RESUMEN
To overcome the limitations of molecular assemblies, the development of novel supramolecular building blocks and self-assembly modes is essential to create more sophisticated, complex, and controllable aggregates. The self-assembly of peptide-DNA conjugates (PDCs), in which two orthogonal self-assembly modes, that is, ß-sheet formation and Watson-Crick base pairing, are covalently combined in one supramolecular system, is reported. Despite extensive research, most self-assembly studies have focused on using only one type of building block, which restricts structural and functional diversity compared to multicomponent systems. Multicomponent systems, however, suffer from poor control of self-assembly behaviors. Covalently conjugated PDC building blocks are shown to assemble into well-defined and controllable nanostructures. This controllability likely results from the decrease in entropy associated with the restriction of the molecular degrees of freedom by the covalent constraints. Using this strategy, the possibility to thermodynamically program nano-assemblies to exert gene regulation activity with low cytotoxicity is demonstrated.
Asunto(s)
ADN/química , Desoxirribonucleoproteínas/química , Nanoestructuras/química , Péptidos/química , ADN/genética , ADN sin Sentido/química , ADN sin Sentido/genética , Desoxirribonucleoproteínas/genética , Células HeLa , Humanos , Nanoestructuras/ultraestructura , Nanotecnología , Conformación Proteica en Lámina beta , TermodinámicaRESUMEN
An increasingly important problem in genome sequencing is the failure of the commonly used shotgun assembly programs to correctly assemble repetitive sequences. The assembly of non-repetitive regions or regions containing repeats considerably shorter than the average read length is in practice easy to solve, while longer repeats have been a difficult problem. We here present a statistical method to separate arbitrarily long, almost identical repeats, which makes it possible to correctly assemble complex repetitive sequence regions. The differences between repeat units may be as low as 1% and the sequencing error may be up to ten times higher. The method is based on the realization that a comparison of only a part of all overlapping sequences at a time in a data set does not generate enough information for a conclusive analysis. Our method uses optimal multi-alignments consisting of all the overlaps of each read. This makes it possible to determine defined nucleotide positions, DNPs, which constitute the differences between the repeat units. Differences between repeats are distinguished from sequencing errors using statistical methods, where the probabilities of obtaining certain combinations of candidate DNPs are calculated using the information from the multi-alignments. The use of DNPs and combinations of DNPs will allow for optimal and rapid assemblies of repeated regions. This method can solve repeats that differ in only two positions in a read length, which is the theoretical limit for repeat separation. We predict that this method will be highly useful in shotgun sequencing in the future.
Asunto(s)
Biología Computacional/métodos , Simulación por Computador , Modelos Estadísticos , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/estadística & datos numéricos , Algoritmos , Secuencia de Bases , Análisis por Conglomerados , Desoxirribonucleoproteínas/genética , Estudios de Factibilidad , Modelos Genéticos , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos/genética , Sensibilidad y Especificidad , Alineación de Secuencia/métodos , Alineación de Secuencia/estadística & datos numéricosRESUMEN
To study the DNA sequences contacting the nuclear lamina (NL) in vivo, Ehrlich ascites tumor cells were UV-irradiated. The NL was purified, and the DNA fragments covalently linked to the lamina proteins in vivo were cloned and sequenced. Although heterogeneous in length and composition, the sequences displayed homology to the introns and/or flanking regions of different genes, suggesting that functionally distinct regions are organized in a topologically defined manner at the nuclear periphery.
