体内枯草芽孢杆菌核糖核酸酶活性分析。

Analysis of Bacillus subtilis Ribonuclease Activity In Vivo.

机构信息

Expression Génétique Microbienne, UMR8261, CNRS, Institut de Biologie Physico-Chimique, Université de Paris, Paris, France.

出版信息

Methods Mol Biol. 2021;2209:387-401. doi: 10.1007/978-1-0716-0935-4_24.

DOI:10.1007/978-1-0716-0935-4_24

PMID:33201482

Abstract

Ribonucleases remodel RNAs to render them functional or to send them on their way toward degradation. In our laboratory, we study these pathways in detail using a plethora of different techniques. These can range from the isolation of RNAs in various RNase mutants to determine their implication in maturation or decay pathways by Northern blot, to proving their direct roles in RNA cleavage reactions using purified enzymes and transcribed substrates in vitro. In this chapter, we provide in-depth protocols for the techniques we use daily in the laboratory to assay RNase activity in vivo, with detailed notes on how to get these methods to work optimally. This chapter complements Chapter 25 on assays of ribonuclease action in vitro.

摘要

核糖核酸酶重塑 RNA，使它们具有功能或使其降解。在我们的实验室中，我们使用大量不同的技术详细研究这些途径。这些技术可以从各种核糖核酸酶突变体中分离 RNA，以通过 Northern blot 确定它们在成熟或降解途径中的作用，到使用纯化的酶和体外转录的底物证明它们在 RNA 切割反应中的直接作用。在本章中，我们提供了深入的方案，介绍了我们在实验室中日常用于测定体内核糖核酸酶活性的技术，并详细说明了如何使这些方法达到最佳效果。本章补充了第 25 章关于体外核糖核酸酶作用测定的内容。