Página 27 dos resultados de 20101 itens digitais encontrados em 0.011 segundos

‣ Stability of St. Louis encephalitis viral antigen detected by enzyme immunoassay in infected mosquitoes.

Tsai, T F; Happ, C M; Bolin, R A; Montoya, M; Campos, E; Francy, D B; Hawkes, R A; Roehrig, J T
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1988 Português
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The use of enzyme immunoassay to detect St. Louis encephalitis (SLE) viral antigen in vector mosquitoes enhances the effectiveness of surveillance because infected mosquitoes can be identified more rapidly than with conventional virus isolation systems and because it is a simple and accessible procedure. Infectivity among mosquitoes experimentally infected with SLE virus was lost within 24 h after the mosquitoes were stored at 27 degrees C and 80% relative humidity; however, viral antigen remained stable under these conditions and could be detected by enzyme immunoassay 2 weeks later. Desiccation further extended the period during which antigen could be detected to 6 weeks. Absorbances were higher in infected mosquitoes stored at 27 degrees C than in mosquitoes frozen continuously. Absorbances in infected mosquitoes also increased after repeated freezing and thawing and sonication. Both phenomena may be related to the release of antigen from decaying or disrupted cells. The relative stability of SLE viral antigen at ambient temperatures lends flexibility to schemes which use direct antigen detection to identify vectors. Surveillance systems can be designed without regard to collecting living mosquitoes, and a cold chain in unnecessary to preserve specimens...

‣ A synthetic analog of the 3-deoxy-D-manno-2-octulosonic acid disaccharide moiety of rough-type endotoxins does not bind to mouse peritoneal macrophages and human monocytes.

Girard, R; Pedron, T; Kosma, P; Chaby, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1993 Português
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Strong evidence supports the concept that lipid A is the main biologically active region of endotoxins and is recognized by specific binding sites of different cell types. However, receptors for carbohydrates are also present on mononuclear phagocytes, and it has been suggested that one of these lectin-like proteins may be specific for the 3-deoxy-D-manno-2-octolosonic acid (Kdo) residues of endotoxins. To reexamine this hypothesis, we prepared a 125I-labeled conjugate consisting of a synthetic Kdo-2,4-Kdo disaccharide covalently linked to bovine serum albumin (125I-Kdo2-BSA). The Kdo disaccharide residues of this radiolabeled conjugate were fully accessible to a monoclonal antibody which reacts specifically with this epitope. However, 125I-Kdo2-BSA did not exhibit any detectable specific binding on thioglycolate-elicited mouse peritoneal macrophages or on human monocytes. Furthermore, the specific binding of biotin-labeled lipopolysaccharide derivatives to mouse macrophages and human monocytes was not inhibited by a soluble synthetic Kdo-2,4-Kdo-polyacrylamide copolymer or by a synthetic glycolipid consisting of an alpha-Kdo residue glycosidically linked to O-6 of allyl-4-O-phosphoryl-N-3-hydroxytetradecanoyl-beta-D-glucosaminide. These results indicate that binding sites specific for Kdo are not present (or not accessible) on the surface of mouse macrophages and human monocytes.

‣ The role of DNA dependent protein kinase in synapsis of DNA ends

Weterings, Eric; Verkaik, Nicole S.; Brüggenwirth, Hennie T.; Hoeijmakers, Jan H. J.; van Gent, Dik C.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 15/12/2003 Português
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DNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PKCS) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks the action of exonucleases and ligases. The DNA termini become accessible after autophosphorylation of DNA-PKCS, which we demonstrate to require synapsis of DNA ends. Interestingly, the presence of DNA-PK prevents ligation of the two synapsed termini, but allows ligation to another DNA molecule. This alteration of the ligation route is independent of the type of ligase that we used, indicating that the intrinsic architecture of the DNA-PK complex itself is not able to support ligation of the synapsed DNA termini. We present a working model in which DNA-PK creates a stable molecular bridge between two DNA ends that is remodeled after DNA-PK autophosphorylation in such a way that the extreme termini become accessible without disrupting synapsis. We infer that joining of synapsed DNA termini would require an additional protein factor.

‣ Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol.

