.

Trends Biochem Sci, 2000 Nov, 25(11), 548 - 55
What does 'chromatin remodeling' mean?
Aalfs JD, Kingston RE.
The regulated alteration of chromatin structure, termed 'chromatin remodeling', can be accomplished by covalent modification of histones or by the action of ATP-dependent remodeling complexes . A variety of mechanisms can be used to remodel chromatin; some act locally on a single nucleosome and others act more broadly . It is critical to establish a direct connection between the remodeling events observed in vivo and the mechanistic capabilities of remodeling complexes in vitro.

Curr Biol, 2000 Nov 2, 10(21), R781 - 4
Mitosis: shutting the door behind when you leave; Li R; Yeast cells must segregate sister chromosomes to the opposite sides of the bud neck during mitosis . A pathway has been identified, involving a small GTPase, which prevents the onset of cytokinesis until one of the spindle poles has migrated into the bud.

Curr Biol, 2000 Nov 2, 10(21), 1379 - 82
The Bfa1/Bub2 GAP complex comprises a universal checkpoint required to prevent mitotic exit; Wang Y et al.; At the end of the cell cycle, cyclin-dependent kinase (CDK) activity is inactivated to allow mitotic exit {1} . A protein phosphatase, Cdc14, plays a key role during mitotic exit in budding yeast by activating the Cdh1 component of the anaphase-promoting complex to degrade cyclin B (Clb) and inducing the CDK inhibitor Sic1 to inactivate Cdk1 {2} . To prevent mitotic exit when the cell cycle is arrested at G2/M, cells must prevent CDK inactivation . In the spindle checkpoint pathway, this is accomplished through Bfa1/Bub2, a heteromeric GTPase-activating protein (GAP) that inhibits Clb degradation by keeping the G protein Tem1 inactive {3-5} . Tem1 is required for Cdc14 activation . Here we show that in budding yeast, BUB2 and BFA1 are also required for the maintenance of G2/M arrest in response to DNA damage and to spindle misorientation . cdc13-1 bub2 and cdc13-1 bfa1 but not cdc13-1 mad2 double mutants rebud and reduplicate their DNA at the restrictive temperature . We also found that the delay in mitotic exit in mutants with misoriented spindles depended on BUB2 and BFA1, but not on MAD2 . We propose that Bfa1/Bub2 checkpoint pathway functions as a universal checkpoint in G2/M that prevents CDK inactivation in response to cell-cycle delay in G2/M.

Infect Immun, 2000 Dec, 68(12), 6618 - 23
Antibodies to malaria vaccine candidates Pvs25 and Pvs28 completely block the ability of Plasmodium vivax to infect mosquitoes; Hisaeda H et al.; Transmission-blocking vaccines are one strategy for controlling malaria, whereby sexual-stage parasites are inhibited from infecting mosquitoes by human antibodies . To evaluate whether the recently cloned Plasmodium vivax proteins Pvs25 and Pvs28 are candidates for a transmission-blocking vaccine, the molecules were expressed in yeast as secreted recombinant proteins . Mice vaccinated with these proteins adsorbed to aluminum hydroxide developed strong antibody responses against the immunogens, although for Pvs28, this response was genetically restricted . Antisera against both recombinant Pvs25 and Pvs28 recognized the corresponding molecules expressed by cultured sexual-stage parasites isolated from patients with P . vivax malaria . The development of malaria parasites in mosquitoes was completely inhibited when these antisera were ingested with the infected blood meal . Pvs25 and Pvs28, expressed in Saccharomyces cerevisiae, are as yet the only fully characterized transmission-blocking vaccine candidates against P . vivax that induce such a potent antiparasite response.

Ann N Y Acad Sci, 2000, 919, 33 - 47
Quantitative proteome analysis: methods and applications; Aebersold R et al.; With the completion of a rapidly increasing number of complete genomic sequences, much attention is currently focused on how the information contained in sequence databases might be interpreted in terms of the structure, function, and control of biological systems . Quantitative proteome analysis, the global analysis of protein expression, has been proposed as a method to study steady-state gene expression and perturbation-induced changes . Here, we discuss the rationale for quantitative proteome analysis, highlight the limitations in the current standard technology, and introduce a new experimental approach to quantitative proteome analysis.

Exp Cell Res, 2000 Nov 25, 261(1), 166 - 79
hNMP 200: a novel human common nuclear matrix protein combining structural and regulatory functions; Gotzmann J et al.; Previously we have reported about human nuclear matrix proteins (hNMPs) with increased reassembling and potential filament-forming capability {C . Gerner et al., 1999, J . Cell . Biochem . 74, 145-151} . Here, we cloned the cDNA of one of these proteins, hNMP 200, following partial amino acid sequencing of the novel 56-kDa nuclear protein . Sequence alignments show hNMP 200-related proteins in metazoans, plants, and yeast, the homologous Saccharomyces cerevisiae protein prp19 being an accessory, but essential, factor for pre-mRNA processing . Evidence for any enzymatic activity was not detected . However, the hNMP 200 primary sequence contained five consensus WD-repeat sequences, indicative of participation and regulatory function in larger protein assemblies . Northern blot analysis and 2D protein electrophoresis showed ubiquitous expression of hNMP 200 in a variety of cell types . (35)S labeling studies indicated a high metabolic stability of the protein . The hNMP 200 gene was assigned to chromosomal region 11q12.2 . Confocal laser scanning microscopy revealed that the intracellular localization conformed with that reported for other structural nuclear proteins . In interphase cells, green fluorescent protein-tagged hNMP 200 was predominantly nucleoplasmic . Structures with speckled appearance extended through several sections of in situ-isolated nuclear matrices . During cell division hNMP 200 became irregularly distributed in prophase, sparing regions of condensing chromatin . In anaphase it was concentrated in the spindle midzone . The putative dual function of the novel NMP is discussed . Being a component of the nuclear framework, it may provide structural support for components of the RNA-processing machinery, thereby also modulating splicing activities .

Eur J Biochem, 2000 Dec, 267(23), 6753 - 61
Interplay between repressing and activating domains defines the transcriptional activity of IRF-1; Kirchhoff S et al.; Interferon regulatory factor-1 (IRF-1) is a transcriptional activator with weak activation capacity . By defining the transcriptional activation domain of IRF-1 we identified two activator fragments located between amino acids 185 and 256 functioning in an additive manner . Another fragment of IRF-1, which has no activator function alone, acts as a strong enhancer element of these activator sequences . This enhancer element resides between the activator domains and the C-terminus . In addition, we identified a novel type of inhibitory domain in the N-terminal 60 amino acids of IRF-1 which strongly inhibits its transcriptional activity . Because this fragment is conserved in all interferon regulatory factors, we found similar repression effects in the corresponding fragments in IRF-2, IRF-3 and interferon consensus sequence binding protein (ICSBP/IRF-8) . Interestingly, the corresponding sequence in p48/IRF-9 is divergent, so that it does not show this inhibitory activity . A five-amino-acid sequence distinguishes the p48/IRF-9 N-terminus from the homologous parts in other interferon regulatory factors containing the repressing function . Replacing the diverged amino acids in IRF-1 with the corresponding sequence of p48/IRF-9 resulted in a loss of inhibitory activity within IRF-1 . The opposing activities within interferon regulatory factors may contribute to balanced or tuned regulation of gene activation, depending on the promoter context.

Br J Pharmacol, 2000 Nov, 131(6), 1105 - 12
Direct interaction of Na-azide with the KATP channel; Trapp S et al.; 1 . The effects of the metabolic inhibitor sodium azide were tested on excised macropatches from XENOPUS: oocytes expressing cloned ATP-sensitive potassium (KATP) channels of the Kir6.2/SUR1 type . 2 . In inside-out patches from oocytes expressing Kir6.2 delta C36 (a truncated form of Kir6.2 that expresses in the absence of SUR), intracellular Na-azide inhibited macroscopic currents with an IC50 of 11 mM . The inhibitory effect of Na-azide was mimicked by the same concentration of NaCl, but not by sucrose . 3 . Na-azide and NaCl blocked Kir6.2/SUR1 currents with IC50 of 36 mM and 19 mM, respectively . Inhibition was abolished in the absence of intracellular Mg2+ . In contrast, Kir6.2 delta C36 currents were inhibited by Na-azide both in the presence or absence of intracellular Mg2+ . 4 . Kir6.2/SUR1 currents were less sensitive to 3 mM Na-azide in the presence of MgATP . This apparent reduction in sensitivity is caused by a small activatory effect of Na-azide conferred by SUR . 5 . We conclude that, in addition to its well-established inhibitory effect on cellular metabolism, which leads to activation of KATP channels in intact cells, intracellular Na-azide has direct effects on the KATP channel . Inhibition is intrinsic to Kir6.2, is mediated by Na+, and is modulated by SUR . There is also a small, ATP-dependent, stimulatory effect of Na-azide mediated by the SUR subunit . The direct effects of 3 mM Na-azide on KATP channels are negligible in comparison to the metabolic activation produced by the same Na-azide concentration.

J Cell Sci, 2000 Dec, 113 Pt 24, 4379 - 89
How motor proteins influence microtubule polymerization dynamics; Hunter AW et al.; The interplay between microtubules and microtubule-based motors is fundamental to basic aspects of cellular function, such as the intracellular transport of organelles and alterations in cellular morphology during cell locomotion and division . Motor proteins are unique in that they couple nucleotide hydrolysis to force production that can do work . The force transduction by proteins belonging to the kinesin and dynein superfamilies has been thought only to power movement of these motors along the surface of microtubules; however, a growing body of evidence, both genetic and biochemical, suggests that motors can also directly influence the polymerization dynamics of microtubules . For example, at the vertebrate kinetochore, motors interact directly with microtubule ends and modulate polymerization dynamics to orchestrate chromosome movements during mitosis . Although a role for motors in regulating microtubule length has been established, the mechanisms used by motors to promote microtubule growth or shrinkage are unclear, as is an understanding of why cells might choose motors to control dynamics rather than a variety of non-motor proteins known to affect microtubule stability . Elucidation of the exact mechanisms by which motors alter the exchange of tubulin subunits at microtubule ends in vitro may shed light on how microtubule stability is regulated to produce the array of dynamic behavior seen in cells.

Cell, 2000 Oct 27, 103(3), 449 - 56
Structural basis for the recognition of DNA repair proteins UNG2, XPA, and RAD52 by replication factor RPA; Mer G et al.; Replication protein A (RPA), the nuclear ssDNA-binding protein in eukaryotes, is essential to DNA replication, recombination, and repair . We have shown that a globular domain at the C terminus of subunit RPA32 contains a specific surface that interacts in a similar manner with the DNA repair enzyme UNG2 and repair factors XPA and RAD52, each of which functions in a different repair pathway . NMR structures of the RPA32 domain, free and in complex with the minimal interaction domain of UNG2, were determined, defining a common structural basis for linking RPA to the nucleotide excision, base excision, and recombinational pathways of repairing damaged DNA . Our findings support a hand-off model for the assembly and coordination of different components of the DNA repair machinery.

Cell, 2000 Oct 27, 103(3), 411 - 22
Histone H2A.Z regulats transcription and is partially redundant with nucleosome remodeling complexes; Santisteban MS et al.; Nucleosomes impose a block to transcription that can be overcome in vivo by remodeling complexes such as SNF/SWI and histone modification complexes such as SAGA . Mutations in the major core histones relieve transcriptional repression and bypass the requirement for SNF/SWI and SAGA . We have found that the variant histone H2A.Z regulates gene transcription, and deletion of the gene encoding H2A.Z strongly increases the requirement for SNF/SWI and SAGA . This synthetic genetic interaction is seen at the level of single genes and acts downstream of promoter nucleosome reorganization . H2A.Z is preferentially crosslinked in vivo to intergenic DNA at the PH05 and GAL1 loci, and this association changes with transcriptional activation . These results describe a novel pathway for regulating transcription using variant histones to modulate chromatin structure.

Cell, 2000 Oct 27, 103(3), 387 - 98
Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin; Buonomo SB et al.; It has been proposed but never proven that cohesion between sister chromatids distal to chiasmata is responsible for holding homologous chromosomes together while spindles attempt to pull them toward opposite poles during metaphase of meiosis I . Meanwhile, the mechanism by which disjunction of homologs is triggered at the onset of anaphase I has remained a complete mystery . In yeast, cohesion between sister chromatid arms during meiosis depends on a meiosis-specific cohesin subunit called Rec8, whose mitotic equivalent, Sccl, is cleaved at the metaphase to anaphase transition by an endopeptidase called separin . We show here that cleavage of Rec8 by separin at one of two different sites is necessary for the resolution of chiasmata and the disjunction of homologous chromosomes during meiosis.

Gene, 2000 Oct 31, 257(2), 291 - 8
Characterization of CAX-like genes in plants: implications for functional diversity; Shigaki T et al.; Transporter-mediated Ca(2+) efflux from the cytoplasm is an important component of plant signal transduction . To elucidate the diversity and role of Ca(2+)/H(+) in controlling plant cytosolic Ca(2+) concentrations, homologs of CAX (for calcium exchanger) genes were cloned from Zea mays and Arabidopsis thaliana cDNA libraries . The A . thaliana homolog of CAX (AtHCX1) is 77% identical to CAX1 while the Z . mays homolog of CAX (ZmHCX1) is 64% identical to CAX1 in amino acid sequence . AtHCX1 transcripts appeared to be expressed in all tissues, and levels of AtHCX1 RNA increased after Ca(2+) or Na(+) treatment . When expressed in yeast mutants defective in vacuolar Ca(2+) uptake, ZmHCX1 and AtHCX1 failed to suppress the Ca(2+) sensitivity of these strains . These results imply that CAX-like genes may have functions in plant ion homeostasis that differ from those of previously characterized CAX genes.

Gene, 2000 Oct 31, 257(2), 233 - 42
Genomic cloning and characterization of mitochondrial elongation factor Tu (EF-Tu) gene (tufM) from maize (Zea mays L.); Choi KR et al.; We have cloned and characterized a mitochondrial elongation factor Tu (EF-Tu) gene (tufM) in maize (Zea mays L.) . This maize tufM gene encoded a polypeptide of 452 amino acid residues, consisting of a putative transit peptide of 55 residues and a mature EF-Tu of 397 residues . The coding region was composed of 12 exons and 11 introns that ranged from 76 to 1673bp in length . The deduced amino acid sequence showed 85.9% and 61.2% identity with Arabidopsis mitochondrial EF-Tu and Arabidopsis chloroplast EF-Tu sequence respectively . The transcription initiation site was determined to be 165bp upstream of the AUG initiation codon by primer extension analysis . Southern blot analysis revealed that the cloned EF-Tu gene was encoded by the members of small gene family in maize . Although this gene does not resemble the Arabidopsis nuclear tufA gene, which encodes the plastid EF-Tu, and does not contain sequence elements found in all cyanobacterial and plastid tufA genes, the predicted amino acid sequence includes an N-terminal extension that resembles a mitochondrial targeting sequence, and shares three unique sequence elements with mitochondrial EF-Tu's from Arabidopsis thaliana, Saccharomyces cerevisiae, and Homo sapiens . Therefore, we concluded that this gene encodes the maize mitochondrial EF-Tu.

Gene, 2000 Oct 31, 257(2), 177 - 85
Identification of two catalytic subunits of tRNA splicing endonuclease from Arabidopsis thaliana; Akama K et al.; tRNA splicing endonuclease is essential for the correct removal of introns from precursor tRNA molecules of Archaea and Eucarya . The only well-characterized eucaryotic enzyme until now is the endonuclease from yeast (Saccharomyces cerevisiae) . This protein has a heterotetrameric structure . Two of the four subunits, i.e . Sen34 and Sen44, contain the active sites for cleavage at the 3'- and 5'-splice sites, respectively . We have identified three novel genes from Arabidopsis thaliana, encoding putative subunits of tRNA splicing endonuclease . They are designated as AtSen1, AtSen2, and AtpsSen1 . Both genes AtSen1 and AtSen2 seem to be functionally active, as deduced from corresponding cDNA sequences . Comparison of the amino acid sequences of the these two Arabidopsis proteins revealed 72% identity . However, AtpsSen1 is more similar to AtSen1, but is very likely a pseudogene, as concluded from extended stretches of deletions and the presence of in-frame stop codons . All putative proteins contain a conserved domain at their C-terminus common to counterparts from other organisms . Interestingly, they are more similar to the yeast catalytic subunit Sen44 than to Sen34 . Southern analysis with various probes revealed that each gene is present as single copies in the nuclear genome . The evolutionary implications of these findings are discussed.