Asunto(s)
ADN de Neoplasias/genética , Desoxirribonucleoproteínas/genética , Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Homología de Secuencia de Ácido Nucleico , Animales , Carcinoma de Ehrlich/genética , Cromatina/metabolismo , Clonación Molecular , Reactivos de Enlaces Cruzados , ADN de Neoplasias/efectos de la radiación , Intrones , Laminas , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/efectos de la radiación , Proteínas Nucleares/efectos de la radiación , Secuencias Repetitivas de Ácidos Nucleicos , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
Using cytofluorimetry with acridine orange staining and a modified thermal denaturation technique of cellular DNP, it has been shown that chromatin melting profiles of normal human nuclei (from lymphocytes and granulocytes) have distinct regularities. It is believed that these regularities reflect specific supramolecular chromatin organization. Parallel comparative analysis performed using electrophoretic fractionation and isoelectric focussing of nuclear proteins has revealed that: 1) peculiarities of chromatin melting profiles are independent of the quantity and molecular weights of chromatin proteins; 2) the lack of principal differences in chromatin melting profiles and the data on isoelectric points of nuclear proteins of granulocytes and lymphocytes from the same patient indicate that specific supramolecular organization of DNP-complex depends on the chromatin protein charge.
Asunto(s)
Desoxirribonucleoproteínas/genética , Adulto , Composición de Base , Cromatina/análisis , Cromatina/genética , Desoxirribonucleoproteínas/análisis , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Granulocitos/metabolismo , Humanos , Focalización Isoeléctrica , Linfocitos/metabolismo , Desnaturalización ProteicaRESUMEN
We report that two types of stable protein-DNA complexes, or transpososomes, are generated in vitro during the Mu DNA strand transfer reaction. The Type 1 complex is an intermediate in the reaction. Its formation requires a supercoiled mini-Mu donor plasmid, Mu A and HU protein, and Mg2+. In the Type 1 complex the two ends of Mu are held together, creating a figure eight-shaped molecule with two independent topological domains; the Mu sequences remain supercoiled while the vector DNA is relaxed because of nicking. In the presence of Mu B protein, ATP, target DNA, and Mg2+, the Type 1 complex is converted into the protein-associated product of the strand transfer reaction. In this Type 2 complex, the target DNA has been joined to the Mu DNA ends held in the synaptic complex at the center of the figure eight. Supercoils are not required for the latter reaction.
Asunto(s)
Bacteriófago mu/genética , Elementos Transponibles de ADN , ADN Viral/genética , Proteínas de Unión al ADN/genética , Desoxirribonucleoproteínas/genética , ADN Superhelicoidal/genética , Sustancias Macromoleculares , Microscopía ElectrónicaRESUMEN
Conditions that favor the complete coating of single-stranded DNA by RecA protein promote the association of these presynaptic filaments with naked double-stranded DNA to form large nucleoprotein networks before homologous pairing occurs. These RecA nucleoprotein networks sequester virtually all of the DNA in the reaction mixture. Conditions that are suboptimal for the formation of the RecA presynaptic filament rendered both the formation of RecA-DNA networks and the subsequent formation of joint molecules sensitive to inhibition by excess ATP or by pyrophosphate when these were added during synapsis. The rate of homologous pairing was directly related to the degree of inhibition of network formation. Various multivalent cations added during synapsis restored both the formation of networks and the pairing of homologous molecules. These observations support the view that the nucleoprotein network is a synaptic intermediate by means of which RecA protein facilitates the conjunction of DNA molecules and the subsequent processive search for homology. Inhibition by multivalent anions and restoration by multivalent cations suggests in addition, that negative charge repulsion inhibits the binding of naked duplex DNA to presynaptic filaments.