Mendez, A J; Anantharamaiah, G M; Segrest, J P; Oram, J F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1994 Português
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Clearance of excess cholesterol from cells by HDL is facilitated by the interaction of HDL apolipoproteins with cell-surface binding sites or receptors, a process that may be important in preventing atherosclerosis. In this study, synthetic peptides containing 18-mer amphipathic helices of the class found in HDL apolipoproteins (class A) were tested for their abilities to remove cholesterol and phospholipid from cultured sterol-laden fibroblasts and macrophages and to interact with cell-surface HDL binding sites. Lipid-free peptides containing two identical tandem repeats of class A amphipathic helices promoted cholesterol and phospholipid efflux from cells and depleted cellular cholesterol accessible for esterification by acyl CoA/cholesterol acyltransferase, similar to what was observed for purified apolipoprotein A-I. Peptide-mediated removal of plasma membrane cholesterol and depletion of acyl CoA/cholesterol acyltransferase-accessible cholesterol appeared to occur by separate mechanisms, as the latter process was less dependent on extracellular phospholipid. The dimeric amphipathic helical peptides also competed for high-affinity HDL binding sites on cholesterol-loaded fibroblasts and displayed saturable high-affinity binding to the cell surface. In contrast...

‣ Rapid and reversible changes in nucleosome structure accompany the activation, repression, and superinduction of murine fibroblast protooncogenes c-fos and c-myc.

Chen, T A; Allfrey, V G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1987 Português
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A procedure for the isolation of transcriptionally active nucleosomes was used to monitor changes in chromatin structure during the activation, repression, and superinduction of the protooncogenes c-fos and c-myc. Nuclei were isolated from murine fibroblasts at successive times after stimulation of quiescent cell cultures with serum or platelet-derived growth factor. The nucleosomes released by a brief micrococcal nuclease digestion were fractionated by HgII-affinity chromatography to separate the unfolded nucleosomes of transcriptionally active genes (in which the sulfhydryl groups of histone H3 are accessible for binding to HgII) from the compactly beaded nucleosomes of transcriptionally inert DNA sequences (in which the H3 sulfhydryl groups are not accessible). The DNA sequence contents of the HgII-bound and unbound nucleosome fractions were compared by slot-blot hybridizations to 32P-labeled cloned probes for c-fos and c-myc. The binding of the c-fos and c-myc nucleosomes to the HgII column accurately reflected both the timing and the degree of their expression, as determined by run-off transcription assays with the isolated nuclei. The superinduction of c-fos and c-myc expression by an inhibitor of protein synthesis (cycloheximide) was reflected in the persistence of the unfolded...

‣ The PEPR GeneChip data warehouse, and implementation of a dynamic time series query tool (SGQT) with graphical interface

Chen, Josephine; Zhao, Po; Massaro, Donald; Clerch, Linda B.; Almon, Richard R.; DuBois, Debra C.; Jusko, William J.; Hoffman, Eric P.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 01/01/2004 Português
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Publicly accessible DNA databases (genome browsers) are rapidly accelerating post-genomic research (see http://www.genome.ucsc.edu/), with integrated genomic DNA, gene structure, EST/ splicing and cross-species ortholog data. DNA databases have relatively low dimensionality; the genome is a linear code that anchors all associated data. In contrast, RNA expression and protein databases need to be able to handle very high dimensional data, with time, tissue, cell type and genes, as interrelated variables. The high dimensionality of microarray expression profile data, and the lack of a standard experimental platform have complicated the development of web-accessible databases and analytical tools. We have designed and implemented a public resource of expression profile data containing 1024 human, mouse and rat Affymetrix GeneChip expression profiles, generated in the same laboratory, and subject to the same quality and procedural controls (Public Expression Profiling Resource; PEPR). Our Oracle-based PEPR data warehouse includes a novel time series query analysis tool (SGQT), enabling dynamic generation of graphs and spreadsheets showing the action of any transcript of interest over time. In this report, we demonstrate the utility of this tool using a 27 time point...