J Mol Evol, 2000 Nov, 51(5), 446 - 58
Evolution of the 14-3-3 protein family: does the large number of isoforms in multicellular organisms reflect functional specificity?
Rosenquist M, Sehnke P, Ferl RJ, Sommarin M, Larsson C.
14-3-3 proteins constitute a family of eukaryotic proteins that are key regulators of a large number of processes ranging from mitosis to apoptosis . 14-3-3s function as dimers and bind to particular motifs in their target proteins . To date, 14-3-3s have been implicated in regulation or stabilization of more than 35 different proteins . This number is probably only a fraction of the number of proteins that 14-3-3s bind to, as reports of new target proteins have become more frequent . An examination of 14-3-3 entries in the public databases reveals 153 isoforms, including alleloforms, reported in 48 different species . The number of isoforms range from 2, in the unicellular organism Saccharomyces cerevisiae, to 12 in the multicellular organism Arabidopsis thaliana . A phylogenetic analysis reveals that there are four major evolutionary lineages: Viridiplantae (plants), Fungi, Alveolata, and Metazoa (animals) . A close examination of the aligned amino acid sequences identifies conserved amino acid residues and regions of importance for monomer stabilization, dimer formation, target protein binding, and the nuclear export function . Given the fact that 53% of the protein is conserved, including all amino acid residues in the target binding groove of the 14-3-3 monomer, one might expect little to no isoform specificity for target protein binding . However, using surface plasmon resonance we show that there are large differences in affinity between nine 14-3-3 isoforms of A . thaliana and a target peptide representing a novel binding motif present in the C terminus of the plant plasma membrane H(+)ATPase . Thus, our data suggest that one reason for the large number of isoforms found in multicellular organisms is isoform-specific functions.

EMBO J, 2000 Nov 15, 19(22), 6207 - 17
Structure and mechanism of activity of the cyclic phosphodiesterase of Appr>p, a product of the tRNA splicing reaction; Hofmann A et al.; The crystal structure of the cyclic phosphodiesterase (CPDase) from Arabidopsis thaliana, an enzyme involved in the tRNA splicing pathway, was determined at 2.5 A resolution . CPDase hydrolyzes ADP-ribose 1",2"-cyclic phosphate (Appr>p), a product of the tRNA splicing reaction, to the monoester ADP-ribose 1"-phosphate (Appr-1"p) . The 181 amino acid protein shows a novel, bilobal arrangement of two alphabeta modules . Each lobe consists of two alpha-helices on the outer side of the molecule, framing a three- or four-stranded antiparallel beta-sheet in the core of the protein . The active site is formed at the interface of the two beta-sheets in a water-filled cavity involving residues from two H-X-T/S-X motifs . This previously noticed motif participates in coordination of a sulfate ion . A solvent-exposed surface loop (residues 100-115) is very likely to play a flap-like role, opening and closing the active site . Based on the crystal structure and on recent mutagenesis studies of a homologous CPDase from Saccharomyces cerevisiae, we propose an enzymatic mechanism that employs the nucleophilic attack of a water molecule activated by one of the active site histidines.

EMBO J, 2000 Nov 15, 19(22), 6141 - 9
The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p; Owen DJ et al.; The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes . We report the crystal structure at 1.9 A resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16 . We show that this bromodomain preferentially binds to peptides containing an N:-acetyl lysine residue . Only residues 16-19 of the acetylated peptide interact with the bromodomain . The primary interaction is the N:-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group . A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding . Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other . These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.

EMBO J, 2000 Nov 15, 19(22), 6131 - 40
Chromosomal localization links the SIN3-RPD3 complex to the regulation of chromatin condensation, histone acetylation and gene expression; Pile LA et al.; Acetylation of core histone N-terminal tails influences chromatin condensation and transcription . To examine how the SIN3-RPD3 deacetylase complex contributes to these events in vivo, we examined binding of SIN3 and RPD3 to DROSOPHILA: salivary gland polytene chromosomes . The binding patterns of SIN3 and RPD3 were highly coincident, suggesting that the SIN3-RPD3 complex is the most abundant chromatin-bound RPD3 complex in salivary gland cells . SIN3- RPD3 binding was restricted to less condensed, hypoacetylated euchromatic interbands and was absent from moderately condensed, hyperacetylated euchromatic bands and highly condensed, differentially acetylated centric heterochromatin . Consistent with its demonstrated role in transcriptional repression, SIN3-RPD3 did not co-localize with RNA polymer ase II . Chromatin binding of the complex, mediated by SMRTER, decreased upon ecdysone-induced transcriptional activation but was restored when transcription was reduced . These results implicate SIN3-RPD3 in maintaining histone acetylation levels or patterns within less condensed chromatin domains and suggest that SIN3-RPD3 activity is required, in the absence of an activation signal, to repress transcription of particular genes within transcriptionally active chromatin domains.

EMBO J, 2000 Nov 15, 19(22), 6085 - 97
Nuclear-specific degradation of Far1 is controlled by the localization of the F-box protein Cdc4; Blondel M et al.; Far1 is a bifunctional protein that is required to arrest the cell cycle and establish cell polarity during yeast mating . Here we show that SCF(Cdc4) ubiquitylates Far1 in the nucleus, which in turn targets the multi-ubiquitylated protein to 26S proteasomes most likely located at the nuclear envelope . In response to mating pheromones, a fraction of Far1 was stabilized after its export into the cytoplasm by Ste21/Msn5 . Preventing nuclear export destabilized Far1, while conversely cytoplasmic Far1 was stabilized, although the protein was efficiently phosphorylated in a Cdc28-Cln-dependent manner . The core SCF subunits Cdc53, Hrt1 and Skp1 were distributed in the nucleus and the cytoplasm, whereas the F-box protein Cdc4 was exclusively nuclear . A cytoplasmic form of Cdc4 was unable to complement the growth defect of cdc4-1 cells, but it was sufficient to degrade Far1 in the cytoplasm . Our results illustrate the importance of subcellular localization of F-box proteins, and provide an example of how an extracellular signal regulates protein stability at the level of substrate localization.

EMBO J, 2000 Nov 15, 19(22), 6000 - 10
Exocytosis requires asymmetry in the central layer of the SNARE complex; Ossig R et al.; Assembly of SNAREs (soluble N:-ethylmaleimide- sensitive factor attachment protein receptors) mediates membrane fusions in all eukaryotic cells . The synaptic SNARE complex is represented by a twisted bundle of four alpha-helices . Leucine zipper-like layers extend through the length of the complex except for an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R) . We have examined the functional consequences of Q-R exchanges in the conserved middle layer using the exocytotic SNAREs of yeast as a model . Exchanging Q for R in Sso2p drastically reduces cell growth and protein secretion . When a 3Q/1R ratio is restored by a mirror R-->Q substitution in the R-SNARE Snc2p, wild-type functionality is observed . Secretion is near normal when all four helices contain Q, but defects become apparent when additional mutations are present in other layers . Using molecular dynamics free energy perturbation simulations, these findings are rationalized in structural and energetic terms . We conclude that the asymmetric arrangement of the polar amino acids in the central layer is essential for normal function of SNAREs in membrane fusion.

Protist, 2000 Oct, 151(3), 201 - 24
Acclimation of Chlamydomonas reinhardtii to its nutrient environment; Grossman A; To cope with low nutrient availability in nature, organisms have evolved inducible systems that enable them to scavenge and efficiently utilize the limiting nutrient . Furthermore, organisms must have the capacity to adjust their rate of metabolism and make specific alterations in metabolic pathways that favor survival when the potential for cell growth and division is reduced . In this article I will focus on the acclimation of Chlamydomonas reinhardtii, a unicellular, eukaryotic green alga to conditions of nitrogen, sulfur and phosphorus deprivation . This organism has a distinguished history as a model for classical genetic analyses, but it has recently been developed for exploitation using an array of molecular and genomic tools . The application of these tools to the analyses of nutrient limitation responses (and other biological processes) is revealing mechanisms that enable Chlamydomonas to survive harsh environmental conditions and establishing relationships between the responses of this morphologically simple, photosynthetic eukaryote and those of both nonphotosynthetic organisms and vascular plants.

J Photochem Photobiol B, 2000 Jul, 56(2-3), 145 - 53
Recombinant phytochrome of the moss Ceratodon purpureus (CP2): fluorescence spectroscopy and photochemistry; Sineshchekov V et al.; The recombinant phytochrome of the moss Ceratodon purpureus (CP2) expressed in Saccharomyces cerevisiae and reconstituted with phycocyanobilin (PCB) was investigated using fluorescence spectroscopy . The pigment had an emission maximum at 670 nm at low temperature (85 K) and at 667 nm at room temperature (RT) and an excitation maximum at 650-652 nm at 85 K (excitation spectra could not be measured at RT) . Both spectra had a half-band width of approx . 30-35 nm at 85 K . The fluorescence intensity revealed a steep temperature dependence with an activation energy of fluorescence decay (Ea) of 5.9-6.4 and 12.6-14.7 kJ mol(-1) in the interval from 85 to 210 K and from 210 to 275 K, respectively . The photochemical properties of CP2/PCB were characterised by the extent of the red-induced (lambda(a) = 639 nm) Pr conversion into the first photoproduct lumi-R at 85 K (gamma1) of approximately 0.07 and into Pfr at RT (gamma2) of approximately 0.7 . From these characteristics, CP2/PCB can be attributed to the Pr" photochemical type with gamma1 < or = 0.05, which comprises the minor phyA fraction (phyA"), phyB, Adiantum phy1 and Synechocystis Cph1 in contrast to the major phyA' fraction (Pr' type with gamma1 = 0.5) . Within the Pr" type, it is closer to phyA" than to phyB and Cph1.

J Biol Chem, 2001 Feb 16, 276(7), 5213 - 21 Epub 2000 Nov 16.
Induction-independent recruitment of CREB-binding protein to the c-fos serum response element through interactions between the bromodomain and Elk-1; Nissen LJ et al.; Proliferative signals lead to the rapid and transient induction of the c-fos proto-oncogene by targeting the ternary complex assembled on the serum response element (SRE) . Transactivation by both components of this complex, serum response factor (SRF) and the ternary complex factor Elk-1, can be potentiated by the coactivator CREB-binding protein (CBP) . We report a novel interaction between the bromodomain of CBP, amino acids 1100-1286, and Elk-1 . DNA binding and glutathione S-transferase pull-down assays demonstrate that binding requires Elk-1(1-212) but not the C-terminal transactivation domain . Competition and antibody controls show that the bromocomplex involves both SRF and Elk-1 on the c-fos SRE and uniquely Elk-1 on the E74 Ets binding site . Interestingly, methylation interference and DNA footprinting analyses show almost indistinguishable patterns between ternary and bromocomplexes, suggesting that CBP-(1100-1286) interacts via Elk-1 and does not require specific DNA contacts . Functionally, the bromocomplex blocks activation, because cotransfection of CBP-(1100-1286) reduces RasV12-driven activation of SRE and E74 luciferase reporters . Repression is relieved moderately or strongly by linking the bromodomain to the N- or C-terminal transactivation domains of CBP, respectively . These results are consistent with a model in which CBP is constitutively bound to the SRE in a higher order complex that would facilitate the rapid transcriptional activation of c-fos by signaling-driven phosphorylation.

J Biol Chem, 2001 Mar 2, 276(9), 6200 - 6 Epub 2000 Nov 15.
Inhibition of the Ca(2+)-ATPase Pmc1p by the v-SNARE protein Nyv1p; Takita Y et al.; Pmc1p, the Ca(2+)-ATPase of budding yeast related to plasma membrane Ca(2+)-ATPases of animals, is transcriptionally up-regulated in response to signaling by the calmodulin-calcineurin-Tcn1p/Crz1p signaling pathway . Little is known about post-translational regulation of Pmc1p . In a genetic screen for potential negative regulators of Pmc1p, a vacuolar v-SNARE protein, Nyv1p, was recovered . Cells overproducing Nyv1p show decreased Ca(2+) tolerance and decreased accumulation of Ca(2+) in the vacuole, similar to pmc1 null mutants . Overexpression of Nyv1p had no such effects on pmc1 mutants, suggesting that Nyv1p may inhibit Pmc1p function . Overexpression of Nyv1p did not decrease Pmc1p levels but decreased the specific ATP-dependent Ca(2+) transport activity of Pmc1p in purified vacuoles by at least 2-fold . The effect of Nyv1p on Pmc1p function is likely to be direct because native immunoprecipitation experiments showed that Pmc1p coprecipitated with Nyv1p . Complexes between Nyv1p and its t-SNARE partner Vam3p were also isolated, but these complexes lacked Pmc1p . We conclude that Nyv1p can interact physically with Pmc1p and inhibit its Ca(2+) transport activity in the vacuole membrane . This is the first example of a Ca(2+)-ATPase regulation by a v-SNARE protein involved in membrane fusion reactions.

J Biol Chem, 2001 Mar 9, 276(10), 7017 - 22 Epub 2000 Nov 15.
The two major plant plasma membrane H+-ATPases display different regulatory properties; Dambly S et al.; The major plant plasma membrane H(+)-ATPases fall into two gene categories, subfamilies I and II . However, in many plant tissues, expression of the two subfamilies overlaps, thus precluding individual characterization . Yeast expression of PMA2 and PMA4, representatives of the two plasma membrane H(+)-ATPase subfamilies in Nicotiana plumbaginifolia, has previously shown that (i) the isoforms have distinct enzymatic properties and that (ii) PMA2 is regulated by phosphorylation of its penultimate residue (Thr) and binds regulatory 14-3-3 proteins, resulting in the displacement of the autoinhibitory C-terminal domain . To obtain insights into regulatory differences between the two subfamilies, we have constructed various chimeric proteins in which the 110-residue C-terminal-encoding region of PMA2 was progressively substituted by the corresponding sequence from PMA4 . The PMA2 autoinhibitory domain was localized to a region between residues 851 and 915 and could not be substituted by the corresponding region of PMA4 . In contrast to PMA2, PMA4 was poorly phosphorylated at its penultimate residue (Thr) and bound 14-3-3 proteins weakly . The only sequence difference around the phosphorylation site is located two residues upstream of the phosphorylated Thr . It is Ser in PMA2 (as in most members of subfamily I) and His in PMA4 (as in most members of subfamily II) . Substitution of His by Ser in PMA4 resulted in an enzyme showing increased phosphorylation status, 14-13-3 binding, and ATPase activity, as well as improved yeast growth . The reverse substitution of Ser by His in PMA2 resulted in the failure of this enzyme to complement the absence of yeast H(+)-ATPases . These results show that the two plant H(+)-ATPase subfamilies differ functionally in their regulatory properties.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13138 - 43
Targeted chromatin binding and histone acetylation in vivo by thyroid hormone receptor during amphibian development; Sachs LM et al.; Amphibian metamorphosis is marked by dramatic, thyroid hormone (TH)-induced changes involving gene regulation by TH receptor (TR) . It has been postulated that TR-mediated gene regulation involves chromatin remodeling . In the absence of ligand, TR can repress gene expression by recruiting a histone deacetylase complex, whereas liganded TR recruits a histone acetylase complex for gene activation . Earlier studies have led us to propose a dual function model for TR during development . In premetamorphic tadpoles, unliganded TR represses transcription involving histone deacetylation . During metamorphosis, endogenous TH allows TR to activate gene expression through histone acetylation . Here using chromatin immunoprecipitation assay, we directly demonstrate TR binding to TH response genes constitutively in vivo in premetamorphic tadpoles . We further show that TH treatment leads to histone deacetylase release from TH response gene promoters . Interestingly, in whole animals, changes in histone acetylation show little correlation with the expression of TH response genes . On the other hand, in the intestine and tail, where TH response genes are known to be up-regulated more dramatically by TH than in most other organs, we demonstrate that TH treatment induces gene activation and histone H4 acetylation . These data argue for a role of histone acetylation in transcriptional regulation by TRs during amphibian development in some tissues, whereas in others changes in histone acetylation levels may play no or only a minor role, supporting the existence of important alternative mechanisms in gene regulation by TR.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13227 - 32
A chemical genomics approach toward understanding the global functions of the target of rapamycin protein (TOR); Chan TF et al.; The target of rapamycin protein (TOR) is a highly conserved ataxia telangiectasia-related protein kinase essential for cell growth . Emerging evidence indicates that TOR signaling is highly complex and is involved in a variety of cellular processes . To understand its general functions, we took a chemical genomics approach to explore the genetic interaction between TOR and other yeast genes on a genomic scale . In this study, the rapamycin sensitivity of individual deletion mutants generated by the Saccharomyces Genome Deletion Project was systematically measured . Our results provide a global view of the rapamycin-sensitive functions of TOR . In contrast to conventional genetic analysis, this approach offers a simple and thorough analysis of genetic interaction on a genomic scale and measures genetic interaction at different possible levels . It can be used to study the functions of other drug targets and to identify novel protein components of a conserved core biological process such as DNA damage checkpoint/repair that is interfered with by a cell-permeable chemical compound.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13281 - 5
Posttranslational modification of TEL and TEL/AML1 by SUMO-1 and cell-cycle-dependent assembly into nuclear bodies; Chakrabarti SR et al.; The E-26 transforming specific (ETS)-related gene, TEL, also known as ETV6, encodes a strong transcription repressor that is rearranged in several recurring chromosomal rearrangements associated with leukemia and congenital fibrosarcoma . TEL is a nuclear phosphoprotein that is widely expressed in all normal tissues . TEL contains a DNA-binding domain at the C terminus and a helix-loop-helix domain (also called a pointed domain) at the N terminus . The pointed domain is necessary for homotypic dimerization and for interaction with the ubiquitin-conjugating enzyme UBC9 . Here we show that the interaction with UBC9 leads to modification of TEL by conjugating it to SUMO-1 . The SUMO-1-modified TEL localizes to cell-cycle-specific nuclear speckles that we named TEL bodies . We also show that the leukemia-associated fusion protein TEL/AML1 is modified by SUMO-1 and found in the TEL bodies, in a pattern quite different from what we observe and report for AML1 . Therefore, SUMO-1 modification of TEL could be a critical signal necessary for normal functioning of the protein . In addition, the modification by SUMO-1 of TEL/AML1 could lead to abnormal localization of the fusion protein, which could have consequences that include contribution to neoplastic transformation.