Asunto(s)
Desoxirribonucleoproteínas/genética , Rec A Recombinasas/genética , Recombinación Genética , Adenosina Trifosfato/farmacología , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/metabolismo , Difosfatos/farmacología , Magnesio/farmacología , Cloruro de MagnesioRESUMEN
Middle-mode RNA synthesis in T4-infected cells takes place before replication of phage DNA commences. What distinguishes it from early-mode RNA synthesis is that initiation of middle RNA depends on T4-coded proteins, in particular on the mot gene product. mot protein is localized in a DNA-protein complex which forms during the first few minutes of infection. All of the cell's mot protein is bound in this complex, and it continues to be bound long after the synthesis of mot protein has stopped. When we infect Escherichia coli with T4 carrying a temperature-sensitive mutation in the mot gene, we find a correlation between the physiology of this mot mutant and the amount of mot protein bound in the DNA-protein complex. Although there is some host RNA polymerase in the complex, mot protein does not seem to bind to this enzyme. Two other T4-coded proteins, of molecular weights 17,600 and 15,000, are also found in the pre-replicative DNA-protein complex. One of these, p17,600, is coded for by a 750-base pair region located between genes 39 and 56; p17,600 appears to be the recently described motB gene product. The other protein, p15,000, is not an RNA polymerase-binding protein; it is characterized by its strong binding to the DNA-protein complex.
Asunto(s)
Replicación del ADN , Desoxirribonucleoproteínas/genética , Escherichia coli/genética , Fagos T/genética , Proteínas Virales/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Desoxirribonucleoproteínas/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Cinética , Peso Molecular , Concentración OsmolarRESUMEN
Transcription in whole HeLa cell extracts of the nucleoprotein core complexes released from adenovirus type 2 or type 5 virions has been examined. The average length of transcripts from deproteinized DNA templates increased steadily during a 90-min reaction in vitro, exhibiting an elongation rate of approximately 70 nucleotides per minute. On the other hand, transcripts made from viral core templates were restricted to a length of less than 2000 nucleotides. Accordingly, efficient transcription of cores (50 nucleotides elongated/min) ceased after 10-20 min of incubation in whole-cell extracts. Deproteinized viral DNA and viral nucleoprotein complexes appeared to support the initiation of a similar number of transcripts per template molecule, but the rate of initiation was faster when cores were provided as templates. Deproteinized viral DNA supported the synthesis of VA-RNA and of transcripts that hybridized to the region of the viral genome containing the 5' portion of the major late transcriptional. Viral cores also directed the synthesis of RNA products which hybridized to fragments of the viral genome containing E1A, E1B, and E4 regions. The results of nuclease protection experiments indicated that the presence of core proteins did not preclude accurate initiation of transcription from the E4 region.
Asunto(s)
Adenovirus Humanos/genética , Transcripción Genética , Sistema Libre de Células , ADN Viral/genética , Desoxirribonucleoproteínas/genética , Femenino , Regulación de la Expresión Génica , Células HeLa , Humanos , Peso MolecularRESUMEN
Two temperature-sensitive mutants of herpes simplex virus type 1 in complementation group 1-1 were analyzed to determine if the major DNA-binding protein they produced was thermolabile. Cells infected with these mutants were analyzed for deoxyribonucleoprotein complexes containing the DNA-binding protein. These complexes were found in cells infected at the permissive temperature but not at the nonpermissive temperature. In temperature shift-up experiments with mutant virus infected cells, the levels of the deoxyribonucleoprotein complexes decreased with time of incubation at the nonpermissive temperature. Viral DNA synthesis terminated in cells infected with these mutants after temperature shift-up. The kinetics of termination of viral DNA synthesis were similar to the kinetics of dissociation of the deoxyribonucleoprotein complexes. These results indicate that two mutants in complementation group 1-1 produce a thermolabile DNA-binding protein and that this protein is required for viral DNA synthesis. Furthermore, they suggest that the major DNA-binding protein of herpes simplex virus type 1 functions in viral DNA synthesis as a component of deoxyribonucleoprotein complexes.