‣ Long-Range Comparison of Human and Mouse SCL Loci: Localized Regions of Sensitivity to Restriction Endonucleases Correspond Precisely with Peaks of Conserved Noncoding Sequences

Göttgens, Berthold; Gilbert, James G.R.; Barton, Linda M.; Grafham, Darren; Rogers, Jane; Bentley, David R.; Green, Anthony R.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /01/2001 Português
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Long-range comparative sequence analysis provides a powerful strategy for identifying conserved regulatory elements. The stem cell leukemia (SCL) gene encodes a bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis, and it displays a highly conserved expression pattern. We present here a detailed sequence comparison of 193 kb of the human SCL locus to 234 kb of the mouse SCL locus. Four new genes have been identified together with an ancient mitochondrial insertion in the human locus. The SCL gene is flanked upstream by the SIL gene and downstream by the MAP17 gene in both species, but the gene order is not collinear downstream from MAP17. To facilitate rapid identification of candidate regulatory elements, we have developed a new sequence analysis tool (SynPlot) that automates the graphical display of large-scale sequence alignments. Unlike existing programs, SynPlot can display the locus features of more than one sequence, thereby indicating the position of homology peaks relative to the structure of all sequences in the alignment. In addition, high-resolution analysis of the chromatin structure of the mouse SCL gene permitted the accurate positioning of localized zones accessible to restriction endonucleases. Zones known to be associated with functional regulatory regions were found to correspond precisely with peaks of human/mouse homology...

‣ Creating the Gene Ontology Resource: Design and Implementation

Consortium, The Gene Ontology
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /08/2001 Português
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The exponential growth in the volume of accessible biological information has generated a confusion of voices surrounding the annotation of molecular information about genes and their products. The Gene Ontology (GO) project seeks to provide a set of structured vocabularies for specific biological domains that can be used to describe gene products in any organism. This work includes building three extensive ontologies to describe molecular function, biological process, and cellular component, and providing a community database resource that supports the use of these ontologies. The GO Consortium was initiated by scientists associated with three model organism databases: SGD, the Saccharomyces Genome database; FlyBase, the Drosophila genome database; and MGD/GXD, the Mouse Genome Informatics databases. Additional model organism database groups are joining the project. Each of these model organism information systems is annotating genes and gene products using GO vocabulary terms and incorporating these annotations into their respective model organism databases. Each database contributes its annotation files to a shared GO data resource accessible to the public at http://www.geneontology.org/. The GO site can be used by the community both to recover the GO vocabularies and to access the annotated gene product data sets from the model organism databases. The GO Consortium supports the development of the GO database resource and provides tools enabling curators and researchers to query and manipulate the vocabularies. We believe that the shared development of this molecular annotation resource will contribute to the unification of biological information.

‣ Conformational alterations of transcription termination protein rho induced by ATP and by RNA.

Engel, D; Richardson, J P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/10/1984 Português
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Transcription termination protein rho from Escherichia coli possesses an RNA-dependent ATP hydrolysis activity necessary for expression of its termination function. We have used the rate of trypsin-mediated inactivation of ATPase activity as a conformational probe to test for ligand binding-induced conformational changes in the rho polypeptide. When present in molar excess over rho polypeptide, trypsin inactivates rho ATPase by a first order process that correlates well with the loss of intact rho polypeptide. When rho protein binds poly(C) or poly(dC), its susceptible bonds become more accessible to trypsin action. On the other hand, when rho binds either ATP or ADP those bonds become less accessible. These results suggest that rho protein assumes an altered conformation when an RNA cofactor is bound and that is assumes a distinctly different conformation when a nucleotide substrate or product is bound. A special change in the accessibility of trypsin-susceptible bonds is also detected when rho in its complex with poly(C) is catalyzing the hydrolysis of ATP.

‣ Transcribed and non-transcribed regions of Tetrahymena ribosomal gene chromatin have different accessibilities to micrococcal nuclease.