J Cell Biol, 2000 Nov 13, 151(4), 863 - 78
Mechanism of metabolic control . Target of rapamycin signaling links nitrogen quality to the activity of the Rtg1 and Rtg3 transcription factors; Komeili A et al.; De novo biosynthesis of amino acids uses intermediates provided by the TCA cycle that must be replenished by anaplerotic reactions to maintain the respiratory competency of the cell . Genome-wide expression analyses in Saccharomyces cerevisiae reveal that many of the genes involved in these reactions are repressed in the presence of the preferred nitrogen sources glutamine or glutamate . Expression of these genes in media containing urea or ammonia as a sole nitrogen source requires the heterodimeric bZip transcription factors Rtg1 and Rtg3 and correlates with a redistribution of the Rtg1p/Rtg3 complex from a predominantly cytoplasmic to a predominantly nuclear location . Nuclear import of the complex requires the cytoplasmic protein Rtg2, a previously identified upstream regulator of Rtg1 and Rtg3, whereas export requires the importin-beta-family member Msn5 . Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases . We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment . Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors.

J Cell Biol, 2000 Nov 13, 151(4), 847 - 62
Identification of ubiquilin, a novel presenilin interactor that increases presenilin protein accumulation; Mah AL et al.; Mutations in the highly homologous presenilin genes encoding presenilin-1 and presenilin-2 (PS1 and PS2) are linked to early-onset Alzheimer's disease (AD) . However, apart from a role in early development, neither the normal function of the presenilins nor the mechanisms by which mutant proteins cause AD are well understood . We describe here the properties of a novel human interactor of the presenilins named ubiquilin . Yeast two-hybrid (Y2H) interaction, glutathione S-transferase pull-down experiments, and colocalization of the proteins expressed in vivo, together with coimmunoprecipitation and cell fractionation studies, provide compelling evidence that ubiquilin interacts with both PS1 and PS2 . Ubiquilin is noteworthy since it contains multiple ubiquitin-related domains typically thought to be involved in targeting proteins for degradation . However, we show that ubiquilin promotes presenilin protein accumulation . Pulse-labeling experiments indicate that ubiquilin facilitates increased presenilin synthesis without substantially changing presenilin protein half-life . Immunohistochemistry of human brain tissue with ubiquilin-specific antibodies revealed prominent staining of neurons . Moreover, the anti-ubiquilin antibodies robustly stained neurofibrillary tangles and Lewy bodies in AD and Parkinson's disease affected brains, respectively . Our results indicate that ubiquilin may be an important modulator of presenilin protein accumulation and that ubiquilin protein is associated with neuropathological neurofibrillary tangles and Lewy body inclusions in diseased brain.

Genome Res, 2000 Nov, 10(11), 1743 - 56
Fusion of the human gene for the polyubiquitination coeffector UEV1 with Kua, a newly identified gene; Thomson TM et al.; UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains . In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway . In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle . Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon-intron structure . We also show that the human UEV1 gene is fused with the previously unknown gene Kua . In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins . In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua-UEV transcript, encoding a two-domain protein . Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs . Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua-UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua-UEV . Therefore, the addition of a Kua domain to UEV in the fused Kua-UEV protein confers new biological properties to this regulator of variant polyubiquitination.

Genome Res, 2000 Nov, 10(11), 1690 - 6
Single nucleotide polymorphisms in wild isolates of Caenorhabditis elegans; Koch R et al.; Caenorhabditis elegans (isolate N2 from Bristol, UK) is the first animal of which the complete genome sequence was available . We sampled genomic DNA of natural isolates of C . elegans from four different locations (Australia, Germany, California, and Wisconsin) and found single nucleotide polymorphisms (SNPs) by comparing with the Bristol strain . SNPs are under-represented in coding regions, and many were found to be third base silent codon mutations . We tested 19 additional natural isolates for the presence and distribution of SNPs originally found in one of the four strains . Most SNPs are present in isolates from around the globe and thus are older than the latest contact between these strains . An exception is formed by an isolate from an island (Hawaii) that contains many unique SNPs, absent in the tested isolates from the rest of the world . It has been noticed previously that conserved genes (as defined by homology to genes in Saccharomyces cerevisiae) cluster in the chromosome centers . We found that the SNP frequency outside these regions is 4.5 times higher, supporting the notion of a higher rate of evolution of genes on the chromosome arms.

Biol Chem, 2000 Sep-Oct, 381(9-10), 815 - 25
Interdependence of filamentous actin and microtubules for asymmetric cell division; Schaerer-Brodbeck C et al.; Asymmetric cell divisions are crucial to the generation of cell fate diversity . They contribute to unequal distribution of cellular factors to the daughter cells . Asymmetric divisions are characterized by a 90 degrees rotation of the mitotic spindle . There is increasing evidence that a tight cooperation between cortical, filamentous actin and astral microtubules is indispensable for successful spindle rotation . Over the past years, the dynactin complex has emerged as a key candidate to mediate actin/microtubule interaction at the cortex . This review discusses our current understanding of how spindle rotation is accomplished by the interplay of filamentous actin and microtubules in a variety of experimental systems.

Mol Endocrinol, 2000 Nov, 14(11), 1849 - 71
Function of N-terminal transactivation domain of the estrogen receptor requires a potential alpha-helical structure and is negatively regulated by the A domain; Metivier R et al.; Transcriptional activation by the estrogen receptor (NR3A1, or ER) requires specific ligand-inducible activation functions located in the amino (AF-1) and the carboxyl (AF-2 and AF-2a) regions of the protein . Although several detailed reports of ER structure and function describe mechanisms whereby AF-2 activates transcription, less precise data exist for AF-1 . We recently reported that the rainbow trout and human estrogen receptors (rtERs and hERs, respectively), two evolutionary distant proteins, exhibit comparable AF-1 activities while sharing only 20% homology in their N-terminal region . These data suggested that the basic mechanisms whereby AF-1 and the ER N-terminal region activate transactivation might be evolutionary conserved . Therefore, a comparative approach between rtER and hER could provide more detailed information on AF-1 function . Transactivation analysis of truncated receptors and Gal4DBD (DNA binding domain of the Gal4 factor) fusion proteins in Saccharomyces cerevisiae defined a minimal region of 11 amino acids, located at the beginning of the B domain, necessary for AF-1 activity in rtER . Hydrophobic cluster analysis (HCA) indicated the presence of a potential alpha-helix within this minimal region that is conserved during evolution . Both rtER and hER sequences corresponding to this potential alpha-helical structure were able to induce transcription when fused to the Gal4DBD, indicating that this region can transactivate in an autonomous manner . Furthermore, point mutations in this 11-amino acid region of the receptors markedly reduced their transcriptional activity either within the context of a whole ER or a Gal4DBD fusion protein . Data were confirmed in mammalian cells and, interestingly, ERs with an inverted alpha-helix were as active as their corresponding wild-type proteins, indicating a conserved role in AF-1 for these structures . Moreover, using two naturally occurring rtER N-terminal variants possessing or not the A domain (rtER(L) and rtER(S), respectively), together with A domain-truncated hER and chimeric rtER/hER receptors, we demonstrated that the A domain of the ER plays an inhibitory role in ligand-independent activity of the receptor . In vitro and in vivo protein-protein interaction assays using both rtER and hER demonstrated that this repression is likely to be mediated by a ligand-sensitive direct interaction between the A domain and the C-terminal region of the ER.

Trends Genet, 2000 Nov, 16(11), 506 - 11
Metabolic control and ageing; Jazwinski SM; There appear to be multiple processes that are limiting for longevity and the associated mechanisms of ageing . Among these processes, metabolic control is coming to the forefront, because it has surfaced in studies in several model systems and because of its relevance to mammalian ageing . The genetic and molecular dissection of ageing in yeast points to mechanisms involving three aspects of metabolism . First, dysfunctional mitochondria signal many changes in nuclear gene expression that result in metabolic adjustments that extend life span . Second, manipulation of nutritional status can also increase longevity in a separate caloric-restriction pathway . Finally, protein synthesis is a third aspect, which depends on the transcriptional state of chromatin and the histone deacetylases that modulate it.

Trends Genet, 2000 Nov, 16(11), 475 - 7
A genetic uncertainty problem; Tautz D; The existence of genes that, when knocked out, result in no obvious phenotype has puzzled biologists for many years . The phenomenon is often ascribed to redundancy in regulatory networks, caused by duplicated genes . However, a recent systematic analysis of data from the yeast genome projects does not support a link between gene duplications and redundancies . An alternative explanation suggests that genes might also evolve by very weak selection, which would mean that their true function cannot be studied in normal laboratory experiments . This problem is comparable to Heisenberg's uncertainty relationship in physics . It is possible to formulate an analogous relationship for biology, which, at its extreme, predicts that the understanding of the full function of a gene might require experiments on an evolutionary scale, involving the entire effective population size of a given species.

Mol Cell Biol, 2000 Dec, 20(23), 9076 - 83
Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality; Zhao X et al.; In budding yeast, MEC1 and RAD53 are essential for cell growth . Previously we reported that mec1 or rad53 lethality is suppressed by removal of Sml1, a protein that binds to the large subunit of ribonucleotide reductase (Rnr1) and inhibits RNR activity . To understand further the relationship between this suppression and the Sml1-Rnr1 interaction, we randomly mutagenized the SML1 open reading frame . Seven mutations were identified that did not affect protein expression levels but relieved mec1 and rad53 inviability . Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed in mec1 and rad53 strains . The mutant residues all cluster within the 33 C-terminal amino acids of the 104-amino-acid-long Sml1 protein . Four of these residues reside within an alpha-helical structure that was revealed by nuclear magnetic resonance studies . Moreover, deletions encompassing the N-terminal half of Sml1 do not interfere with its RNR inhibitory activity . Finally, the seven sml1 mutations also disrupt the interaction with yeast Rnr3 and human R1, suggesting a conserved binding mechanism between Sml1 and the large subunit of RNR from different species.

Mol Cell Biol, 2000 Dec, 20(23), 9028 - 40
Human H/ACA small nucleolar RNPs and telomerase share evolutionarily conserved proteins NHP2 and NOP10; Pogacic V et al.; The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs . In the yeast Saccharomyces cerevisiae, four common proteins are associated with H/ACA snoRNAs: Gar1p, Cbf5p, Nhp2p, and Nop10p . In vitro reconstitution studies showed that four proteins also specifically interact with H/ACA snoRNAs in mammalian cell extracts . Two mammalian proteins, NAP57/dyskerin (the ortholog of Cbf5p) and hGAR1, have been characterized . In this work we describe properties of hNOP10 and hNHP2, human orthologs of yeast Nop10p and Nhp2p, respectively, and further characterize hGAR1 . hNOP10 and hNHP2 complement yeast cells depleted of Nhp2p and Nop10p, respectively . Immunoprecipitation experiments with extracts from transfected HeLa cells indicated that epitope-tagged hNOP10 and hNHP2 specifically associate with hGAR1 and H/ACA RNAs; they also interact with the RNA subunit of telomerase, which contains an H/ACA-like domain in its 3' moiety . Immunofluorescence microscopy experiments showed that hGAR1, hNOP10, and hNHP2 are localized in the dense fibrillar component of the nucleolus and in Cajal (coiled) bodies . Deletion analysis of hGAR1 indicated that its evolutionarily conserved core domain contains all the signals required for localization, but progressive deletions from either the N or the C terminus of the core domain abolish localization in the nucleolus and/or the Cajal bodies.

Mol Cell Biol, 2000 Dec, 20(23), 8903 - 15
A novel Rb- and p300-binding protein inhibits transactivation by MyoD; MacLellan WR et al.; The retinoblastoma protein (Rb) regulates both the cell cycle and tissue-specific transcription, by modulating the activity of factors that associate with its A-B and C pockets . In skeletal muscle, Rb has been reported to regulate irreversible cell cycle exit and muscle-specific transcription . To identify factors interacting with Rb in muscle cells, we utilized the yeast two-hybrid system, using the A-B and C pockets of Rb as bait . A novel protein we have designated E1A-like inhibitor of differentiation 1 (EID-1), was the predominant Rb-binding clone isolated . It is preferentially expressed in adult cardiac and skeletal muscle and encodes a 187-amino-acid protein, with a classic Rb-binding motif (LXCXE) in its C terminus . Overexpression of EID-1 in skeletal muscle inhibited tissue-specific transcription . Repression of skeletal muscle-restricted genes was mediated by a block to transactivation by MyoD independent of G(1) exit and, surprisingly, was potentiated by a mutation that prevents EID-1 binding to Rb . Inhibition of MyoD may be explained by EID-1's ability to bind and inhibit p300's histone acetylase activity, an essential MyoD coactivator . Thus, EID-1 binds both Rb and p300 and is a novel repressor of MyoD function.

Mol Cell Biol, 2000 Dec, 20(23), 8889 - 902
Cells degrade a novel inhibitor of differentiation with E1A-like properties upon exiting the cell cycle; Miyake S et al.; Control of proliferation and differentiation by the retinoblastoma tumor suppressor protein (pRB) and related family members depends upon their interactions with key cellular substrates . Efforts to identify such cellular targets led to the isolation of a novel protein, EID-1 (for E1A-like inhibitor of differentiation 1) . Here, we show that EID-1 is a potent inhibitor of differentiation and link this activity to its ability to inhibit p300 (and the highly related molecule, CREB-binding protein, or CBP) histone acetylation activity . EID-1 is rapidly degraded by the proteasome as cells exit the cell cycle . Ubiquitination of EID-1 requires an intact C-terminal region that is also necessary for stable binding to p300 and pRB, two proteins that bind to the ubiquitin ligase MDM2 . A pRB variant that can bind to EID1, but not MDM2, stabilizes EID-1 in cells . Thus, EID-1 may act at a nodal point that couples cell cycle exit to the transcriptional activation of genes required for differentiation.

