RESUMEN
A microsatellite-containing 359-bp restriction fragment, isolated from the rat Pigr gene (murine polymeric immunoglobulin receptor gene) 3'-untranslated region (3'-UTR) and inserted into 3'-UTR or 3' flanking positions in transcription units of supercoiled plasmids, attenuates luciferase reporter gene expression in orientation- and position-dependent ways following transient transfection of human 293 cells. The same fragment stimulates orientation-dependent gene expression in a 5' flanking position. Plasmid linearization abrogates both orientation- and position-dependent responses. Cell-free translation reveals that 5' and 3' flanking expression responses are proportional to increased and decreased luciferase mRNA levels, whereas 3'-UTR expression is associated with control mRNA levels. Hypersensitivity to nucleases S1 and P1, gel mobility differences between supercoiled plasmids carrying opposing microsatellite orientations, and anomalous melting profiles of this fragment are also observed. These results suggest that functional pleiotropy of this fragment depends on the DNA context of its purine-rich microsatellite strand and on DNA supercoiling. Intramolecular triplexes stabilized by supercoiling and secondary structures of purine repeat-rich mRNAs may also confer regulatory properties to similar genomic elements.
Asunto(s)
Regiones no Traducidas 3'/genética , ADN/química , ADN/genética , Regulación de la Expresión Génica , Repeticiones de Microsatélite/genética , Conformación de Ácido Nucleico , Receptores de Inmunoglobulina Polimérica/genética , Regiones no Traducidas 3'/metabolismo , Animales , Secuencia de Bases , Línea Celular , ADN Superhelicoidal/química , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , Elementos de Facilitación Genéticos/genética , Genes Reporteros/genética , Humanos , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , Especificidad de Órganos , Plásmidos/química , Plásmidos/genética , Plásmidos/metabolismo , Biosíntesis de Proteínas , Ratas , Reproducibilidad de los Resultados , Especificidad de la Especie , Termodinámica , Transcripción Genética/genéticaRESUMEN
A systematic study of agarose gel electrophoresis of double-stranded RNA in the kilobase range of sizes was performed. The dsRNA to dsDNA relative mobility was found to depend on gel concentration: in low density gels RNA moves slower and in high density gels - faster than DNA of the same molecular size. The electrophoretic differences were interpreted within the reptation theory to be mainly due to the molecular stiffness differences. The dsRNA persistence length was roughly estimated to be about twice as great as that of DNA.
Asunto(s)
Bacteriófagos/genética , ADN , ARN Bicatenario , Saccharomyces cerevisiae/genética , Fenómenos Químicos , Química Física , ADN Bacteriano , Electroforesis en Gel de Agar , Conformación de Ácido Nucleico , Polimorfismo de Longitud del Fragmento de Restricción , ARN de Hongos , ARN ViralRESUMEN
Double-stranded RNAs (M and L molecules) of two strains of the killer system Saccharomyces cerevisiae M437 (wild type) and ski-5 (superkiller mutant) were studied by means of electron microscopy and high resolution thermal melting. The M molecules of the ski-5 mutant were by 100 b.p. shorter than those of M437. L molecules were of the same length for both strains. Analysis of the differential melting curves of L molecules showed that L molecules differ significantly in their nucleotide sequences, whereas M molecules were practically identical. It was found that M molecules contained a long AU region: that of M molecules of M 437 was 170-180 b.p. long and contained almost no GC pairs, whereas the AU region of M molecules of the ski-5 mutant was three times shorter and contained GC pairs.
Asunto(s)
ARN Bicatenario/análisis , ARN de Hongos/análisis , Saccharomyces cerevisiae/genética , Microscopía Electrónica , Mutación , ARN Bicatenario/ultraestructura , ARN de Hongos/ultraestructura , Saccharomyces cerevisiae/ultraestructuraAsunto(s)
Neutrófilos/fisiología , Neumonía/etiología , Enfermedad Aguda , Enfermedad Crónica , HumanosRESUMEN
A significant level of correction of the mutation responsible for sickle cell anemia has been achieved in monkey COS-7 cells on a plasmid containing a beta-globin gene fragment. The plasmid was treated in vitro with a nucleic acid 'third strand' bearing a terminal photoreactive psoralen moiety that binds immediately adjacent to the mutant base pair. Following covalent attachment of the psoralen by monoadduct or diadduct formation to the mutant T-residue on the coding strand, the treated plasmid was transfected into the cells, which were then incubated for 48 h to allow the cellular DNA repair mechanisms to remove the photoadducts. Upon re-isolation and amplification of the transfected plasmid, sickle cell mutation correction, as determined by sequence analysis of both complementary strands, was established in a full 1%. This result encourages extension of the approach to correct the mutation directly on the chromosome.