Asunto(s)
Replicación del ADN , Desoxirribonucleoproteínas/genética , Simplexvirus/genética , Animales , Línea Celular , Núcleo Celular/metabolismo , Transformación Celular Viral , Chlorocebus aethiops , ADN Viral/aislamiento & purificación , Prueba de Complementación Genética , Riñón , Mutación , TemperaturaRESUMEN
The 5' ends of Cp-1 DNA, which have a covalently linked terminal protein, can be partially unblocked by treatment with 1 M NaOH (E. Garcia, A. Gomez, C. Ronda, C. Escarmis, and R. Lopez (1983) Virology 128, 92-104) and labeled with polynucleotide kinase and [gamma-32P]ATP. The sequence of the first 444 and 520 nucleotides at the termini of Cp-1 DNA has been determined. A 236-nucleotide-long inverted terminal repeat was found and, in addition, the 116 nucleotides following the repeat show 93% homology. The first 352 nucleotides at both ends have an adenine plus thymine content of 75%. More than 50% of the nucleotides of the sequenced regions are involved in repeats of a minimum of 8 nucleotides. Three promoter-like sequences were also found at each end of Cp-1 DNA.
Asunto(s)
Bacteriófagos/genética , ADN Viral/genética , Secuencia de Bases , Desoxirribonucleoproteínas/genética , Oligodesoxirribonucleótidos/aislamiento & purificación , Secuencias Repetitivas de Ácidos Nucleicos , Streptococcus pneumoniaeAsunto(s)
Adenoviridae/genética , Bacteriófagos/genética , Replicación del ADN , Proteínas Virales/genética , Replicación Viral , Bacillus subtilis/genética , Secuencia de Bases , Clonación Molecular , Desoxirribonucleoproteínas/genética , Genes , Genes Virales , Mutación , Especificidad por Sustrato , Proteínas Virales/aislamiento & purificaciónRESUMEN
Under restrictive conditions, the 220 S SV40 virions are not assembled in tsB201-infected cells. Instead, a new class of SV40 DNA-containing particles is isolated in addition to the 75 S chromatin. This new class of nucleoprotein complex sediments heterogeneously between 100 to 160 S with a peak at 130 S. Under an electron microscope, these complexes appear predominantly as SV40 chromatin associated with a shell-like protein cluster. These structures resemble the wild type assembly intermediates previously observed by Coca-Prados and Hsu (Coca-Prados, N., and Hsu, M.-T. (1979) J. Virol. 31, 199-208). Like the wild type assembly intermediates, the tsB201 DNA-protein complexes are unstable in high salt. In CsCl, they yield a protein species with a density characteristic of empty shells. In 1 M NaCl, they release heterogeneous 55-110 S protein polymers which consist of the capsid proteins VP1, VP2, and VP3. Our results indicate that the tsB201 nucleoproteins consist of capsid proteins, with varying extents of polymerization, held to chromatin by electrostatic bonds. The accumulation of these nucleoproteins is consistent with a simian virus 40 morphogenetic pathway wherein the capsid proteins are added gradually to the 75 S chromatin.
Asunto(s)
ADN Viral/genética , Desoxirribonucleoproteínas/genética , Nucleoproteínas/genética , Virus 40 de los Simios/genética , Proteínas Virales/genética , Transformación Celular Neoplásica , Replicación del ADN , ADN Viral/aislamiento & purificación , Histonas/genética , Histonas/aislamiento & purificación , Morfogénesis , Proteínas Virales/aislamiento & purificación , Virión/genética , Replicación ViralRESUMEN
Interaction of SV40 DNA with three different HeLa cell extracts capable of directing correct initiation of transcription leads to the formation of ordered nucleoprotein complexes that are structurally similar to SV40 minichromosomes and eukaryotic chromatin. These nucleoprotein complexes can be conveniently purified by band sedimentation or gel filtration. Their sedimentation and elution properties resemble those of SV40 minichromosomes. Electron microscopy of purified complexes shows beaded structures that are sensitive to proteases, resulting in recovery of naked, largely undegraded DNA. Contour lengths and compaction ratios of these nucleoprotein complexes are similar to those of authentic SV40 minichromosomes. Their digestion patterns with micrococcal nuclease and pancreatic DNase I resemble those of SV40 minichromosomes. Such nucleosome-like structures can also be obtained with linear SV40 DNA. Unlike nucleosomes, no histones could be detected in the purified nucleoprotein complexes. Non-histone chromosomal protein fractions (high mol. wt. and free of high mobility group proteins) prepared from the HeLa cell extracts can also generate similar ordered structures. We conclude that ordered nucleoprotein structures with certain common characteristics can be formed by interaction of DNA with non-histone chromosomal proteins as well as with histones. Only the former structures are generated in currently used cell-free transcription systems. It appears that only those purified nucleoprotein complexes containing the promoter can be actively transcribed in the presence of additional cell-free extract, suggesting that such structures and their protein components may be important in transcription.
