RESUMO
The cum1 and cum2 mutations of Arabidopsis thaliana inhibit cucumber mosaic virus (CMV) multiplication. In cum1 and cum2 protoplasts, CMV RNA and the coat protein accumulated to wild-type levels, but the accumulation of the 3a protein of CMV, which is necessary for cell-to-cell movement of the virus, was strongly reduced compared with that in wild-type protoplasts. In cum2 protoplasts, the accumulation of turnip crinkle virus (TCV)-related RNA and proteins was also reduced. Positional cloning demonstrated that CUM1 and CUM2 encode eukaryotic translation initiation factors 4E and 4G, respectively. Unlike most cellular mRNA, the CMV RNA lacks a poly(A) tail, whereas the TCV RNA lacks both a 5'-terminal cap and a poly(A) tail. In vivo translation analyses, using chimeric luciferase mRNA carrying the terminal structures and untranslated sequences of the CMV or TCV RNA, demonstrated that these viral untranslated sequences contain elements that regulate the expression of encoded proteins positively or negatively. The cum1 and cum2 mutations had different effects on the action of these elements, suggesting that the cum1 and cum2 mutations cause inefficient production of CMV 3a protein and that the cum2 mutation affects the production of TCV-encoded proteins.
Assuntos
Arabidopsis/virologia , Cucumovirus/patogenicidade , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Mutação , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Carmovirus/genética , Carmovirus/metabolismo , Mapeamento Cromossômico , Cucumovirus/genética , Cucumovirus/fisiologia , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação Eucariótico 4G/genética , Dados de Sequência Molecular , Proteínas do Movimento Viral em Plantas , Protoplastos/virologia , RNA Viral/biossíntese , RNA Viral/genética , Análise de Sequência de DNA , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Optical imaging of electrical activity using voltage-sensitive dyes has been envisaged for many years as a powerful method to investigate multineuronal representation of information processing in brain tissue. This article describes the advent of novel genetically targeted voltage-sensitive fluorescent proteins. This new class of membrane voltage sensors overcomes previous limitations related to the nonselective staining of membranes associated with conventional voltage-sensitive dyes. Here, we discuss the methodology, applications, and potential advantages of this novel technique.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas Luminescentes/fisiologia , Animais , Linhagem Celular , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Potenciais da Membrana/fisiologia , Óptica e Fotônica , Canais de Potássio/química , Canais de Potássio/fisiologiaRESUMO
The TOM1 gene of Arabidopsis thaliana encodes a putative multipass transmembrane protein which is necessary for the efficient multiplication of tobamoviruses. We have previously shown that mutations severely destructive to the TOM1 gene reduce tobamovirus multiplication to low levels but do not impair it completely. In this report, we subjected one of the tom1 mutants (tom1-1) to another round of mutagenesis and isolated a new mutant which did not permit a detectable level of tobamovirus multiplication. In addition to tom1-1, this mutant carried a mutation referred to as tom3-1. Positional cloning showed that TOM3 was one of two TOM1-like genes in Arabidopsis. Based on the similarity between the amino acid sequences of TOM1 and TOM3, together with the results of a Sos recruitment assay suggesting that both TOM1 and TOM3 bind tobamovirus-encoded replication proteins, we propose that TOM1 and TOM3 play parallel and essential roles in the replication of tobamoviruses.
