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Recombineering Homologous Recombination Constructs in Drosophila
Published on: July 13, 2013
La amplificación y la escisión de secuencias de ADN de polioma integrado requieren un origen funcional de la
Cell
|April 1, 1984
Resumen
El antígeno T grande del virus del polioma (T-Ag) promueve la escisión o amplificación del ADN viral iniciando la replicación en el origen integrado. Este proceso dependiente de la replicación facilita eventos de recombinación posteriores, destacando el papel crucial del T-Ag.
Área de la Ciencia:
- Biología Molecular Biología Molecular
- Virología Virología.
- Genética La genética.
Sus antecedentes:
- La integración del ADN del virus del polioma (Py) en las células huésped puede conducir a la extirpación o la amplificación.
- Estos procesos requieren homología del ADN viral y un antígeno T grande funcional (T-Ag).
- El papel preciso de la T-Ag (replicante frente a la promoción de la recombinación) en estos eventos sigue sin estar claro.
Objetivo del estudio:
- Aclarar el mecanismo por el cual el antígeno T grande del virus del polioma (T-Ag) media la escisión y la amplificación del ADN viral integrado.
- Para determinar si la función primaria de T-Ag en estos fenómenos es la replicación o la promoción de la recombinación.
Principales métodos:
- Se estudiaron líneas celulares de ratas transformadas con inserciones de virus de polioma ts-a que carecen de un origen de replicación.
- Evaluación de la escisión/amplificación a las temperaturas permitidas para el T-Ag. térmolable.
- Introdujo un plásmido recombinante con un origen de replicación viral y el gen de resistencia G418 en células defectuosas de origen.
- Se analizaron clones resistentes al G418 para su amplificación en condiciones permisivas de T-Ag.
Principales resultados:
- Las células con inserciones de virus de polioma defectuosas de origen no mostraron excisión ni amplificación detectables, incluso con T-Ag funcional.
- La introducción de un plásmido que contiene el origen de la replicación del virus del polioma permitió la amplificación dependiente de T-Ag de las secuencias integradas.
- T-Ag facilitó la amplificación en trans de las secuencias de ADN que albergan el origen del virus del polioma.
Conclusiones:
- El antígeno T grande del virus del polioma (T-Ag) promueve la escisión y la amplificación del ADN viral principalmente iniciando la replicación en el origen viral integrado.
- La iniciación de la replicación por T-Ag genera un sustrato propicio para eventos de recombinación posteriores.
- Esto sugiere un papel replicativo para el T-Ag en la inestabilidad del ADN viral dentro de las células transformadas.
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The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with the...
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