Mol Cell Biol, 2000 Dec, 20(23), 8879 - 88
A specificity and targeting subunit of a human SWI/SNF family-related chromatin-remodeling complex; Nie Z et al.; The SWI/SNF family of chromatin-remodeling complexes facilitates gene activation by assisting transcription machinery to gain access to targets in chromatin . This family includes BAF (also called hSWI/SNF-A) and PBAF (hSWI/SNF-B) from humans and SWI/SNF and Rsc from Saccharomyces cerevisiae . However, the relationship between the human and yeast complexes is unclear because all human subunits published to date are similar to those of both yeast SWI/SNF and Rsc . Also, the two human complexes have many identical subunits, making it difficult to distinguish their structures or functions . Here we describe the cloning and characterization of BAF250, a subunit present in human BAF but not PBAF . BAF250 contains structural motifs conserved in yeast SWI1 but not in any Rsc components, suggesting that BAF is related to SWI/SNF . BAF250 is also a homolog of the Drosophila melanogaster Osa protein, which has been shown to interact with a SWI/SNF-like complex in flies . BAF250 possesses at least two conserved domains that could be important for its function . First, it has an AT-rich DNA interaction-type DNA-binding domain, which can specifically bind a DNA sequence known to be recognized by a SWI/SNF family-related complex at the beta-globin locus . Second, BAF250 stimulates glucocorticoid receptor-dependent transcriptional activation, and the stimulation is sharply reduced when the C-terminal region of BAF250 is deleted . This region of BAF250 is capable of interacting directly with the glucocorticoid receptor in vitro . Our data suggest that BAF250 confers specificity to the human BAF complex and may recruit the complex to its targets through either protein-DNA or protein-protein interactions.

Mol Cell Biol, 2000 Dec, 20(23), 8855 - 65
Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis; Pagliari LJ et al.; NF-kappaB is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophage survival has yet to be elucidated . Here, we demonstrate that constitutive NF-kappaB activation is essential for macrophage survival . Blocking the constitutive activation of NF-kappaB with pyrrolidine dithiocarbamate or expression of IkappaBalpha induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages . This apoptosis was independent of additional death-inducing stimuli, including Fas ligation . Suppression of NF-kappaB activation induced a time-dependent loss of mitochondrial transmembrane potential (DeltaPsi(m)) and DNA fragmentation . Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3 . Addition of a general caspase inhibitor, z-VAD . fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on DeltaPsi(m) collapse, indicating this event was caspase independent . To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to DeltaPsi(m) loss and that ectopic expression of A1 protected against cell death following inactivation of NF-kappaB . These data suggest that inhibition of NF-kappaB in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction . Thus, constitutive NF-kappaB activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.

Mol Cell Biol, 2000 Dec, 20(23), 8826 - 35
Localization and signaling of G(beta) subunit Ste4p are controlled by a-factor receptor and the a-specific protein Asg7p; Kim J et al.; Haploid yeast cells initiate pheromone signaling upon the binding of pheromone to its receptor and activation of the coupled G protein . A regulatory process termed receptor inhibition blocks pheromone signaling when the a-factor receptor is inappropriately expressed in MATa cells . Receptor inhibition blocks signaling by inhibiting the activity of the G protein beta subunit, Ste4p . To investigate how Ste4p activity is inhibited, its subcellular location was examined . In wild-type cells, alpha-factor treatment resulted in localization of Ste4p to the plasma membrane of mating projections . In cells expressing the a-factor receptor, alpha-factor treatment resulted in localization of Ste4p away from the plasma membrane to an internal compartment . An altered version of Ste4p that is largely insensitive to receptor inhibition retained its association with the membrane in cells expressing the a-factor receptor . The inhibitory function of the a-factor receptor required ASG7, an a-specific gene of previously unknown function . ASG7 RNA was induced by pheromone, consistent with increased inhibition as the pheromone response progresses . The a-factor receptor inhibited signaling in its liganded state, demonstrating that the receptor can block the signal that it initiates . ASG7 was required for the altered localization of Ste4p that occurs during receptor inhibition, and the subcellular location of Asg7p was consistent with its having a direct effect on Ste4p localization . These results demonstrate that Asg7p mediates a regulatory process that blocks signaling from a G protein beta subunit and causes its relocalization within the cell.

Mol Cell Biol, 2000 Dec, 20(23), 8815 - 25
Asg7p-Ste3p inhibition of pheromone signaling: regulation of the zygotic transition to vegetative growth; Roth AF et al.; The inappropriate expression of the a-factor pheromone receptor (Ste3p) in the MATa cell leads to a striking inhibition of the yeast pheromone response, the result of a functional interaction between Ste3p and some MATa-specific protein . The present work identifies this protein as Asg7p . Normally, expression of Ste3p and Asg7p is limited to distinct haploid mating types, Ste3p to MATalpha cells and Asg7p to MATa cells . Artificial coexpression of the two in the same cell, either a or alpha, leads to dramatic inhibition of the pheromone response . Ste3p-Asg7p coexpression also perturbs the membrane trafficking of Ste3p: Ste3p turnover is slowed, a result of an Asg7p-mediated retardation of the secretory delivery of the newly synthesized receptor to the plasma membrane . However, in the absence of ectopic Ste3p expression, the asg7Delta mutation is without consequence either for pheromone signaling or overall mating efficiency of a cells . Indeed, the sole phenotype that can be assigned to MATa asg7Delta cells is observed following zygotic fusion to its alpha mating partner . Though formed at wild-type efficiency, zygotes from these pairings are morphologically abnormal . The pattern of growth is deranged: emergence of the first mitotic bud is delayed, and, in its place, growth is apparently diverted into a novel structure superficially resembling the polarized mating projection characteristic of haploid cells responding to pheromone . Together these results suggest a mechanism in which, following the zygotic fusion event, Ste3p and Asg7p gain access to one another and together act to repress the pheromone response, promoting the transition of the new diploid cell to vegetative growth.

Mol Cell Biol, 2000 Dec, 20(23), 8709 - 19
In vivo requirement of activator-specific binding targets of mediator; Park JM et al.; There has been no unequivocal demonstration that the activator binding targets identified in vitro play a key role in transcriptional activation in vivo . To examine whether activator-Mediator interactions are required for gene transcription under physiological conditions, we performed functional analyses with Mediator components that interact specifically with natural yeast activators . Different activators interact with Mediator via distinct binding targets . Deletion of a distinct activator binding region of Mediator completely compromised gene activation in vivo by some, but not all, transcriptional activators . These demonstrate that the activator-specific targets in Mediator are essential for transcriptional activation in living cells, but their requirement was affected by the nature of the activator-DNA interaction and the existence of a postrecruitment activation process.

J Bacteriol, 2000 Dec, 182(23), 6673 - 8
Novel role for an HPt domain in stabilizing the phosphorylated state of a response regulator domain; Janiak-Spens F et al.; Two-component regulatory systems that utilize a multistep phosphorelay mechanism often involve a histidine-containing phosphotransfer (HPt) domain . These HPt domains serve an essential role as histidine-phosphorylated protein intermediates during phosphoryl transfer from one response regulator domain to another . In Saccharomyces cerevisiae, the YPD1 protein facilitates phosphoryl transfer from a hybrid sensor kinase, SLN1, to two distinct response regulator proteins, SSK1 and SKN7 . Because the phosphorylation state largely determines the functional state of response regulator proteins, we have carried out a comparative study of the phosphorylated lifetimes of the three response regulator domains associated with SLN1, SSK1, and SKN7 (R1, R2, and R3, respectively) . The isolated regulatory domains exhibited phosphorylated lifetimes within the range previously observed for other response regulator domains (i.e., several minutes to several hours) . However, in the presence of YPD1, we found that the half-life of phosphorylated SSK1-R2 was dramatically extended (almost 200-fold longer than in the absence of YPD1) . This stabilization effect was specific for SSK1-R2 and was not observed for SLN1-R1 or SKN7-R3 . Our findings suggest a mechanism by which SSK1 is maintained in its phosphorylated state under normal physiological conditions and demonstrate an unprecedented regulatory role for an HPt domain in a phosphorelay signaling system.

Am J Pathol, 2000 Nov, 157(5), 1447 - 52
Physiological expression of the gene for PrP-like protein, PrPLP/Dpl, by brain endothelial cells and its ectopic expression in neurons of PrP-deficient mice ataxic due to Purkinje cell degeneration; Li A et al.; Recently, a novel gene encoding a prion protein (PrP)-like glycoprotein, PrPLP/Dpl, was identified as being expressed ectopically by neurons of the ataxic PrP-deficient (PRNP(-/-)) mouse lines exhibiting Purkinje cell degeneration . In adult wild-type mice, PrPLP/Dpl mRNA was physiologically expressed at a high level by testis and heart, but was barely detectable in brain . However, transient expression of PrPLP/Dpl mRNA was detectable by Northern blotting in the brain of neonatal wild-type mice, showing maximal expression around 1 week after birth . In situ hybridization paired with immunohistochemistry using anti-factor VIII serum identified brain endothelial cells as expressing the transcripts . Moreover, in the neonatal wild-type mice, the PrPLP/Dpl mRNA colocalized with factor VIII immunoreactivities in spleen and was detectable on capillaries in lamina propria mucosa of gut . These findings suggested a role of PrPLP/Dpl in angiogenesis, in particular blood-brain barrier maturation in the central nervous system . Even in the ataxic Ngsk PRNP(-/-) mice, the physiological regulation of PrPLP/Dpl mRNA expression in brain endothelial cells was still preserved . This strongly supports the argument that the ectopic expression of PrPLP/Dpl in neurons, but not deregulation of its physiological expression in endothelial cells, is involved in the neuronal degeneration in ataxic PRNP(-/-) mice.

Science, 2000 Nov 10, 290(5494), 1151 - 5
The evolutionary fate and consequences of duplicate genes; Lynch M et al.; Gene duplication has generally been viewed as a necessary source of material for the origin of evolutionary novelties, but it is unclear how often gene duplicates arise and how frequently they evolve new functions . Observations from the genomic databases for several eukaryotic species suggest that duplicate genes arise at a very high rate, on average 0.01 per gene per million years . Most duplicated genes experience a brief period of relaxed selection early in their history, with a moderate fraction of them evolving in an effectively neutral manner during this period . However, the vast majority of gene duplicates are silenced within a few million years, with the few survivors subsequently experiencing strong purifying selection . Although duplicate genes may only rarely evolve new functions, the stochastic silencing of such genes may play a significant role in the passive origin of new species.

J Med Microbiol, 2000 Nov, 49(11), 977 - 84
In-vivo selection of an azole-resistant petite mutant of Candida glabrata; Bouchara JP et al.; Two isolates of Candida glabrata from the same stool sample from a bone marrow transplant recipient treated with fluconazole, and designated 1084-L for large colonies on yeast extract-peptone-dextrose-agar and 1084-S for small colonies, were analysed . In-vitro susceptibility tests with a commercially available disk diffusion procedure showed that isolate 1084-L had a susceptibility pattern typical of wild-type strains of C . glabrata with sensitivity to polyenes and the presence of resistant colonies randomly distributed within the inhibition zones for all azole compounds except tioconazole . In contrast, isolate 1084-S, which was found by pulsed-field gel electrophoresis and random amplification of polymorphic DNA to be genetically closely related to isolate 1084-L, exhibited cross-resistance to the azole compounds except tioconazole . Determination of MICs by the E-test method confirmed these results, showing that isolate 1084-S had greater sensitivity to amphotericin B and complete resistance to ketoconazole and fluconazole . Growth on agar plates containing glucose or glycerol as the sole carbon source suggested that the resistant isolate had a respiratory deficiency, which was further demonstrated by flow cytometric analysis of the fluorescence of rhodamine 123-stained blastoconidia . Restriction endonuclease analysis of mitochondrial DNA (mtDNA) established the mitochondrial origin of the respiratory deficiency . However, PCR amplification of the mtDNA with primers ML1 and ML6, as well as transmission electron microscopy, suggested a partial deletion of the mtDNA analogous to that described for rho- petite mutants of Saccharomyces cerevisiae . Together, these results provided evidence that the selection of azole-resistant petite mutants of C . glabrata may occur in vivo after fluconazole administration, which might explain, therefore, clinical failure of antifungal therapy.

Mol Biol Cell, 2000 Nov, 11(11), 3977 - 91
Membrane potential-driven protein import into mitochondria . The sorting sequence of cytochrome b(2) modulates the deltapsi-dependence of translocation of the matrix-targeting sequence; Geissler A et al.; The transport of preproteins into or across the mitochondrial inner membrane requires the membrane potential Deltapsi across this membrane . Two roles of Deltapsi in the import of cleavable preproteins have been described: an electrophoretic effect on the positively charged matrix-targeting sequences and the activation of the translocase subunit Tim23 . We report the unexpected finding that deletion of a segment within the sorting sequence of cytochrome b(2), which is located behind the matrix-targeting sequence, strongly influenced the Deltapsi-dependence of import . The differential Deltapsi-dependence was independent of the submitochondrial destination of the preprotein and was not attributable to the requirement for mitochondrial Hsp70 or Tim23 . With a series of preprotein constructs, the net charge of the sorting sequence was altered, but the Deltapsi-dependence of import was not affected . These results suggested that the sorting sequence contributed to the import driving mechanism in a manner distinct from the two known roles of Deltapsi . Indeed, a charge-neutral amino acid exchange in the hydrophobic segment of the sorting sequence generated a preprotein with an even better import, i.e . one with lower Deltapsi-dependence than the wild-type preprotein . The sorting sequence functioned early in the import pathway since it strongly influenced the efficiency of translocation of the matrix-targeting sequence across the inner membrane . These results suggest a model whereby an electrophoretic effect of Deltapsi on the matrix-targeting sequence is complemented by an import-stimulating activity of the sorting sequence.

Mol Biol Cell, 2000 Nov, 11(11), 3859 - 71
Sec62p, a component of the endoplasmic reticulum protein translocation machinery, contains multiple binding sites for the Sec-complex; Wittke S et al.; SEC62 encodes an essential component of the Sec-complex that is responsible for posttranslational protein translocation across the membrane of the endoplasmic reticulum in Saccharomyces cerevisiae . The specific role of Sec62p in translocation was not known and difficult to identify because it is part of an oligomeric protein complex in the endoplasmic reticulum membrane . An in vivo competition assay allowed us to characterize and dissect physical and functional interactions between Sec62p and components of the Sec-complex . We could show that Sec62p binds via its cytosolic N- and C-terminal domains to the Sec-complex . The N-terminal domain, which harbors the major interaction site, binds directly to the last 14 residues of Sec63p . The C-terminal binding site of Sec62p is less important for complex stability, but adjoins the region in Sec62p that might be involved in signal sequence recognition.

Mol Biochem Parasitol, 2000 Oct, 110(2), 323 - 31
Cloning and functional expression of Rpn1, a regulatory-particle non-ATPase subunit 1, of proteasome from Trypanosoma cruzi; Zou CB et al.; Non-lysosomal protein degradation in eukaryotic cells involves a proteolytic complex referred to as 26S proteasome that consists of a 20S core particle and one or two 19S regulatory particles . We have cloned the gene RPN1 encoding Rpnl (regulatory-particle non-ATPase subunit 1), one of the largest subunits of proteasome, from Trypanosoma cruzi . It contains 2712 bp and encodes 904 amino acid residues with a calculated molecular mass of 98.2 kDa and an isoelectric point of 5.2 . The predicted amino acid sequence of the trypanosomatid Rpn1 shares 39.0 and 32.0% overall identities with human Rpn1 and Saccharomyces cerevisiae Nas1 (non-ATPase subunit 1), an Rpn1 homolog, respectively, while the sequence identities among T . cruzi, Plasmodium falciparum, and Entamoeba histolytica Rpnl are approximately 30% . T . cruzi Rpn1 contains nine repeats of about 36 amino acid residues conserved in Rpn1s from various organisms . T . cruzi RPN1 is located on the 2300- and 1900-kb chromosomal DNA, displays a putative allelic variation as RPN1-1 and RPN1-2 with 98.8% identity between these two putative gene products, and is transcribed from both alleles at a comparable level throughout the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes . The expression of the trypanosomatid Rpnl in the temperature-sensitive nas1 yeast mutant rescued the growth defect at the restrictive temperature, indicating that Rpn1 functions as a Nas1 and probably assembles into the 19S regulatory particle of the yeast 26S proteasome.