Asunto(s)
Anemia de Células Falciformes/terapia , Furocumarinas/genética , Terapia Genética/métodos , Globinas/genética , Plásmidos/farmacología , Transfección/métodos , Anemia de Células Falciformes/sangre , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Aductos de ADN , Reparación del ADN , Ingeniería Genética , Humanos , Mutación PuntualRESUMEN
Eight base analogs were tested as third strand residues in otherwise homopyrimidine strands opposite each of the 'direct' (A.T and G.C) and 'inverted' (T.A and C.G) Watson-Crick base pairs, using UV melting profiles to assess triplex stability. The target duplexes contained 20 A.T base pairs and a central test base pair X.Y, while the third strand contained 20 T residues and a central Z test base. Z included 5-bromo-uracil, 5-propynyluracil, 5-propynylcytosine, 5-methyl-cytosine, 5-bromocytosine, hypoxanthine, 2-amino-purine and 2,6-diaminopurine. Some of the base analogs enhanced third strand binding to the target duplex with one or other 'inverted' central base pair relative to the binding afforded by any of the canonical bases. Other analogs did the same for the duplexes with the 'direct' target pairs. The increasing order of triplex stabilization by these base analogs is: opposite the 'inverted' base pairs, for T.A, A < C < 5-pC < 5-pU < T < 5-BrC < 5-meC < 5-BrU < 2-AP < 2,6-DAP < Hy < G, for C.G, 2-AP < A < Hy < G < 5-pC < 5-BrC < 5-meC < C < 2,6-DAP < T < 5-BrU < 5-pU; opposite the 'direct' base pairs, for A.T, 2-AP < A < 5-meC < C < G < Hy < 2,6-DAP < 5-pU < T = 5-BrU < 5-BrC < 5-pC, for G.C, G < 2,6-DAP < 2-AP < A < Hy < T < 5-BrU < 5-pU < 5-pC < 5-BrC < C < 5-meC.
Asunto(s)
Emparejamiento Base , ADN/química , Nucleótidos de Pirimidina/química , TemperaturaRESUMEN
3-Nitropyrrole (M) was introduced as a non-discriminating 'universal' base in nucleic acid duplexes by virtue of small size and a presumed tendency to stack but not hydrogen bond with canonical bases. However, the absence of thermally-induced hyperchromic changes by single-stranded deoxyoligomers in which M alternates with A or C residues shows that M does not stack strongly with A or C nearest neighbors. Yet, the insertion of a centrally located M opposite any canonical base in a duplex is sometimes even less destabilizing than that of some mismatches, and the variation in duplex stability is small. In triplexes, on the other hand, an M residue centrally located in the third strand reduces triplex stability drastically even when the X.Y target base pair is A.T or G. C in a homopurine. homopyrimidine segment. But, when the target duplex opposition is M-T and the third strand residue is T, the presence of M in the test triplet has little effect on triplex stability. Therefore, a lack of hydrogen bonding in an otherwise helix-compatible test triplet cannot be responsible for triplex destabilization when M is the third strand residue. Thus, M is non-discriminating and none-too-destabilizing in a duplex, but in a triplex it is extremely destabilizing when in the third strand.
Asunto(s)
ADN/química , Desoxirribonucleósidos , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Adenina , Composición de Base , Secuencia de Bases , Calorimetría , Citosina , Guanina , Desnaturalización de Ácido Nucleico , Pirroles , Relación Estructura-Actividad , Termodinámica , TiminaRESUMEN
A DNA third strand with a 3'-psoralen substituent was designed to form a triplex with the sequence downstream of the T.A mutant base pair of the human sickle cell beta-globin gene. Triplex-mediated psoralen modification of the mutant T residue was sought as an approach to gene repair. The 24-nucleotide purine-rich target sequence switches from one strand to the other and has four pyrimidine interruptions. Therefore, a third strand sequence favorable to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third strand binding, 5-methylcytosine and 5-propynyluracil were used in the third strand. Further, a six residue "hook" complementary to an overhang of a linear duplex target was added to the 5'-end of the third strand via a T(4) linker. In binding to the overhang by Watson-Crick pairing, the hook facilitates triplex formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3'-end of the third strand forms photoadducts to the targeted T with high efficiency. Such monoadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.