Asunto(s)
Transformación Celular Viral , ADN Viral/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Desoxirribonucleoproteínas/genética , Nucleoproteínas/genética , ARN Polimerasa II/metabolismo , Virus 40 de los Simios/genética , Transcripción Genética , Animales , Línea Celular , Chlorocebus aethiops , Desoxirribonucleasa I , Endodesoxirribonucleasas , Células HeLa/enzimología , Humanos , Riñón , Nucleasa Microcócica , Páncreas/enzimología , Virus 40 de los Simios/enzimologíaAsunto(s)
ADN Viral/genética , Genes Virales , Virus 40 de los Simios/genética , Animales , Células Cultivadas , Chlorocebus aethiops , Cromatina/ultraestructura , Enzimas de Restricción del ADN , Elementos Transponibles de ADN , Desoxirribonucleoproteínas/genética , Mutación , Nucleosomas/ultraestructuraRESUMEN
The major product of in vitro translation of early RNA prepared from H5ts125-infected cells and selected by hybridization to adenoviral DNA fragments spanning the region from 14.7 to 31.5 map units had been shown to be identical to the 87-kilodalton terminal protein precursor. A 72- to 75-kilodalton polypeptide whose rRNA can be selected by DNA from this same region and made in the presence of anisomycin was indistinguishable from the 72-kilodalton single-stranded DNA-binding protein encoded by the region from 60.1 to 66.6 map units. The accumulation of cytoplasmic RNA sequences complementary to these l-strand genes under various conditions of infection and in certain lines of transformed cells has been investigated by solution hybridization of cytoplasmic RNA to the separated strands of restriction endonuclease fragments of adenoviral DNA. During the early phase, RNA sequences complementary to the region from 11.6 to 36.7 map units were present at a concentration of 10 to 60 copies per cell, regardless of the nature of the block used to inhibit viral DNA synthesis. By 24 h after infection in the absence of any such block, sequences complementary to the regions from 11.6 to 18.2 map units (IVa2) and from 18.6 to 36.7 map units (E2B) accumulated to concentrations of 4,800 and 280 copies per cell, respectively. The ratio of cytoplasmic E2A RNA sequences to E2B RNA sequences remained close to 10:1 throughout the time period investigated. Of the transformed cell lines which retained E2B DNA sequences that were examined, only the T2C4 line expressed these sequences in cytoplasmic RNA. The implications of these observations for regulation of expression of the adenoviral early l-strand genes are discussed.
