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Iniciación de la recombinación V(D) J en un sistema libre de células

D C van Gent¹, J F McBlane, D A Ramsden

¹Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

|June 16, 1995

Resumen

Los extractos nucleares de las células pre-B dividen el ADN en sitios específicos durante la recombinación V(D) J. Las proteínas RAG1 y RAG2 recombinantes son componentes clave de este proceso de corte de ADN, esencial para el desarrollo del sistema inmunológico.

Área de la Ciencia:

Inmunología Inmunología.
Biología Molecular Biología Molecular
Genética La genética.

Sus antecedentes:

La recombinación de J es un proceso crucial para la inmunidad adaptativa, generando diversos genes de receptores de anticuerpos y células T.
Este proceso implica la escisión específica del ADN en las secuencias de señales de recombinación (RSS).

Objetivo del estudio:

Para investigar los componentes moleculares responsables de la escisión del ADN durante la recombinación V(D) J en las células pre-B.
Para caracterizar la actividad bioquímica de la V(D) J recombinasa in vitro.

Principales métodos:

Utilizados extractos nucleares de líneas celulares pre-B.
Realizó ensayos bioquímicos para detectar la actividad de escisión del ADN.
Extractos nucleares fraccionados y probados para la presencia de proteína del gen de activación de la recombinación (RAG).
Extractos inactivos complementados de células deficientes de RAG1 con proteína RAG1 recombinante.

Principales resultados:

Los extractos nucleares de las células pre-B exhibieron una escisión específica del ADN en RSS.
Los productos de escisión fueron idénticos a los observados en los timocitos: extremos de señal contundentes, 5'-fosforilados y extremos de codificación de horquilla.
La proteína RAG1 recombinante mejoró significativamente la actividad de escisión y la actividad rescatada en extractos con deficiencia de RAG1.
La actividad de la hendidura se correlacionó con la presencia de la proteína RAG2 durante la fraccionamiento del extracto.

Conclusiones:

Las proteínas RAG1 y RAG2 son componentes esenciales del complejo de la recombinasa V(D) J.
Estas proteínas median la escisión específica del ADN requerida para la recombinación V(D) J.
Los hallazgos confirman el papel de RAG1 y RAG2 en el inicio de la recombinación V(D) J en el desarrollo de los linfocitos.

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