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Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay
Published on: February 22, 2018
Secuencias requeridas para la activación transcripcional in vitro de un gen hsp 70 de Drosophila
Cell
|September 1, 1985
Resumen
El factor de transcripción del gen de choque térmico (HSTF) que se une a Drosophila hsp 70 implica interacciones cooperativas en múltiples sitios aguas arriba. La activación máxima del gen requiere la unión en el sitio proximal de TATA, pero esto por sí solo es insuficiente.
Área de la Ciencia:
- Biología Molecular Biología Molecular
- Genética La genética.
- La bioquímica es la bioquímica.
Sus antecedentes:
- El gen Drosophila hsp 70 está regulado por el factor de transcripción específico del gen de choque térmico (HSTF).
- La unión de HSTF ocurre en múltiples sitios dentro de la región reguladora del gen.
Objetivo del estudio:
- Para investigar la afinidad de unión y las interacciones cooperativas de HSTF en el gen Drosophila hsp 70.
- Para determinar el papel de los sitios de unión HSTF individuales en la activación de la transcripción.
Principales métodos:
- Estudios de huellas utilizando mutaciones de deleción 5' y 3'.
- Prueba de gel de agarosa-acrilamida para determinar las afinidades de unión.
- Ensayos de transcripción in vitro con extractos nucleares y experimentos de reconstitución.
Principales resultados:
- Se identificaron dos sitios de unión HSTF contiguos con diferentes afinidades intrínsecas dentro del dominio 55 bp proximal a la homología TATA.
- HSTF exhibe una afinidad 12,5 veces mayor para el sitio proximal TATA en comparación con el sitio más distal.
- La unión al sitio distal parece cooperativa, dependiendo de la ocupación del sitio proximal.
Conclusiones:
- El sitio de unión proximal HSTF de TATA es crucial pero insuficiente para la activación transcripcional máxima del gen hsp 70.
- La unión cooperativa de HSTF a múltiples sitios es esencial para una regulación eficiente del gen de choque térmico en Drosophila.
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