Palen, T E; Cech, T R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/04/1983 Português
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DNA renaturation kinetics was used to examine the relative accessibility of various regions of the Tetrahymena ribosomal RNA gene (rDNA) chromatin to micrococcal nuclease. In nuclei from cells active in rRNA transcription, the transcribed region of the rDNA chromatin was as much as 5-fold more accessible than the average of the total chromatin. As few as 20% inactive genes in the population could have accounted for all of the hybridization, so the transcribed region of the active units may be totally unprotected from nuclease degradation. The terminal non-transcribed spacer downstream from the transcription unit was also preferentially digested, but to a smaller degree. The central non-transcribed spacer was degraded to the same extent as total chromatin after a high degree of nuclease digestion. In nuclei from starved cells, which have 96% reduced rRNA transcription, the transcribed and terminal spacer regions of the rDNA were again more accessible than the total chromatin from the same nuclei, but the difference did not exceed 2-fold. We conclude that transcriptional activation is accompanied by major changes in the structure of the ribosomal gene chromatin, and that the extent and/or type of structural alteration differs in each functionally defined region of the rDNA.

‣ Isolation and characterization of the DNA fraction of rat liver chromatin which binds polylysine.

Arnold, E A; Wahn, U; Young, K E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1975 Português
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The structure of eukaryotic chromatin has been investigated by isolating and analyzing the "accessible" DNA fraction of rat liver chromatin. This DNA fraction has been isolated by titrating the chromatin with the protese-resistant D isomer of polylysine to bind the "accessible" DNA sites. After removal of chromosomal proteins by digestion with pronase, all DNA not protected from attack by bound polylysine was removed by digestion with DNase. Even after exhaustive treatment with pronase and DNase approximately 30% of the chromatin DNA remains resistant to nuclease attack. Analysis of the isolated DNA shows it to be mainly double-stranded with an average size of 200-250 base pairs. The DNA is slightly A-T rich and contains both repetitive and "single-copy" nuleotide sequences. The results suggest that there are extensive regions in chromatin where the DNA is not tightly complexed with protein. Furthermore, the DNA of these regions is similar in gross properties to the DNA of the total genome.

‣ Quantitation of aflatoxin B1 adduction within the ribosomal RNA gene sequences of rat liver DNA.

Irvin, T R; Wogan, G N
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1984 Português
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The in vivo formation of covalent aflatoxin B1 (AFB1)-DNA adducts within the rRNA gene sequences of nuclear DNA has been studied in AFB1-treated rats. Liver nuclear DNA, enriched in ribosomal DNA (rDNA) by one round of cesium salt density gradient centrifugation, was treated under buffered alkaline conditions to convert unstable AFB1-N7-guanine adducts to stable AFB1-formamidopyrimidine derivatives. The alkali-treated DNA was hybridized to 18S and 28S rRNA in 70% formamide buffer to form rRNA X rDNA hybrids. These hybrids were separated from the bulk of nuclear DNA by two rounds of centrifugation in CsCl, and the level of AFB1 adduction to rDNA versus total nuclear DNA was compared as a function of dose 2 hr after AFB1 administration. Over an 8-fold dose range (0.25-2.0 mg of AFB1 per kg of body weight), rDNA contained 4- to 5-fold more AFB1 residues than nuclear DNA, indicating that rDNA is preferentially accessible to carcinogen modification in vivo. While aflatoxin B1 forms adducts with DNA principally at guanine residues, the guanine enrichment of rDNA was insufficient to explain the magnitude of observed preferential AFB1 modification of rDNA. These results support the hypothesis that rDNA regions are preferentially accessible to carcinogen modification because of the diffuse conformation maintained within transcribed genes. This experimental approach permits the quantitative description of carcinogen modification within a defined gene sequence; further refinement of this approach may be useful in defining the precise relationships between covalent chemical-DNA interactions and the alterations in gene expression that result.

‣ Photochemically induced dynamic nuclear polarization investigation of complex formation of the NH2-terminal DNA-binding domain of lac repressor with poly[d(AT)].

Buck, F; Rüterjans, H; Kaptein, R; Beyreuther, K
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1980 Português
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The interaction of the NH2-terminal DNA-binding domain of lac repressor with synthetic oligo[d(AT)] was studied by a photo-CIDNP technique (CIDNP is chemically induced dynamic nuclear polarization). Three of the four tyrosines of the NH2-terminal region were found to be accessible to the photosensitive dye. The corresponding ring proton resonances were enhanced in the photo-CIDNP 1H NMR spectrum, and the only histidine (histidine 29) was located at the surface of the domain, which is supposed to be linked to the core protein of lac repressor by a flexible hinge region. After complex formation of the NH2-terminal region with oligo[d(AT)], two of the three tyrosine residues were no longer accessible to solvent or to photosensitive dye, which is strong evidence that the two tyrosines are part of the contact region.