J Virol, 2000 Dec, 74(23), 11278 - 85
DNA-Dependent protein kinase is not required for efficient lentivirus integration; Baekelandt V et al.; How DNA is repaired after retrovirus integration is not well understood . DNA-dependent protein kinase (DNA-PK) is known to play a central role in the repair of double-stranded DNA breaks . Recently, a role for DNA-PK in retroviral DNA integration has been proposed (R . Daniel, R . A . Katz, and A . M . Skalka, Science 284:644-647, 1999) . Reduced transduction efficiency and increased cell death by apoptosis were observed upon retrovirus infection of cultured scid cells . We have used a human immunodeficiency virus (HIV) type 1 (HIV-1)-derived lentivirus vector system to further investigate the role of DNA-PK during integration . We measured lentivirus transduction of scid mouse embryonic fibroblasts (MEF) and xrs-5 or xrs-6 cells . These cells are deficient in the catalytic subunit of DNA-PK and in Ku, the DNA-binding subunit of DNA-PK, respectively . At low vector titers, efficient and stable lentivirus transduction was obtained, excluding an essential role for DNA-PK in lentivirus integration . Likewise, the efficiency of transduction of HIV-derived vectors in scid mouse brain was as efficient as that in control mice, without evidence of apoptosis . We observed increased cell death in scid MEF and xrs-5 or xrs-6 cells, but only after transduction with high vector titers (multiplicity of infection {MOI}, >1 transducing unit {TU}/cell) and subsequent passage of the transduced cells . At an MOI of <1 TU/cell, however, transduction efficiency was even higher in DNA-PK-deficient cells than in control cells . Taken together, the data suggest a protective role of DNA-PK against cellular toxicity induced by high levels of retrovirus integrase or integration . Another candidate cellular enzyme that has been claimed to play an important role during retrovirus integration is poly(ADP-ribose) polymerase (PARP) . However, no inhibition of lentivirus vector-mediated transduction or HIV-1 replication by 3-methoxybenzamide, a known PARP inhibitor, was observed . In conclusion, DNA-PK and PARP are not essential for lentivirus integration.

Genes Dev, 2000 Nov 1, 14(21), 2737 - 44
Ssn6-Tup1 interacts with class I histone deacetylases required for repression; Watson AD et al.; Ssn6-Tup1 regulates multiple genes in yeast, providing a paradigm for corepressor functions . Tup1 interacts directly with histones H3 and H4, and mutation of these histones synergistically compromises Ssn6-Tup1-mediated repression . In vitro, Tup1 interacts preferentially with underacetylated isoforms of H3 and H4, suggesting that histone acetylation may modulate Tup1 functions in vivo . Here we report that histone hyperacetylation caused by combined mutations in genes encoding the histone deacetylases (HDACs) Rpd3, Hos1, and Hos2 abolishes Ssn6-Tup1 repression . Unlike HDAC mutations that do not affect repression, this combination of mutations causes concomitant hyperacetylation of both H3 and H4 . Strikingly, two of these class I HDACs interact physically with Ssn6-Tup1 . These findings suggest that Ssn6-Tup1 actively recruits deacetylase activities to deacetylate adjacent nucleosomes and promote Tup1-histone interactions.

Genes Dev, 2000 Nov 1, 14(21), 2725 - 36
The endoribonuclease activity of mammalian IRE1 autoregulates its mRNA and is required for the unfolded protein response; Tirasophon W et al.; The unfolded protein response (UPR) is a signal transduction pathway that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) . In Saccharomyces cerevisiae the ER transmembrane receptor, Ire1p, transmits the signal to the nucleus culminating in the transcriptional activation of genes encoding an adaptive response . Yeast Ire1p requires both protein kinase and site-specific endoribonuclease (RNase) activities to signal the UPR . In mammalian cells, two homologs, Ire1 alpha and Ire1 beta, are implicated in signaling the UPR . To elucidate the RNase requirement for mammalian Ire1 function, we have identified five amino acid residues within IRE1 alpha that are essential for RNase activity but not kinase activity . These mutants were used to demonstrate that the RNase activity is required for UPR activation by IRE1 alpha and IRE1 beta . In addition, the data support that IRE1 RNase is activated by dimerization-induced trans-autophosphorylation and requires a homodimer of catalytically functional RNase domains . Finally, the RNase activity of wild-type IRE1 alpha down-regulates hIre1 alpha mRNA expression by a novel mechanism involving cis-mediated IRE1 alpha-dependent cleavage at three specific sites within the 5' end of Ire1 alpha mRNA.

Genes Dev, 2000 Nov 1, 14(21), 2689 - 94
Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin; Oldham S et al.; The adaptation of growth in response to nutritional changes is essential for the proper development of all organisms . Here we describe the identification of the Drosophila homolog of the target of rapamycin (TOR), a candidate effector for nutritional sensing . Genetic and biochemical analyses indicate that dTOR impinges on the insulin signaling pathway by autonomously affecting growth through modulating the activity of dS6K . However, in contrast to other components in the insulin signaling pathway, partial loss of dTOR function preferentially reduces growth of the endoreplicating tissues . These results are consistent with dTOR residing on a parallel amino acid sensing pathway.

J Cell Sci, 2000 Dec, 113 Pt 23, 4313 - 8
Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling; Varnum-Finney B et al.; Cell-cell interactions mediated by Notch and its ligands are known to effect many cell fate decisions in both invertebrates and vertebrates . However, the mechanisms involved in ligand induced Notch activation are unknown . Recently it was shown that, in at least some cases, endocytosis of the extracellular domain of Notch and ligand by the signaling cell is required for signal induction in the receptive cell . These results imply that soluble ligands (ligand extracellular domains) although capable of binding Notch would be unlikely to activate it . To test the potential activity of soluble Notch ligands, we generated monomeric and dimeric forms of the Notch ligand Delta-1 by fusing the extracellular domain to either a series of myc epitopes (Delta-1(ext-myc)) or to the Fc portion of human IgG-1 (Delta-1(ext-IgG)), respectively . Notch activation, assayed by inhibition of differentiation in C2 myoblasts and by HES1 transactivation in U20S cells, occurred when either Delta-1(ext-myc) or Delta-1(ext-IgG) were first immobilized on the plastic surface . However, Notch was not activated by either monomeric or dimeric ligand in solution (non-immobilized) . Furthermore, both non-immobilized Delta-1(ext-myc) and Delta-1(ext-IgG) blocked the effect of immobilized Delta . These results indicate that Delta-1 extracellular domain must be immobilized to induce Notch activation in C2 or U20S cells and that non-immobilized Delta-1 extracellular domain is inhibitory to Notch function . These results imply that ligand stabilization may be essential for Notch activation.

Mol Microbiol, 2000 Oct, 38(2), 411 - 22
Regulation of the Pcl7-Pho85 cyclin-cdk complex by Pho81; Lee M et al.; Saccharomyces cerevisiae strains lacking a functional Pho85 cyclin-dependent kinase (cdk) exhibit a complex phenotype, including deregulation of phosphatase genes controlled by the transcription factor Pho4, slow growth on rich media, failure to grow using galactose, lactate or glycerol as a carbon source and hyperaccumulation of glycogen . The ability of Pho85 to regulate the transcription factor Pho4 is mediated by its association the Pho80 cyclin . Some other regulatory functions of the Pho85 cdk have been shown to be mediated via its interaction with a recently identified family of Pho80-related cyclins (Pcls) . Here, we show that the poorly characterized Pho80-like protein Pcl7 forms a functional kinase complex with the Pho85 cdk, and that the activity of this complex is inhibited in response to phosphate starvation . Additionally, we show that Pcl7 interacts with the phosphate-regulated cyclin-cdk inhibitor Pho81, and that the regulation of the Pcl7-Pho85 complex in response to changes in phosphate levels is dependent on Pho81 . Thus, we demonstrate for the first time that the Pho81 regulator is not dedicated to regulating Pho80, but may act to co-ordinate the activity of both the Pho80-Pho85 and Pcl7-Pho85 cyclin-cdk complexes in response to phosphate levels . We also demonstrate that expression of Pcl7 is cell cycle regulated, with maximal activity occurring in mid to late S-phase, perhaps suggesting a role for Pcl7 in cell cycle progression . Finally, we describe the phenotype of pcl7Delta and pcl6Delta yeast strains that have defects in carbon source utilization.

Br J Dermatol, 2000 Oct, 143(4), 733 - 40
Expression of small proline rich proteins in neoplastic and inflammatory skin diseases; De Heller-Milev M et al.; BACKGROUND: The formation of the cornified cell envelope (CE) during the late stages of epidermal differentiation is essential for epidermal barrier function and protects the body against environmental attack and water loss . Formation of the CE involves the replacement of the plasma membrane by cross-linkage of precursor proteins such as involucrin, small proline rich proteins (SPRR) and loricrin . In normal epidermis, SPRR1 is restricted to appendages, SPRR2 is also expressed in interfollicular areas, while SPRR3 is completely absent; the latter is most abundant in oral epithelium . This differential expression indicates an important part for SPRRs in specific barrier requirements, and reflects their importance in the biomechanical properties of the CE . OBJECTIVES: We report here on the expression of SPRR1, SPRR2 and SPRR3 in a wide range of cutaneous neoplastic and inflammatory diseases . METHODS: We used immunohistochemistry; in addition, Northern blot analysis of malignant tumours was performed . RESULTS: Increased suprabasal expression of SPRR1 and SPRR2, but no SPRR3 expression, was noted in inflammatory dermatoses with orthokeratotic and parakeratotic squamous differentiation . By contrast, differentiating epidermal tumours such as Bowen's disease, keratoacanthoma and squamous cell carcinoma expressed SPRR3 . CONCLUSIONS: As SPRRs were originally cloned on the basis of their expression in ultraviolet light-irradiated keratinocytes, the expression of SPRR3 in actinic lesions is of interest, and might serve as a diagnostic tool.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 12985 - 90
A mutant allele of essential, general translation initiation factor DED1 selectively inhibits translation of a viral mRNA; Noueiry AO et al.; Positive-strand RNA virus genomes are substrates for translation, RNA replication, and encapsidation . To identify host factors involved in these functions, we used the ability of brome mosaic virus (BMV) RNA to replicate in yeast . We report herein identification of a mutation in the essential yeast gene DED1 that inhibited BMV RNA replication but not yeast growth . DED1 encodes a DEAD (Asp-Glu-Ala-Asp)-box RNA helicase required for translation initiation of all yeast mRNAs . Inhibition of BMV RNA replication by the mutant DED1 allele (ded1-18) resulted from inhibited expression of viral polymerase-like protein 2a, encoded by BMV RNA2 . Inhibition of RNA2 translation was selective, with no effect on general cellular translation or translation of BMV RNA1-encoded replication factor 1a, and was independent of p20, a cellular antagonist of DED1 function in translation . Inhibition of RNA2 translation in ded1-18 yeast required the RNA2 5' noncoding region (NCR), which also conferred a ded1-18-specific reduction in expression on a reporter gene mRNA . Comparison of the similar RNA1 and RNA2 5' NCRs identified a 31-nucleotide RNA2-specific region that was required for the ded1-18-specific RNA2 translation block and attenuated RNA2 translation in wild-type yeast . Further comparisons and RNA structure predictions suggest a modular arrangement of replication and translation signals in RNA1 and RNA2 5' NCRs that appears conserved among bromoviruses . The 5' attenuator and DED1 dependence of RNA2 suggest that, despite its divided genome, BMV regulates polymerase translation relative to other replication factors, just as many single-component RNA viruses use translational read-through and frameshift mechanisms to down-regulate polymerase . The results show that a DEAD-box helicase can selectively activate translation of a specific mRNA and may provide a paradigm for translational regulation by other members of the ubiquitous DEAD-box RNA helicase family.

Nature, 2000 Oct 26, 407(6807), 1022 - 6
Matrix proteins can generate the higher order architecture of the Golgi apparatus; Seemann J et al.; The Golgi apparatus in animal cells comprises a reticulum of linked stacks in the pericentriolar and often in the juxtanuclear regions of the cell . The unique architecture of this organelle is thought to depend on the cytoskeleton and cytoplasmic matrix proteins--the best characterized being the golgin family of fibrous, coiled-coil proteins and the GRASP family of stacking proteins . Here we show that these matrix proteins can be separated from oligosaccharide-modifying enzymes in the Golgi stack without affecting their ability to form a ribbon-like reticulum in the correct location near to the nucleus . Our data suggest that the Golgi is a structural scaffold that can exist independently of, but is normally populated by, the enzyme-containing membranes that modify transiting cargo . This new concept of the Golgi further indicates that the Golgi may be an autonomous organelle rather than one that is in simple dynamic equilibrium with the endoplasmic reticulum.

Curr Biol, 2000 Oct 19, 10(20), R750 - 2
Membrane transport: deciphering fusion; Clague MJ et al.; Membrane fusion events that occur in yeast have been reconstituted with a minimal set of SNARE protein components . This system has been exploited to establish the syntax underlying specificity of intracellular fusion events from yeast to mammals.

Arch Biochem Biophys, 2000 Oct 15, 382(2), 262 - 74
Interaction of insulin-like growth factor binding protein-4, Miz-1, leptin, lipocalin-type prostaglandin D synthase, and granulin precursor with the N-terminal half of type III hexokinase; Sui D et al.; Insulin-like growth factor binding protein-4, Miz-1, leptin, prostaglandin D synthase, and granulin precursor were identified as proteins interacting with the N-terminal half of mammalian Type III hexokinase (HKIII) in the yeast two-hybrid method . These interactions were confirmed by in vitro binding studies . All five of these proteins, and their mRNAs, were present in PC12 cells, as shown by immunoblotting and RT-PCR, respectively . All were coimmunoprecipitated from PC12 extracts with an antibody against HKIII, but not with anti-Type I hexokinase . Moreover, all of these proteins were coimmunoprecipitated using antileptin as precipitating antibody, indicating the existence of a macromolecular complex including these five proteins and HKIII . Transfection of M+R 42 cells with HKIII-green fluorescent protein (GFP) reporter constructs gave a diffuse intracellular fluorescence . Cotransfection with leptin or Miz-1 resulted in distinctly different localization of the HKIII-GFP fusion protein, at intracellular sites coincident with localization of leptin-GFP or Miz-1-GFP reporter constructs.

Arch Biochem Biophys, 2000 Oct 15, 382(2), 219 - 23
Expression and functional characterization of human protein X variants in SV40-immortalized protein X-deficient and E2-deficient human skin fibroblasts; Seyda A et al.; To gain further insight into the nature and function of the domains of the human protein X (a pyruvate dehydrogenase complex component also known as the E3-binding protein), we expressed the wild-type as well as two artificially created variants, K37E and S422H, in SV40-immortalized protein X-deficient and E2-deficient human skin fibroblasts . The former mutant does not carry the lipoic acid moiety, the latter mutant was designed to investigate the possibility that protein X could exhibit an intrinsic acetyltransferase activity and use either its own catalytic center or the catalytic center of E2 . Similar experiments have been performed in the past using the Saccharomyces cerevisiae expression system . However, lack of sequence similarity between the mammalian and the yeast protein X homologues suggests they are not biochemically equivalent . Mutant cells transfected with the wild-type gene for protein X produced a PDH complex that exhibited about 50% overall activity of the control cells . None of the expressed protein X variants had an effect on the specific activity of the PDH complex, suggesting that the human protein X plays a purely structural role in the functioning of the pyruvate dehydrogenase complex.

