Assuntos
Nicotiana/citologia , Nicotiana/genética , Plantas Tóxicas , RNA de Plantas/isolamento & purificação , Sonicação , Triticum/citologia , Triticum/genética , Northern Blotting , Técnicas de Cultura de Células , Técnicas de Cultura , Eletroforese em Gel de Ágar , Vetores Genéticos/genética , Vetores Genéticos/isolamento & purificação , Guanidinas/química , Reação em Cadeia da Polimerase , Frações Subcelulares/química , Tiocianatos/química , Nicotiana/crescimento & desenvolvimento , Triticum/crescimento & desenvolvimentoRESUMO
A series of in-frame deletion mutants was used to identify a domain within the 3a protein of cucumber mosaic virus (CMV) that is required for RNA-binding activity. Deletions in the 3a gene were generated by PCR and restriction digestion, and the resulting mutated 3a sequences were cloned either in pT7-7 or in pGEX-5X3 expression vectors. The mutated 3a proteins or fusions with glutathione S-transferase (GST) were expressed in E. coli, purified, and their nucleic acid-binding activities analysed by photochemical UV cross-linking assays using digoxigenin-UTP-labelled RNA probes. Comparative analyses of seven mutated 3a proteins obtained from inclusion bodies and eight GST fusion proteins revealed that there is an RNA-binding domain located between amino acids 174 and 233. This RNA-binding domain is able to bind single-stranded RNA out of the context of the complete 3a movement protein and is highly conserved within both subgroups of CMV.
Assuntos
Cucumovirus/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Glutationa Transferase , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas do Movimento Viral em Plantas , Reação em Cadeia da Polimerase , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Raios UltravioletaRESUMO
Pure samples and as-prepared mixtures of Rh9 and Rh10 carbonyl clusters with interstitial P atoms have been studied quantitatively by 31P MAS and 1H-31P CP/MAS NMR. Information on the 31P chemical shift tensor of the Rh9 and Rh10 clusters has been derived from spinning sideband simulations. The chemical shift anisotropy is slightly larger in the Rh10 clusters (340-400 ppm) than in the Rh9 clusters (230-300 ppm), while the asymmetry parameters are similar (eta = 0.1-0.4). The results contribute to the understanding of the relationship between the shielding anisotropy and the structure of the cluster cavity.