‣ Mitochondrial carbonic anhydrase.

Dodgson, S J; Forster, R E; Storey, B T; Mela, L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1980 Português
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We have assayed carbonic anhydrase activity (carbonate dehydratase, carbonate hydro-lyase, EC 4.2.1.1) and bicarbonate permeability in suspensions of broken and intact guinea pig mitochondria by monitoring the disappearance of C16O18O. We found significant activity in preparations from liver and skeletal muscle, but not in preparations from heart muscle, brain, and kidney. Intact mitochondria containing carbonic anhydrase produce a two-phase acceleration of the disappearance of the labeled CO2, which indicates that the enzyme is located in a region more accessible to CO2 than to HCO3-. Acetazolamide inhibits the enzyme activity instantly in broken mitochondria but only after a delay in intact mitochondria, indicating that the enzyme is in a region not immediately accessible to the inhibitor. Sonication of mitochondria containing carbonic anhydrase activity releases the enzyme, which remains in the supernatant after sedimentation of the submitochondrial particles. This shows that mitochondrial carbonic anhydrase is in the matrix compartment and not in, or bound to, the inner membrane. The activity of the enzyme increases markedly with increasing pH. The enzyme activity of intact mitochondria is greater than that of the broken mitochondria at the same pH of the suspending fluid...

‣ A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled α-Factor Receptor

Lin, Jennifer C.; Duell, Ken; Konopka, James B.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2004 Português
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The α-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible...

‣ Antibodies to cell envelope proteins of Pseudomonas aeruginosa in cystic fibrosis patients.

Fernandes, P B; Kim, C; Cundy, K R; Haung, N N
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1981 Português
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Many vaccines containing somatic and secreted antigens of Pseudomonas aeruginosa have been reported. The vaccines containing lipopolysaccharide have been found to provide type-specific protection, but the endotoxin content of these vaccines does not make it feasible to use them in patients who are already debilitated. Outer membrane proteins could be effective as vaccines, as they can be purified free of lipopolysaccharide, and also because they are common to all serotypes of P. aeruginosa. To be effective as a vaccine, such proteins must be immunogenic and accessible from the outside of the intact bacterial cell. In this study, we showed that systemic antibodies were produced frequently to two cell envelope proteins with masses of 58,500 and 37,500 daltons and occasionally to 34,000-dalton protein of P. aeruginosa in cystic fibrosis patients with chronic lung infections. In rabbits immunized with whole, fixed cells of P. aeruginosa, antibodies were also produced against the 58,500-dalton proteins. Thus, the 58,500-dalton cell envelope protein of P. aeruginosa was the only immunogenic protein that was accessible to the immune system when whole, fixed cells were used for immunization. These serum antibodies did not protect the cystic fibrosis patients against further lung infection with P. aeruginosa.

‣ Determination of Surface-Exposed, Functional Domains of Gonococcal Transferrin-Binding Protein A

Yost-Daljev, Mary Kate; Cornelissen, Cynthia Nau
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2004 Português
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The gonococcal transferrin receptor is composed of two distinct proteins, TbpA and TbpB. TbpA is a member of the TonB-dependent family of integral outer membrane transporters, while TbpB is lipid modified and thought to be peripherally surface exposed. We previously proposed a hypothetical topology model for gonococcal TbpA that was based upon computer predictions and similarity with other TonB-dependent transporters for which crystal structures have been determined. In the present study, the hemagglutinin epitope was inserted into TbpA to probe the surface topology of this protein and secondarily to test the functional impacts of site-specific mutagenesis. Twelve epitope insertion mutants were constructed, five of which allowed us to confirm the surface exposure of loops 2, 3, 5, 7, and 10. In contrast to the predictions set forth by the hypothetical model, insertion into the plug region resulted in an epitope that was surface accessible, while epitope insertions into two putative loops (9 and 11) were not surface accessible. Insertions into putative loop 3 and β strand 9 abolished transferrin binding and utilization, and the plug insertion mutant exhibited decreased transferrin-binding affinity concomitant with an inability to utilize it. Insertion into putative β strand 16 generated a mutant that was able to bind transferrin normally but that was unable to mediate utilization. Mutants with insertions into putative loops 2...

