RESUMEN
The delivery of DNA to target cells using simple, defined, nonviral systems has become an area of intense interest in gene therapy. We describe here the development and characterization of one such novel system. A recombinant, bifunctional, fusion protein was expressed and purified from Escherichia coli. This protein consists of the DNA-binding domain of the yeast transcription factor GAL4 fused to the cell binding, internalization domain of the Yersinia pseudotuberculosis inv gene product, invasin. This protein, GAL4/Inv, together with poly-L-lysine, formed complexes with a chloramphenicol acetyltransferase (CAT) reporter plasmid that contains eight repeats of the GAL4 consensus recognition sequence. These complexes were shown to transfect target cells in an invasin receptor-dependent manner, resulting in transient CAT expression. A simple, targeted DNA delivery vehicle, as we describe here, represents a viable approach to nonviral gene delivery.
Asunto(s)
Adhesinas Bacterianas , Proteínas Bacterianas/genética , Técnicas de Transferencia de Gen , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/genética , Animales , Anticuerpos/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Proteínas de Unión al ADN , Células HeLa , Humanos , Ratones , Staphylococcus aureus/fisiología , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Plants form their gametes late in somatic development and, as a result, often pass somatic mutations on to their progeny. Classic examples of this process are the germinal revertants of unstable, Ac/Ds transposon-induced kernel mutations in maize: frequent and early reversion events during somatic development are generally correlated with a high frequency of revertant gametes. We have characterized a Ds allele of the maize waxy (wx) gene, wx-m5:CS7, for which the correlation between somatic and germinal reversion frequencies no longer holds. The ability of wx-m5:CS7 (CS7) to produce revertant gametes is suppressed approximately 100-fold in comparison with a second Ds allele, wx-m5:CS8 (CS8), which has an identical insertion at Wx and the same frequent and early somatic reversion pattern in endosperm. The excision of Ds from wx is not reduced 100-fold in the somatic tissues of CS7 plants as compared with CS8 plants. Suppressed formation of CS7 revertant gametes is independent of the Ac transposase source and is heritably passed to the embryos of progeny kernels; however, frequent and early somatic reversion is observed again in endosperms of these progeny kernels. This suppression appears to be caused by a dominant mutation in a trans-acting product that can suppress the germinal reversion of other Ds-induced alleles as well; the mutation is tightly linked to Wx but is not in the CS7 Ds itself. Taken together, the data suggest a novel mode of developmental control of Ac/Ds elements by the host plant, suppressing element excision in the shoot meristem.
Asunto(s)
Proteínas de Plantas/genética , Almidón Sintasa/genética , Zea mays/genética , Alelos , Clonación Molecular , Elementos Transponibles de ADN , ADN de Plantas/análisis , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Genes de Plantas , Células Germinativas/crecimiento & desarrollo , Mutagénesis Insercional , Reacción en Cadena de la Polimerasa , Eliminación de Secuencia , Transposasas/metabolismo , Zea mays/crecimiento & desarrolloRESUMEN
Mobile elements transposing via DNA intermediates often leave small rearrangements, or "transposon footprints," at sites where they excise. Each excision event leaves its own footprint and, at any given site, these vary in size and sequence. Footprint formation involves DNA repair of sequences flanking the element. We have analyzed the footprints formed by a 2-kb Ds element excising from six different sites in exons of the maize waxy (Wx) gene. We find that groups of footprints left at individual sites are surprisingly nonrandom; different excision products predominate consistently at each site. Less frequent footprints left by each insertion appear related to the predominant type. The data suggest that flanking sequences affect the DNA repair processes associated with element excision. Two models have been proposed to explain footprint formation, one featuring a 5' exonuclease and the other featuring hairpin loop formation and an endonuclease. Our data have interesting implications for both these models. Evidence is also presented to support the presence of a separate excision mechanism that can remove Ac/Ds elements without leaving any footprint and that operates in parallel with the footprint-forming mechanism.