Arch Biochem Biophys, 2000 Oct 15, 382(2), 189 - 94
On the reduction of dithiolethiones and dithiolylium ions by NADPH and glutathione reductase; Levron B et al.; Results of in vitro experiments carried out in water at 25 degrees C and at pH 7.56 proved that NADPH in the presence of yeast glutathione reductase did not react with 1,2-dithiole-3-thiones and 1,2-dithiole-3-ones . On the other hand, 3-methylthiodithiolylium ions did react in these conditions . The reaction was identified and methyl 3-mercaptopropenedithioate resulting from a two-electron reduction process was obtained . A kinetic scheme consisting in a biordered mechanism has been found (Km = 2.6 10(-5) mol x l(-1)) . All these results raise the question of a possible in vivo methylation (or alkylation) of dithiolethiones occurring prior to any other reductive biochemical process they may undergo . They also raise the question of the very existence (or in any case the generalization) of a reductive metabolism of dithiolethiones.

FEBS Lett, 2000 Nov 3, 484(2), 164 - 8
EMI, a novel cysteine-rich domain of EMILINs and other extracellular proteins, interacts with the gC1q domains and participates in multimerization; Doliana R et al.; The N-terminal cysteine-rich domain (EMI domain) of EMILIN-1 is a new protein domain that is shared with two proteins (multimerin and EMILIN-2) and with four additional database entries . The EMI domains are always located at the N-terminus, have a common gene organization, and belong to proteins that are forming or are compatible with multimer formation . The potential role of the EMI domain in the assembly of EMILIN-1 was investigated by the two-hybrid system . No reporter gene activity was detected when EMI-1 was co-transformed with the C-terminal gC1q-1 domain excluding a head-to-tail multimerization; conversely, a strong interaction was detected when the EMI-1 domain was co-transformed with the gC1q-2 domain of EMILIN-2.

FEBS Lett, 2000 Nov 3, 484(2), 82 - 6
Binding of coatomer by the PEX11 C-terminus is not required for function; Maier AG et al.; Microbodies are single membrane-bound organelles found in eukaryotes from trypanosomes to man . Although they have diverse roles in metabolism, the mechanisms and molecules involved in membrane biogenesis and matrix protein import are conserved . Similarly, the basic mechanisms and structures involved in vesicular transport are similar throughout eukaryotic evolution . The PEX11 proteins are required for the division of microbodies in trypanosomes, yeast and mammals, and a role of coatomer in this process has been suggested . We show here that the binding of trypanosome, yeast and bovine coatomers to selected peptides is identical . Coatomer binds to the C-termini of trypanosome PEX11 and rat Pex11alpha, but not yeast Pex11p or human Pex11beta . Mutations of the C-terminus of trypanosome PEX11 that eliminated coatomer binding did not affect function in yeast or trypanosomes . Thus binding of coatomer to the C-terminus of PEX11 is not required for PEX11 function.

Anal Biochem, 2000 Nov 15, 286(2), 289 - 94
A chromogenic substrate for a beta-xylosidase-coupled assay of alpha-glucuronidase; Biely P et al.; 4-Nitrophenyl 2-(4-O-methyl-alpha-d-glucopyranuronosyl)-beta-d-xylopyranoside obtained on deesterification of 4-nitrophenyl 2-O-(methyl 4-O-methyl-alpha-d-glucopyranosyluronate)-beta-d-xylopyranoside (Hirsch et al., Carbohydr . Res . 310, 145-149, 1998) was found to be an excellent substrate for the measurement of hemicellulolytic alpha-glucuronidase activity . A new precise alpha-glucuronidase assay was developed by coupling the alpha-glucuronidase-catalyzed formation of 4-nitrophenyl beta-d-xylopyranoside with its efficient hydrolysis by beta-xylosidase . A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the beta-xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, was used as a source of beta-xylosidase . The activity values of beta-xylosidase in the assay required to achieve a steady-state rate of 4-nitrophenol formation shortly after starting the alpha-glucuronidase reaction were obtained both experimentally and by calculation using the kinetics of coupled enzyme reactions .

J Biol Chem, 2001 Feb 9, 276(6), 3811 - 9 Epub 2000 Nov 07.
Role of accessory factors and steroid receptor coactivator 1 in the regulation of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids; Stafford JM et al.; In the liver, glucocorticoids induce a 10-15-fold increase in the rate of transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene, which encodes a key gluconeogenic enzyme . This induction requires a multicomponent glucocorticoid response unit (GRU) comprised of four glucocorticoid accessory factor (AF) elements and two glucocorticoid receptor binding sites . We show that the AFs that bind the gAF1, gAF2, and gAF3 elements (hepatocyte nuclear factor {HNF}4/chicken ovalbumin upstream promoter transcription factor 1 and HNF3beta) all interact with steroid receptor coactivator 1 (SRC1) . This suggests that the AFs function in part by recruiting coactivators to the GRU . The binding of a GAL4-SRC1 chimeric protein completely restores the glucocorticoid induction that is lost when any one of these elements is replaced with a GAL4 binding site . Thus, when SRC1 is recruited directly to gAF1, gAF2, or gAF3, the requirement for the corresponding AF is bypassed . Surprisingly, glucocorticoid receptor is still required when SRC1 is recruited directly to the GAL4 site, suggesting a role for the receptor in activating SRC1 in the context of the GRU . Structural variants of GAL4-SRC1 were used to identify requirements for the basic-helix-loop-helix and histone acetyltransferase domains of SRC1, and these are specific to the region of the promoter to which the coactivator is recruited.

J Biol Chem, 2001 Jan 26, 276(4), 2637 - 43 Epub 2000 Nov 06.
Formation of conjugated delta8,delta10-double bonds by delta12-oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid; Cahoon EB et al.; Divergent forms of the plant Delta(12)-oleic-acid desaturase (FAD2) have previously been shown to catalyze the formation of acetylenic bonds, epoxy groups, and conjugated Delta(11),Delta(13)-double bonds by modification of an existing Delta(12)-double bond in C(18) fatty acids . Here, we report a class of FAD2-related enzymes that modifies a Delta(9)-double bond to produce the conjugated trans-Delta(8),trans-Delta(10)-double bonds found in calendic acid (18:3Delta(8trans,10trans,12cis)), the major component of the seed oil of Calendula officinalis . Using an expressed sequence tag approach, cDNAs for two closely related FAD2-like enzymes, designated CoFADX-1 and CoFADX-2, were identified from a C . officinalis developing seed cDNA library . The deduced amino acid sequences of these polypeptides share 40-50% identity with those of other FAD2 and FAD2-related enzymes . Expression of either CoFADX-1 or CoFADX-2 in somatic soybean embryos resulted in the production of calendic acid . In embryos expressing CoFADX-2, calendic acid accumulated to as high as 22% (w/w) of the total fatty acids . In addition, expression of CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae was accompanied by calendic acid accumulation when induced cells were supplied exogenous linoleic acid (18:2Delta(9cis,12cis)) . These results are thus consistent with a route of calendic acid synthesis involving modification of the Delta(9)-double bond of linoleic acid . Regiospecificity for Delta(9)-double bonds is unprecedented among FAD2-related enzymes and further expands the functional diversity found in this family of enzymes.

J Comp Neurol, 2000 Dec 11, 428(2), 223 - 39
Differential localization of septins in the mouse brain; Kinoshita A et al.; We have carried out a comparative immunohistochemical study on four members of the septin family, CDCrel-1, Septin6, CDC10, and H5, which are abundantly expressed in the adult mouse brain . We found that each septin showed overlapping but distinct distribution at the levels of light and electron microscopy . CDCrel-1 was abundant in inhibitory presynaptic terminals and associated with GABAergic vesicles in the thalamus, globus pallidus, and cerebellar nuclei . Septin6 was associated with synaptic vesicles in various brain regions, including glomeruli of the olfactory bulb . CDC10 was diffusely expressed in the brain and was localized beneath presynaptic membrane and astroglial processes . H5 was localized in the astroglial processes in some specific brain regions . The differential expression and subcellular localization of these septins indicates that a given neuron or glial cell expresses a specific set of septin monomers and that the resulting septin complexes with distinct compositions may play distinct roles in the brain .

Curr Opin Cell Biol, 2000 Dec, 12(6), 690 - 6
Eukaryotic DNA replication: from pre-replication complex to initiation complex; Takisawa H et al.; A common mechanism has emerged for the control of the initiation of eukaryotic DNA replication . The minichromosome maintenance protein complex (MCM) and Cdc45 have now been recognized as central components of the initiation machinery . In addition, two types of S phase promoting kinases conserved between yeast and humans play critical roles in the initiation reaction . At the onset of S phase, S phase kinases promote the association of Cdc45 with MCM at origins . Upon the formation of the MCM-Cdc45 complex at origins, the duplex DNA is unwound and various replication proteins, including DNA polymerases, are recruited onto unwound DNA . The increasing number of newly identified factors involved in the initiation reaction indicates that the control of initiation requires highly evolved machinery in eukaryotic cells.

Biochim Biophys Acta, 2000 Nov 10, 1469(3), 121 - 32
Maturation of HIV envelope glycoprotein precursors by cellular endoproteases; Moulard M et al.; The entry of enveloped viruses into its host cells is a crucial step for the propagation of viral infection . The envelope glycoprotein complex controls viral tropism and promotes the membrane fusion process . The surface glycoproteins of enveloped viruses are synthesized as inactive precursors and sorted through the constitutive secretory pathway of the infected cells . To be infectious, most of the viruses require viral envelope glycoprotein maturation by host cell endoproteases . In spite of the strong variability of primary sequences observed within different viral envelope glycoproteins, the endoproteolytical cleavage occurs mainly in a highly conserved domain at the carboxy terminus of the basic consensus sequence (Arg-X-Lys/Arg-Arg downward arrow) . The same consensus sequence is recognized by the kexin/subtilisin-like serine proteinases (so called convertases) in many cellular substrates such as prohormones, proprotein of receptors, plasma proteins, growth factors and bacterial toxins . Therefore, several groups of investigators have evaluated the implication of convertases in viral envelope glycoprotein cleavage . Using the vaccinia virus overexpression system, furin was first shown to mediate the proteolytic maturation of both human immunodeficiency virus (HIV-1) and influenza virus envelope glycoproteins . In vitro studies demonstrated that purified convertases directly and specifically cleave viral envelope glycoproteins . Although these studies suggested the participation of several enzymes belonging to the convertases family, recent data suggest that other protease families may also participate in the HIV envelope glycoprotein processing . Their role in the physiological maturation process is still hypothetical and the molecular mechanism of the cleavage is not well documented . Crystallization of the hemagglutinin precursor (HA0) of influenza virus allowed further understanding of the molecular interaction between viral precursors and the cellular endoproteases . Furthermore, relationships between differential pathogenicity of influenza strains and their susceptibility to cleavage are molecularly funded . Here we review the most recent data and recent insights demonstrating the crucial role played by this activation step in virus infectivity . We discuss the cellular endoproteases that are implicated in HIV gp160 endoproteolytical maturation into gp120 and gp41.

Biochemistry, 2000 Nov 7, 39(44), 13584 - 94
pH dependence of formation of a partially unfolded state of a Lys 73 --> His variant of iso-1-cytochrome c: implications for the alkaline conformational transition of cytochrome c; Nelson CJ et al.; The alkaline conformational transition of a lysine 73 --> histidine variant of iso-1-cytochrome c has been studied . The transition has been monitored at 695 nm, a band sensitive to the presence of the heme-methionine 80 bond, at the heme Soret band which is sensitive to the nature of the heme ligand, and by NMR methods . The guanidine hydrochloride dependence of the alkaline conformational transition has also been monitored . The histidine 73 protein has an unusual biphasic alkaline conformational transition at both 695 nm and the heme Soret band, consistent with a three-state process . The conformational transition is fully reversible . An equilibrium model has been developed to account for this behavior . With this model, it has been possible to obtain the acid constant for the trigger group, pK(H), of the low-pH phase from the equilibrium data . A pK(H) value of 6.6 +/- 0.1 in H(2)O was obtained, consistent with a histidine acting as the trigger group . The NMR data for the low-pH phase of the alkaline conformational transition are consistent with an imidazole ligand replacing Met 80 . For the high-pH phase of the biphasic alkaline transition, the NMR data are consistent with lysine 79 being the heme ligand . Guanidine hydrochloride m values of 1.67 +/- 0.08 and 1.1 +/- 0.2 kcal mol(-1) M(-1) were obtained for the low- and high-pH phases of the biphasic alkaline transition of the histidine 73 protein, respectively, consistent with a greater structural disruption for the low-pH phase of the transition.

Mamm Genome, 2000 Nov, 11(11), 1006 - 15
HIP12 is a non-proapoptotic member of a gene family including HIP1, an interacting protein with huntingtin; Chopra VS et al.; Huntingtin-interacting protein I (HIP1) is a membrane-associated protein that interacts with huntingtin, the protein altered in Huntington disease . HIP1 shows homology to Sla2p, a protein essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae . We have determined that the HIP1 gene comprises 32 exons spanning approximately 215 kb of genomic DNA and gives rise to two alternate splice forms termed HIP1-1 and HIP1-2 . Additionally, we have identified a novel protein termed HIP12 with significant sequence and biochemical similarities to HIP1 and high sequence similarity to Sla2p . HIP12 differs from HIP1 in its pattern of expression both at the mRNA and protein level . However, HIP1 and HIP12 are both found within the brain and show a similar subcellular distribution pattern . In contrast to HIP1, which is toxic in cell culture, HIP12 does not confer toxicity in the same assay systems . Interestingly, HIP12 does not interact with huntingtin but can interact with HIP1 . suggesting a potential interaction in vivo that may influence the function of each respective protein.

Nat Struct Biol, 2000 Nov, 7(11), 1062 - 7
A gated channel into the proteasome core particle; Groll M et al.; The core particle (CP) of the yeast proteasome is composed of four heptameric rings of subunits arranged in a hollow, barrel-like structure . We report that the CP is autoinhibited by the N-terminal tails of the outer (alpha) ring subunits . Crystallographic analysis showed that deletion of the tail of the alpha 3-subunit opens a channel into the proteolytically active interior chamber of the CP, thus derepressing peptide hydrolysis . In the latent state of the particle, the tails prevent substrate entry by imposing topological closure on the CP . Inhibition by the alpha-subunit tails is relieved upon binding of the regulatory particle to the CP to form the proteasome holoenzyme.

Nat Genet, 2000 Nov, 26(3), 375 - 8
Proliferating cell nuclear antigen and Msh2p-Msh6p interact to form an active mispair recognition complex; Flores-Rozas H et al.; Proliferating cell nuclear antigen (PCNA) is required for mismatch repair (MMR) and has been shown to interact with complexes containing Msh2p or MLH1 (refs 1-4) . PCNA has been implicated to act in MMR before and during the DNA synthesis step, although the biochemical basis for the role of PCNA early in MMR is unclear . Here we observe an interaction between PCNA and Msh2p-Msh6p mediated by a specific PCNA-binding site present in Msh6p . An msh6 mutation that eliminated the PCNA-binding site caused a mutator phenotype and a defect in the interaction with PCNA . The association of PCNA with Msh2p-Msh6p stimulated the preferential binding of Msh2p-Msh6p to DNA containing mispaired bases . Mutant PCNA proteins encoded by MMR-defective pol30 alleles were defective for interaction with Msh2p-Msh6p and for stimulation of mispair binding by Msh2p-Msh6p . Our results suggest that PCNA functions directly in mispair recognition and that mispair recognition requires a higher-order complex containing proteins in addition to Msh2p-Msh6p.

J Cell Biol, 2000 Oct 30, 151(3), 551 - 62
New component of the vacuolar class C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion; Wurmser AE et al.; The class C subset of vacuolar protein sorting (Vps) proteins (Vps11, Vps18, Vps16 and Vps33) assembles into a vacuole/prevacuole-associated complex . Here we demonstrate that the class C-Vps complex contains two additional proteins, Vps39 and Vps41 . The COOH-terminal 148 amino acids of Vps39 direct its association with the class C-Vps complex by binding to Vps11 . A previous study has shown that a large protein complex containing Vps39 and Vps41 functions as a downstream effector of the active, GTP-bound form of Ypt7, a rab GTPase required for the fusion of vesicular intermediates with the vacuole (Price, A., D . Seals, W . Wickner, and C . Ungermann . 2000 . J . Cell Biol . 148:1231-1238) . Here we present data that indicate that this complex also functions to stimulate nucleotide exchange on Ypt7 . We show that Vps39 directly binds the GDP-bound and nucleotide-free forms of Ypt7 and that purified Vps39 stimulates nucleotide exchange on Ypt7 . We propose that the class C-Vps complex both promotes Vps39-dependent nucleotide exchange on Ypt7 and, based on the work of Price et al., acts as a Ypt7 effector that tethers transport vesicles to the vacuole . Thus, the class C-Vps complex directs multiple reactions during the docking and fusion of vesicles with the vacuole, each of which contributes to the overall specificity and efficiency of this transport process.

