RESUMO
We have detected the specific binding in Southern blot hybridization experiments of both complementary antiparallel and parallel 40 bp synthetic DNA probes, corresponding to a cloned Drosophila DNA fragment. The highly cooperative annealing and melting were observed in solution with these probes, which are complementary in the same direction and possess 17 GC pairs. The binding of ethidium bromide is indicative of formation of a perfect parallel DNA duplex. The specific binding was also detected in both genomic and in plaque hybridization experiments.
Assuntos
Southern Blotting/métodos , Sondas de Oligonucleotídeos , Animais , Sequência de Bases , Clonagem Molecular , DNA , Drosophila melanogaster/genética , Etídio , Dados de Sequência Molecular , Hibridização de Ácido NucleicoRESUMO
Ethidium bromide (EB) adsorption isotherms on 30S nuclear RNP particles isolated from liver nuclei has revealed 6% of double-stranded regions in pre-mRNA (dsRNA). It has been established by measurements of the EB fluorescence polarization that the bulk of dsRNA regions in RNP is rigidly attached to RNP. They are longer than 45 degree A. The increase of NaCl concentration from 0.1 up to 0.4 M causes a significant loosening of dsRNA-protein bonds. As a result the dsRNA segments become more flexible. Measurements of energy transfer from fluorescamine (covalently bound to the protein) to EB (adsorbed on dsRNA) have yielded information about dsRNA location. The fact that absorbtion of exciting light by fluorescamine causes pronounced increase of EB fluorescence is consistent with the idea that helical regions of RNA are located outside the RNP particles.
Assuntos
Nucleoproteínas/análise , RNA de Cadeia Dupla/análise , Ribonucleoproteínas/análise , Animais , Núcleo Celular/análise , Transferência de Energia , Etídio , Fluorescamina , Polarização de Fluorescência/métodos , Fígado/análise , Ratos , Espectrometria de Fluorescência/métodosRESUMO
Conformational transitions in several individual tRNAs (tRNAMetf, tRNAPhe from E. coli, tRNAVal1, tRNASer, tRNAPhe from yeast) have been studied under various environmental conditions. The binding isotherms studies for dyes-tRNA complexes exhibited similarities in conformational states of all tRNAs investigated at low ionic strength (0.01 M NaCl). By contrast, at high ionic strength (0.4 M NaCl or 2 X 10(-4) M Mg2+) a marked difference is found in structural features of tRNAMetf as compared with other tRNAs used. The tRNAMetf is the only tRNA species that does not reveal the strong type of complexes with ethidium bromide, acriflavine and acridine orange.