Asunto(s)
Adenovirus Humanos/genética , Transformación Celular Viral , Desoxirribonucleoproteínas/genética , Genes Virales , Nucleoproteínas/genética , Precursores de Proteínas/genética , Proteínas Virales/genética , Humanos , Hibridación de Ácido Nucleico , ARN Mensajero/genética , ARN Viral/genéticaAsunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , ARN Viral/genética , Fagos T/genética , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Desoxirribonucleoproteínas/genética , Prueba de Complementación Genética , Cinética , Mutación , Rifampin/farmacología , TemperaturaRESUMEN
We disrupted bacteriophage Mu particles by freeze-thaw treatment and recovered the DNA by CsCl density gradient centrifugation. This CsCl-purified DNA had a buoyant density which was indistinguishable from that of phenol-extracted Mu DNA. It was, however, 10(3) times more infective than phenol-extracted DNA for spheroplasts of exoV endI Escherichia coli. Infectivity was destroyed by proteinase K as well as by pancreatic DNase, indicating that the infective form was a DNA-protein complex. The infective properties of the complex demonstrated that the protein protects. Mu DNA against degradation by exonuclease V and that it serves at least one other function in bacteriophage Mu infection. The infectivity of the CsCl-purified DNA was due to a small class of highly infective molecules which sedimented 1.2. times faster than phenol-extracted Mu DNA on neutral sucrose gradients. This change in sedimentation rate is best explained by the formation of protein-linked circular monomers or linear dimers of Mu DNA. In vitro labeling of the DNA-protein complex, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that the CsCl-purified DNA contained a noncovalently associated 65,000-dalton polypeptide. A 65,000-dalton protein was also found to be a minor component of the bacteriophage Mu particle. No protein was found in phenol-extracted Mu DNA. These results suggest that the 65,000-dalton protein is necessary for successful phage infection and is normally injected into the host cell with the Mu genome.
Asunto(s)
Bacteriófago mu/genética , ADN Viral/genética , Desoxirribonucleoproteínas/genética , Escherichia coli/genética , Nucleoproteínas/genética , Transfección , Proteínas Virales/genética , Desoxirribonucleoproteínas/aislamiento & purificación , Genes Virales , Esferoplastos/metabolismo , Proteínas Virales/aislamiento & purificaciónRESUMEN
Minute virus of mice (MVM) nucleoprotein complexes were leached from infected cell nuclei in the presence of a hypotonic buffer. Detailed biochemical analyses performed on the extracted complexes revealed nucleoprotein complexes sedimenting together with virions at 110S and defective particles sedimenting at 50S. In contrast to the virions, the nucleoprotein complexes were found to be sensitive to treatment with DNase, Sarkosyl, and heparin. They were found to be composed of replicative forms of MVM DNA and cellular histones. After extensive micrococcal nuclease digestion performed on purified nucleoprotein complexes, a viral nucleosomes core containing a DNA segment of about 140 base pairs in length was identified. These complexes when visualized by electron microscopy revealed the existence of beaded structures (minichromosomes) having 26 and 52 beads per monomer and dimer molecules, respectively. We suggest that the organization of the intracellular viral DNA in a minichromosome structure is an essential step in the virus growth cycle.
Asunto(s)
Cromosomas/ultraestructura , ADN Viral/genética , Virus Diminuto del Ratón/genética , Parvoviridae/genética , Desoxirribonucleoproteínas/genética , Histonas/metabolismo , Microscopía Electrónica , Virus Diminuto del Ratón/ultraestructura , Conformación de Ácido Nucleico , Nucleosomas/análisis , Conformación ProteicaRESUMEN
We have previously shown that the 5'-terminal deoxycytidine residue of each nascent adenovirus 5 DNA strand synthesized in vitro is covalently linked to the 80-kilodalton (kd) terminal protein precursor via a phosphodiester bond to a serine residue in the protein. When extracts prepared from adenovirus 5-infected cells are incubated with [alpha-33P]dCTP as the only added deoxynucleoside triphosphate, complexes consisting of nucleotide covalently linked to the 80-kd protein can be detected. The nucleotide moieties present in such complexes include d(pC) and d(pCpA), the 5'-terminal nucleotide and dinucleotide of adenovirus 5 DNA, respectively, as well as some longer oligonucleotides. The formation of these complexes requires the presence of adenovirus DNA containing the attached 55-kd terminal protein and ATP. Extracts from H5ts125-infected cells which are defective in DNA replication catalyze complex formation to the same extent as extracts prepared from wild-type infected cells; thus, the presence of the adenovirus-coded 72-kd DNA-binding protein is apparently not required. Most, if not all, of the 80-kd protein-nucleotide complexes that are formed are noncovalently bound to the input viral DNA. These observations are consistent with the protein-priming model for the initiation of adenovirus DNA replication.