J Cell Biol, 2000 Oct 30, 151(3), 529 - 38
Cell-free reconstitution of microautophagic vacuole invagination and vesicle formation; Sattler T et al.; Many organelles change their shape in the course of the cell cycle or in response to environmental conditions . Lysosomes undergo drastic changes of shape during microautophagocytosis, which include the invagination of their boundary membrane and the subsequent scission of vesicles into the lumen of the organelle . The mechanism driving these structural changes is enigmatic . We have begun to analyze this process by reconstituting microautophagocytosis in a cell-free system . Isolated yeast vacuoles took up fluorescent dyes or reporter enzymes in a cytosol-, ATP-, and temperature-dependent fashion . During the uptake reaction, vacuolar membrane invaginations, called autophagic tubes, were observed . The reaction resulted in the transient formation of autophagic bodies in the vacuolar lumen, which were degraded upon prolonged incubation . Under starvation conditions, the system reproduced the induction of autophagocytosis and depended on specific gene products, which were identified in screens for mutants deficient in autophagocytosis . Microautophagic uptake depended on the activity of the vacuolar ATPase and was sensitive to GTPgammaS, indicating a requirement for GTPases and for the vacuolar membrane potential . However, microautophagocytosis was independent of known factors for vacuolar fusion and vesicular trafficking . Therefore, scission of the invaginated membrane must occur via a novel mechanism distinct from the homotypic fusion of vacuolar membranes.

J Cell Biol, 2000 Oct 30, 151(3), 519 - 28
Autophagic tubes: vacuolar invaginations involved in lateral membrane sorting and inverse vesicle budding; Muller O et al.; Many intracellular compartments of eukaryotic cells do not adopt a spherical shape, which would be expected in the absence of mechanisms organizing their structure . However, little is known about the principles determining the shape of organelles . We have observed very defined structural changes of vacuoles, the lysosome equivalents of yeast . The vacuolar membrane can form a large tubular invagination from which vesicles bud off into the lumen of the organelle . Formation of the tube is regulated via the Apg/Aut pathway . Its lumen is continuous with the cytosol, making this inverse budding reaction equivalent to microautophagocytosis . The tube is highly dynamic, often branched, and defined by a sharp kink of the vacuolar membrane at the site of invagination . The tube is formed by vacuoles in an autonomous fashion . It persists after vacuole isolation and, therefore, is independent of surrounding cytoskeleton . There is a striking lateral heterogeneity along the tube, with a high density of transmembrane particles at the base and a smooth zone devoid of transmembrane particles at the tip where budding occurs . We postulate a lateral sorting mechanism along the tube that mediates a depletion of large transmembrane proteins at the tip and results in the inverse budding of lipid-rich vesicles into the lumen of the organelle.

J Biol Chem, 2001 Jan 5, 276(1), 12 - 5
The forkhead-associated domain of NBS1 is essential for nuclear foci formation after irradiation but not essential for hRAD50{middle dot}hMRE11{middle dot}NBS1 complex DNA repair activity; Tauchi H et al.; NBS1 (p95), the protein responsible for Nijmegen breakage syndrome, shows a weak homology to the yeast Xrs2 protein at the N terminus region, known as the forkhead-associated (FHA) domain and the BRCA1 C terminus domain . The protein interacts with hMRE11 to form a complex with a nuclease activity for initiation of both nonhomologous end joining and homologous recombination . Here, we show in vivo direct evidence that NBS1 recruits the hMRE11 nuclease complex into the cell nucleus and leads to the formation of foci by utilizing different functions from several domains . The amino acid sequence at 665-693 on the C terminus of NBS1, where a novel identical sequence with yeast Xrs2 protein was found, is essential for hMRE11 binding . The hMRE11-binding region is necessary for both nuclear localization of the complex and for cellular radiation resistance . On the other hand, the FHA domain regulates nuclear foci formation of the multiprotein complex in response to DNA damage but is not essential for nuclear transportation of the complex and radiation resistance . Because the FHA/BRCA1 C terminus domain is widely conserved in eukaryotic nuclear proteins related to the cell cycle, gene regulation, and DNA repair, the foci formation could be associated with many phenotypes of Nijmegen breakage syndrome other than radiation sensitivity.

Carcinogenesis, 2000 Nov, 21(11), 2005 - 10
Differentially expressed genes in asbestos-induced tumorigenic human bronchial epithelial cells: implication for mechanism; Zhao YL et al.; Although exposure to asbestos fibers is associated with the development of lung cancer, the underlying mechanism(s) remains unclear . Using human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells, we previously showed that UICC chrysotiles can malignantly transform these cells in a stepwise fashion before they become tumorigenic in nude mice . In the present study we used cDNA expression arrays to screen differentially expressed genes among the tumorigenic cells . A total of 15 genes were identified, 11 of which were further confirmed by northern blot . Expression levels of these genes were then determined among transformed BEP2D cells at different stages of the neoplastic process, including non-tumorigenic cells that were resistant to serum-induced terminal differentiation, early and late passage transformed BEP2D cells, five representative tumor cell lines and fused tumorigenic-control cell lines which were no longer tumorigenic . A consistent 2- to 3-fold down-regulation of the DCC (deleted in colon cancer), Ku70 and heat shock protein 27 genes were detected in all the independently generated tumor cell lines while expression levels in early transformants as well as in the fusion cell lines remained normal . In contrast, all the tumor cell lines examined demonstrated 2- to 4-fold overexpression of the insulin receptor and its signal transduction genes . Differential expression of these genes was completely restored in the fusion cell lines examined . No alteration in c-jun or EGF receptor expression was found in any of the cell lines . Our data suggest that activation of the insulin receptor pathway and inactivation of DCC and Ku70 may cooperate in malignant transformation of BEP2D cells induced by asbestos.

Virology, 2000 Nov 10, 277(1), 127 - 35
The human T-cell leukemia virus type I (HTLV-I) X region encoded protein p13(II) interacts with cellular proteins; Hou X et al.; Interactions between the Human T-cell leukemia virus type I (HTLV-I) gene product p13(II) and cellular proteins were investigated using the yeast two-hybrid system . Variant forms of p13(II) were derived from two HTLV-I molecular clones, K30p and K34p, that differ in both virus production and in vivo and in vitro infectivity . Two nucleotide differences between the p13 from K30p (p13K30) and K34p (p13K34) result in a Trp-Arg substitution at amino acid 17 and the truncation of the 25 carboxyl-terminal residues of p13K34 . A cDNA library from an HTLV-I-infected rabbit T-cell line was screened with p13K30 and p13K34 as bait . Products of two cDNA clones, C44 and C254, interacted with p13K34 but not with p13K30 . Interactions were further confirmed using the GST-fusion protein coprecipitation assay . Sequence analysis of C44 and C254 cDNA clones revealed similarities to members of the nucleoside monophosphate kinase superfamily and actin-binding protein 280, respectively . Further analysis of the function of these two proteins and the consequence of their interaction with p13 may help elucidate a role for p13 in virus production, infectivity, or the pathogenesis of HTLV-I .

J Mol Biol, 2000 Nov 10, 303(5), 745 - 60
X-ray structures of five renin inhibitors bound to saccharopepsin: exploration of active-site specificity; Cronin NB et al.; Saccharopepsin is a vacuolar aspartic proteinase involved in activation of a number of hydrolases . The enzyme has great structural homology to mammalian aspartic proteinases including human renin and we have used it as a model system to study the binding of renin inhibitors by X-ray crystallography . Five medium-to-high resolution structures of saccharopepsin complexed with transition-state analogue renin inhibitors were determined . The structure of a cyclic peptide inhibitor (PD-129,541) complexed with the proteinase was solved to 2.5 A resolution . This inhibitor has low affinity for human renin yet binds very tightly to the yeast proteinase (K(i)=4 nM) . The high affinity of this inhibitor can be attributed to its bulky cyclic moiety spanning P(2)-P(3)' and other residues that appear to optimally fit the binding sub-sites of the enzyme . Superposition of the saccharopepsin structure on that of renin showed that a movement of the loop 286-301 relative to renin facilitates tighter binding of this inhibitor to saccharopepsin . Our 2.8 A resolution structure of the complex with CP-108,420 shows that its benzimidazole P(3 )replacement retains one of the standard hydrogen bonds that normally involve the inhibitor's main-chain . This suggests a non-peptide lead in overcoming the problem of susceptible peptide bonds in the design of aspartic proteinase inhibitors . CP-72,647 which possesses a basic histidine residue at P(2), has a high affinity for renin (K(i)=5 nM) but proves to be a poor inhibitor for saccharopepsin (K(i)=3.7 microM) . This may stem from the fact that the histidine residue would not bind favourably with the predominantly hydrophobic S(2) sub-site of saccharopepsin .

Development, 2000 Dec, 127(23), 5203 - 12
Synaptogenesis in the giant-fibre system of Drosophila: interaction of the giant fibre and its major motorneuronal target; Jacobs K et al.; The tergotrochanteral (jump) motorneuron is a major synaptic target of the Giant Fibre in Drosophila . These two neurons are major components of the fly's Giant-Fibre escape system . Our previous work has described the development of the Giant Fibre in early metamorphosis and the involvement of the shaking-B locus in the formation of its electrical synapses . In the present study, we have investigated the development of the tergotrochanteral motorneuron and its electrical synapses by transforming Drosophila with a Gal4 fusion construct containing sequences largely upstream of, but including, the shaking-B(lethal) promoter . This construct drives reporter gene expression in the tergotrochanteral motorneuron and some other neurons . Expression of green fluorescent protein in the motorneuron allows visualization of its cell body and its subsequent intracellular staining with Lucifer Yellow . These preparations provide high-resolution data on motorneuron morphogenesis during the first half of pupal development . Dye-coupling reveals onset of gap-junction formation between the tergotrochanteral motorneuron and other neurons of the Giant-Fibre System . The medial dendrite of the tergotrochanteral motorneuron becomes dye-coupled to the peripheral synapsing interneurons between 28 and 32 hours after puparium formation . Dye-coupling between tergotrochanteral motorneuron and Giant Fibre is first seen at 42 hours after puparium formation . All dye coupling is abolished in a shaking-B(neural) mutant . To investigate any interactions between the Giant Fibre and the tergotroachanteral motorneuron, we arrested the growth of the motorneuron's medial neurite by targeted expression of a constitutively active form of Dcdc42 . This results in the Giant Fibre remaining stranded at the midline, unable to make its characteristic bend . We conclude that Giant Fibre morphogenesis normally relies on fasciculation with its major motorneuronal target.

Development, 2000 Dec, 127(23), 5071 - 82
The C . elegans F-box/WD-repeat protein LIN-23 functions to limit cell division during development; Kipreos ET et al.; In multicellular eukaryotes, a complex program of developmental signals regulates cell growth and division by controlling the synthesis, activation and degradation of G(1) cell cycle regulators . Here we describe the lin-23 gene of Caenorhabditis elegans, which is required to restrain cell proliferation in response to developmental cues . In lin-23 null mutants, all postembryonic blast cells undergo extra divisions, creating supernumerary cells that can differentiate and function normally . In contrast to the inability to regulate the extent of blast cell division in lin-23 mutants, the timing of initial cell cycle entry of blast cells is not affected . lin-23 encodes an F-box/WD-repeat protein that is orthologous to the Saccharomyces cerevisiae gene MET30, the Drosophila melanogaster gene slmb and the human gene betaTRCP, all of which function as components of SCF ubiquitin-ligase complexes . Loss of function of the Drosophila slmb gene causes the growth of ectopic appendages in a non-cell autonomous manner . In contrast, lin-23 functions cell autonomously to negatively regulate cell cycle progression, thereby allowing cell cycle exit in response to developmental signals.

EMBO J, 2000 Nov 1, 19(21), 5875 - 83
Tup1p represses Mcm1p transcriptional activation and chromatin remodeling of an a-cell-specific gene; Gavin IM et al.; In yeast, a number of regulatory proteins expressed only in specific cell types interact with general transcription factors in a combinatorial manner to control expression of cell-type-specific genes . We report a detailed analysis of activation and repression events that occur at the promoter of the a-cell-specific STE6 gene fused to a beta-galactosidase gene in a yeast minichromosome, as well as factors that control the chromatin structure of this promoter both in the minichromosome and in the genomic STE6 locus . Mcm1p results in chromatin remodeling and is responsible for all transcriptional activity from the STE6 promoter in both wild-type a and alpha cells . Matalpha2p cooperates with Tup1p to block both chromatin remodeling and Mcm1p-associated activation . While Matalpha2p represses only Mcm1p, the Tup1p-mediated repression involves both Mcm1p-dependent and -independent mechanisms . Swi/Snf and Gcn5p, required for full induction of the STE6 gene, do not contribute to chromatin remodeling . We suggest that Tup1p can contribute to repression by blocking transcriptional activators, in addition to interacting with transcription machinery and stabilizing chromatin.

EMBO J, 2000 Nov 1, 19(21), 5845 - 55
The zinc finger protein Gfi-1 can enhance STAT3 signaling by interacting with the STAT3 inhibitor PIAS3; Rodel B et al.; STAT factors act as signal transducers of cytokine receptors and transcriptionally activate specific target genes . The recently discovered protein PIAS3 binds directly to STAT3 and blocks transcriptional activation . Here, we present experimental evidence implementing the zinc finger protein Gfi-1 as a new regulatory factor in STAT3-mediated signal transduction . The interaction between the two proteins first became evident in a yeast two-hybrid screen but was also seen in coprecipitation experiments from eukaryotic cells . Moreover, we found that both Gfi-1 and PIAS3 colocalize in a characteristic nuclear dot structure . While PIAS3 exerts a profound inhibitory effect on STAT3-mediated transcription of target promoters, Gfi-1 can overcome the PIAS3 block and significantly enhances STAT3-mediated transcriptional activation . In primary T cells, Gfi-1 was able to amplify IL-6-dependent T-cell activation . As Gfi-1 is a known, dominant proto-oncogene, our findings bear particular importance for the recently described ability of STAT3 to transform cells malignantly and offer an explanation of the oncogenic potential of Gfi-1 in T lymphocytes.

EMBO J, 2000 Nov 1, 19(21), 5672 - 81
Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo; Wu J et al.; The phosphoprotein phosphatase 2A (PP2A) catalytic subunit contains a methyl ester on its C-terminus, which in mammalian cells is added by a specific carboxyl methyltransferase and removed by a specific carboxyl methylesterase . We have identified genes in yeast that show significant homology to human carboxyl methyltransferase and methylesterase . Extracts of wild-type yeast cells contain carboxyl methyltransferase activity, while extracts of strains deleted for one of the methyltransferase genes, PPM1, lack all activity . Mutation of PPM1 partially disrupts the PP2A holoenzyme in vivo and ppm1 mutations exhibit synthetic lethality with mutations in genes encoding the B or B' regulatory subunit . Inactivation of PPM1 or overexpression of PPE1, the yeast gene homologous to bovine methylesterase, yields phenotypes similar to those observed after inactivation of either regulatory subunit . These phenotypes can be reversed by overexpression of the B regulatory subunit . These results demonstrate that Ppm1 is the sole PP2A methyltransferase in yeast and that its activity is required for the integrity of the PP2A holoenzyme.

EMBO J, 2000 Nov 1, 19(21), 5599 - 610
tRNA aminoacylation by arginyl-tRNA synthetase: induced conformations during substrates binding; Delagoutte B et al.; The 2.2 A crystal structure of a ternary complex formed by yeast arginyl-tRNA synthetase and its cognate tRNA(Arg) in the presence of the L-arginine substrate highlights new atomic features used for specific substrate recognition . This first example of an active complex formed by a class Ia aminoacyl-tRNA synthetase and its natural cognate tRNA illustrates additional strategies used for specific tRNA selection . The enzyme specifically recognizes the D-loop and the anticodon of the tRNA, and the mutually induced fit produces a conformation of the anticodon loop never seen before . Moreover, the anticodon binding triggers conformational changes in the catalytic center of the protein . The comparison with the 2.9 A structure of a binary complex formed by yeast arginyl-tRNA synthetase and tRNA(Arg) reveals that L-arginine binding controls the correct positioning of the CCA end of the tRNA(Arg) . Important structural changes induced by substrate binding are observed in the enzyme . Several key residues of the active site play multiple roles in the catalytic pathway and thus highlight the structural dynamics of the aminoacylation reaction.