‣ Role of the histone amino termini in facilitated binding of a transcription factor, GAL4-AH, to nucleosome cores.

Vettese-Dadey, M; Walter, P; Chen, H; Juan, L J; Workman, J L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1994 Português
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Facilitated, "cooperative" binding of GAL4-AH to nucleosomal DNA occurred in response to inhibition from the core histone amino termini. The binding of GAL4-AH (which contains the DNA-binding and dimerization domains of GAL4) to nucleosome cores containing multiple binding sites initiated at the end of a nucleosome core and proceeded in a cooperative manner until all sites were occupied. However, following tryptic removal of the core histone amino termini, GAL4-AH binding appeared to be noncooperative, similar to binding naked DNA. Binding of GAL4-AH to nucleosomes bearing a single GAL4 site at different positions indicated that inhibition of GAL4 binding was largely mediated by the histone amino termini and primarily occurred at sites well within the core and not near the end. When the histone amino termini were intact, binding of GAL4-AH to sites near the center of a nucleosome core was greatly enhanced by the presence of additional GAL4 dimers bound to more-accessible positions. These data illustrate that the binding of a factor to more-accessible sites, near the end of a nucleosome, allows facilitated binding of additional factors to the center of the nucleosome, thereby overcoming repression from the core histone amino termini. This mechanism may contribute to the binding of multiple factors to complex promoter and enhancer elements in cellular chromatin.

‣ Methylation-related chromatin structure is associated with exclusion of transcription factors from and suppressed expression of the O-6-methylguanine DNA methyltransferase gene in human glioma cell lines.

Costello, J F; Futscher, B W; Kroes, R A; Pieper, R O
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1994 Português
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There is considerable interest in identifying factors responsible for expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, as MGMT is a major determinant in the response of glioma cells to the chemotherapeutic agent 1,3 bis(2-chloroethyl)-1-nitrosourea. Recently we have shown that MGMT expression is correlated in a direct, graded fashion with methylation in the body of the MGMT gene and in an inverse, graded fashion with promoter methylation in human glioma cell lines. To determine if promoter methylation is an important component of MGMT expression, this study addressed the complex interactions between methylation, chromatin structure, and in vivo transcription factor occupancy in the MGMT promoter of glioma cell lines with different levels of MGMT expression. Our results show that the basal promoter in MGMT-expressing glioma cell lines, which is 100% unmethylated, was very accessible to restriction enzymes at all sites tested, suggesting that this region may be nucleosome free. The basal promoter in glioma cells with minimal MGMT expression, however, which is 75% unmethylated, was much less accessible, and the basal promoter in nonexpressing cells, which is 50% unmethylated, was entirely inaccessible to restriction enzymes. Despite the presence of the relevant transcription factors in all cell lines examined...

‣ Two components of Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) are stereospecifically located upstream of a tRNA gene and interact with the second-largest subunit of TFIIIC.

Bartholomew, B; Kassavetis, G A; Geiduschek, E P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1991 Português
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A novel photocrosslinking method has been used to identify the components of transcription factor IIIB (TFIIIB) and TFIIIC that associate with DNA upstream of the Saccharomyces cerevisiae SUP4 tRNATyr gene and to map these components to specific positions in DNA. When TFIIIC binds to the tRNA gene, only its second-largest subunit (135 kDa) is accessible for reaction with a photoactive nucleotide, 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP, inserted into DNA upstream of the transcriptional start. Formation of TFIII(C + B)-tRNA gene complexes specifically brings two additional polypeptides (90 and 70 kDa) within reach of upstream photoprobes. A collection of 13 probes has been used to map the locations of these three proteins along a 45-bp segment of DNA upstream of the transcriptional start site. Evidence is presented that the 90- and 70-kDa polypeptides are separate and distinct components of yeast TFIIIB, that they are accessible to crosslinking on opposite sides of the DNA helix in a 6-bp segment centered 35 bp upstream of the tRNATyr gene transcriptional start, and that they interact with the second-largest subunit of TFIIIC.