Cancer Res, 2000 Oct 15, 60(20), 5789 - 96
Loss of interferon-gamma inducibility of TAP1 and LMP2 in a renal cell carcinoma cell line; Dovhey SE et al.; The inadequate ability of cancer cells to present antigen on the cell surface via MHC class I molecules is one mechanism by which tumor cells evade antitumor-associated antigen immunity . In many cases, such as in renal cell carcinoma (RCC), the lack of MHC class I antigen presentation can be attributed to the down-regulation of genes needed for antigen processing, such as the transporters associated with antigen processing (TAP)1 and TAP2, and the proteasomal components low molecular weight proteins (LMP)2 and LMP7 . The TAP1 and LMP2 genes are transcribed from a shared bidirectional promoter containing an IFN response factor element that confers IFN-gamma inducibility . Here, we investigate the differential responsiveness to IFN-gamma of RCC cell lines, Caki-1 and Caki-2, which have been reported to have abnormally low expressions of TAP1 and LMP2 . We now demonstrate that the Caki-2 cell line is defective in the IFN-gamma signaling pathway . The effects of IFN-gamma on TAP1 and LMP2 expression revealed a loss of up-regulation in Caki-2 cells, but not in Caki-1 cells . In vivo DNA footprinting shows a specific loss of occupancy at the IFN response factor element site in Caki-2 cells, whereas Caki-1 cells show full promoter occupancy . Furthermore, in vitro DNA-binding studies indicated that Caki-2 cells do not have IFN-regulatory factor 1- or signal transducer and activator of transcription 1 (Stat1)-binding activity after IFN-gamma stimulation . Examination of Stat1, Jak1, and Jak2 proteins demonstrated that the proteins were expressed, however, not phosphorylated, upon IFN-gamma treatment in Caki-2 cells . Also, this cell line expressed both IFN-gamma receptor chains . IFN-gamma inducibility could not be rescued by introduction of normal Jak1 and/or Jak2 proteins . However, overexpression of Jak1 did increase TAP1 and LMP2 expression independent of IFN-gamma, indicating that the Stat1 and IFN-regulatory factor 1 proteins present in Caki-2 can be activated . These findings suggest that the loss of TAP1 and LMP2 induction is a defect in the earliest steps of the IFN-gamma signaling pathway resulting in the inability of Caki-2 cells to up-regulate the MHC class I antigen-processing pathway . Because immunotherapy may be one of the most promising approaches for treating RCC, understanding the mechanisms by which these tumors circumvent cytokine signaling, thereby evading antitumor-specific-antigen immunity, would greatly aid the efficacy of such therapy.

Bioelectrochemistry, 2000 Sep, 52(1), 1 - 7
Application of boundary element method to calculation of the complex permittivity of suspensions of cells in shape of Dinfinityh symmetry; Sekine K; A numerical method using the boundary element method was developed to calculate the complex permittivity of suspensions of particles in the shape of Dinfinityh symmetry covered with a shell phase . It was an extension of the analytical methods based on Maxwell-Wagner-Sillars' effects in suspensions of shelled ellipsoids . This method was applied to particles, which were relevant to budding yeast cells and erythrocytes, to examine the effects of the shape on frequency-dependence of the permittivity and conductivity of their suspensions . Results of the calculations showed that the permittivity and conductivity at high frequencies were insensitive to the change in the shape . The change in shape affected the permittivity and conductivity at low frequencies and their frequency-dependence in the intermediate frequency region . This behavior could not be imitated by the calculation using analytical methods with shelled spheroid models.

J Biol Chem, 2001 Jan 26, 276(4), 2411 - 6 Epub 2000 Oct 31.
Signal peptidase and oligosaccharyltransferase interact in a sequential and dependent manner within the endoplasmic reticulum; Chen X et al.; We demonstrate that the signal peptides of prepro-alpha-factor and preinvertase must be cleaved before Asn-X-Ser/Thr acceptor tripeptides located near the signal peptides of these precursors can be efficiently glycosylated within the endoplasmic reticulum of the yeast Saccharomyces cerevisiae . The data support a model whereby the interaction of a signal peptide with the membrane prevents an acceptor tripeptide juxtaposed to the signal peptide from accessing the oligosaccharyltransferase active site until the signal peptide is cleaved.

Proc Natl Acad Sci U S A, 2000 Nov 7, 97(23), 12631 - 6
Two tandem verprolin homology domains are necessary for a strong activation of Arp2/3 complex-induced actin polymerization and induction of microspike formation by N-WASP; Yamaguchi H et al.; All WASP family proteins share a common C terminus that consists of the verprolin homology domain (V), cofilin homology domain (C), and acidic region (A), through which they activate Arp2/3 complex-induced actin polymerization . In this study, we characterized the Arp2/3 complex-mediated actin polymerization activity of VCA fragments of all of the WASP family proteins: WASP, N-WASP, WAVE1, WAVE2, and WAVE3 . All of the VCA fragments stimulated the nucleating activity of Arp2/3 complex . Among them, N-WASP VCA, which possesses two tandem V motifs, had a more potent activity than other VCA proteins . The chimeric protein experiments revealed that the V motif was more important to the activation potency than the CA region; two V motifs were required for full activity of N-WASP . COS7 cells overexpressing N-WASP form microspikes in response to epidermal growth factor . However, when a chimeric protein in which the VCA region of N-WASP is replaced with WAVE1 VCA was overexpressed, microspike formation was suppressed . Interestingly, when the N-WASP VCA region was replaced with WAVE1 VCA, having two V motifs, this chimeric protein could induce microspike formation . These results indicate that strong activation of Arp2/3 complex by N-WASP is mainly caused by its two tandem V motifs, which are essential for actin microspike formation.

Nucleic Acids Res, 2000 Nov 1, 28(21), 4299 - 305
Archaeal RNA polymerase subunits F and P are bona fide homologs of eukaryotic RPB4 and RPB12; Werner F et al.; The archaeal and eukaryotic evolutionary domains diverged from each other approximately 2 billion years ago, but many of the core components of their transcriptional and translational machineries still display a readily recognizable degree of similarity in their primary structures . The F and P subunits present in archaeal RNA polymerases were only recently identified in a purified archaeal RNA polymerase preparation and, on the basis of localized sequence homologies, tentatively identified as archaeal versions of the eukaryotic RPB4 and RPB12 RNA polymerase subunits, respectively . We prepared recombinant versions of the F and P subunits from Methanococcus jannaschii and used them in in vitro and in vivo protein interaction assays to demonstrate that they interact with other archaeal subunits in a manner predicted from their eukaryotic counterparts . The overall structural conservation of the M . jannaschii F subunit, although not readily recognizable on the primary amino acid sequence level, is sufficiently high to allow the formation of an archaeal-human F-RPB7 hybrid complex.

Nucleic Acids Res, 2000 Nov 1, 28(21), 4189 - 96
Analysis of Groucho-histone interactions suggests mechanistic similarities between Groucho- and Tup1-mediated repression; Flores-Saaib RD et al.; The DROSOPHILA: Groucho (Gro) protein is the defining member of a family of metazoan corepressors that have roles in many aspects of development, including segmentation, dorsal/ventral pattern formation, Notch signaling, and Wnt/Wg signaling . Previous speculation has suggested that Gro may be orthologous to the yeast corepressor Tup1 . In support of this idea, a detailed alignment between the C-terminal WD-repeat domains of these two proteins shows that each Gro WD repeat is most similar to the Tup1 WD repeat occupying the corresponding position in that protein . Our analysis of Gro-histone interactions provides further support for a close evolutionary relationship between Gro and Tup1 . In particular, we show that, as with the N-terminal region of Tup1, the N-terminal region of Gro is necessary and sufficient for direct binding to histones . The highest affinity interaction is with histone H3 and binding is primarily observed with hypoacetylated histones . Using transient transfection assays, we show that a Gal4-Gro fusion protein containing the histone-binding domain is able to repress transcription . Deletions that weaken histone binding also weaken repression . These findings, along with our recent report that Gro interacts with the histone deacetylase Rpd3, suggest a mechanism for Gro-mediated repression.

J Cell Sci, 2000 Nov, 113 ( Pt 22), 4013 - 23
The Dlx3 protein harbors basic residues required for nuclear localization, transcriptional activity and binding to Msx1; Bryan JT et al.; The murine Dlx3 protein is a putative transcriptional activator that has been implicated during development and differentiation of epithelial tissue . Dlx3 contains a homeodomain and mutational analysis has revealed two regions, one N-terminal and one C-terminal to the homeodomain, that act as transcriptional activators in a yeast one-hybrid assay . In addition to transactivation, data are presented to demonstrate specific DNA binding and an association between Dlx3 and the Msx1 protein in vitro . Immunohistochemical analysis confirmed coexpression of Dlx3 and Msx1 proteins in the differentiated layers of murine epidermal tissues . Transcription factor function requires nuclear localization . In this study, the intracellular localization of the green fluorescent protein fused to Dlx3 was examined in keratinocytes induced to differentiate by calcium and is shown to localize to the nucleus . A bipartite nuclear localization signal (NLS) was identified by mutational analysis and shown to be sufficient for nuclear localization . This was demonstrated by insertion of the Dlx3 bipartite NLS sequence into a cytoplasmic fusion protein, GFP-keratin 14, which functionally redirected GFP-keratin 14 expression to the nucleus . Further analysis of Dlx3 NLS mutants revealed that the Dlx3 NLS sequences are required for specific DNA binding, transactivation potential and interactions with the Msx1 protein.

J Cell Sci, 2000 Nov, 113 ( Pt 22), 3889 - 96
Chk1 and Cds1: linchpins of the DNA damage and replication checkpoint pathways; Rhind N et al.; Recent work on the mechanisms of DNA damage and replication cell cycle checkpoints has revealed great similarity between the checkpoint pathways of organisms as diverse as yeasts, flies and humans . However, there are differences in the ways these organisms regulate their cell cycles . To connect the conserved checkpoint pathways with various cell cycle targets requires an adaptable link that can target different cell cycle components in different organisms . The Chk1 and Cds1 protein kinases, downstream effectors in the checkpoint pathways, seem to play just such roles . Perhaps more surprisingly, the two kinases not only have different targets in different organisms but also seem to respond to different signals in different organisms . So, whereas in fission yeast Chk1 is required for the DNA damage checkpoint and Cds1 is specifically involved in the replication checkpoint, their roles seem to be shuffled in metazoans.

Dig Liver Dis, 2000 Aug-Sep, 32(6), 510 - 7
The augmenter of liver regeneration induces mitochondrial gene expression in rat liver and enhances oxidative phosphorylation capacity of liver mitochondria; Polimeno L et al.; BACKGROUND: The mammalian augmenter of liver regeneration gene encodes a protein involved in the unique process of liver regeneration . The augmenter of liver regeneration respective protein stimulates hepatocyte proliferation in hepatectomized rats and inhibits cytotoxic activity of liver-derived Natural Killer cells from intact rats . Augmenter of liver regeneration protein shares homology with a Saccharomyces Cerevisiae protein essential for the viability, oxidative phosphorylation and cell-division cycle . AIMS: To demonstrate if augmenter of liver regeneration protein, like the homologous in the yeast, plays a role in the regulation of biogenesis of mitochondria . METHODS: Augmenter of liver regeneration protein was injected in intact rats and, in the hepatic tissue, the expression of two genes located in two different regions of the mitochondrial genome, mitochondrial ATPase 6/8, and ND1 subunit, and of a nuclear gene, mitochondrial Transcription Factor A, were considered . In addition, cytochrome content and oxidative phosphorylation capacity of liver-derived mitochondria were evaluated . RESULTS: The augmenter of liver regeneration protein administration induces an increase in the mitochondrial gene expression and enhances cytochrome content and oxidative phosphorylation capacity of liver-derived mitochondria . CONCLUSIONS: The present data demonstrate a comparable role in the regulation of mitochondria biogenesis in the eukaryotic cell like the yeast protein . This phenomenon could be part of the complex mechanism through which augmenter of liver regeneration regulates hepatocyte proliferation.

Cell, 2000 Oct 13, 103(2), 253 - 62
TOR, a central controller of cell growth; Schmelzle T et al.; Cell growth (increase in cell mass) and cell proliferation (increase in cell number) are distinct yet coupled processes that go hand-in-hand to give rise to an organ, organism, or tumor . Cyclin-dependent kinase(s) is the central regulator of cell proliferation . Is there an equivalent regulator for cell growth? Recent findings reveal that the target of rapamycin TOR controls an unusually abundant and diverse set of readouts all of which are important for cell growth, suggesting that this conserved kinase is such a central regulator.

Curr Genet, 2000 Oct, 38(3), 126 - 31
Sulfate assimilation in Aspergillus terreus: analysis of genes encoding ATP-sulfurylase and PAPS-reductase; Schierova M et al.; Two genes for the sulfate assimilation pathway in Aspergillus terreus were cloned . The genes sAT (coding for PAPS-reductase) and sCT (coding for ATP-sulfurylase) form a small gene cluster . Both genes are similar to their homologs in A . nidulans (sA and sC), Penicillium chrysogenum (aps) and Saccharomyces cerevisiae (MET3 and MET16) . In the coding sequence of the sCT gene, a typical non-functional APS-kinase-like domain is present . The sCT gene is expressed in A . nidulans, but its expression there is less sensitive to methionine level than in the original species . Two regions 5' upstream of sAT were found to be similar to those of sA.

FEBS Lett, 2000 Oct 27, 484(1), 48 - 54
Srb7p is essential for the activation of a subset of genes; Gromoller A et al.; The mediator complex in the RNA polymerase II holoenzyme is known to be involved in transcriptional activation . The role of the essential mediator component Srb7p has been difficult to investigate, since no conditional lethal allele has been available to date . While the expression of Srb7p under the control of a repressible promoter is not sufficient to reduce the level of Srb7p beneath the threshold for survival, we have been able to isolate a clone termed ts16 which confers a temperature sensitive phenotype . ts16 contains an insertion mutation that requires translational frameshifting for correct expression of Srb7p, leading to extremely low protein levels . Strains bearing the ts16 construct show mild defects in the transcription of constitutive genes like TDH1 but severely affect activated transcription, e.g . of the GAL1 gene . In contrast, CUP1, which is also independent of other holoenzyme components, is not affected by ts16.

Mol Med, 2000 Aug, 6(8), 623 - 44
Histone acetylation modifiers in the pathogenesis of malignant disease; Mahlknecht U et al.; Chromatin structure is gaining increasing attention as a potential target in the treatment of cancer . Relaxation of the chromatin fiber facilitates transcription and is regulated by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which modify the acetylation state of histone proteins and other promoter-bound transcription factors . While HATs, which are frequently part of multisubunit coactivator complexes, lead to the relaxation of chromatin structure and transcriptional activation, HDACs tend to associate with multisubunit core-pressor complexes, which result in chromatin condensation and transcriptional repression of specific target genes . HATs and HDACs are known to be involved both in the pathogenesis as well as in the suppression of cancer . Some of the genes encoding these enzymes have been shown to be rearranged in the context of chromosomal translocations in human acute leukemias and solid tumors, where fusions of regulatory and coding regions of a variety of transcription factor genes result in completely new gene products that may interfere with regulatory cascades controlling cell growth and differentiation . On the other hand, some histone acetylation-modifying enzymes have been located within chromosomal regions that are particularly prone to chromosomal breaks . In these cases gains and losses of chromosomal material may affect the availability of functionally active HATs and HDACs, which in turn disturbs the tightly controlled equilibrium of histone acetylation . We review herein the recent achievements, which further help to elucidate the biological role of histone acetylation modifying enzymes and their potential impact on our current understanding of the molecular changes involved in the development of solid tumors and leukemias.

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