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Sensitivity of the mouse 5' ETS processing reaction to micrococcal nuclease or RNase H treatment of the mouse S-100 processing extract. (A) Micrococcal nuclease sensitivity. Samples of the mouse S-100 processing extract were treated as indicated in the table below the autoradiograph. At the start (t 0), samples received CaCI2 (Ca lanes 3 and 5) or CaCI2 and excess EGTA (Ca,E lane 4), or no addition (lane 2). After 4 min (i 4),micrococcal nuclease (MN) was added (lanes 4 and 5). After...

M

Add the following to the required number of microcentrifuge tubes on ice 2. Incubate the reactions at the desired temperature for the desired time (see Protocol 6). 3. Either store the reaction mixtures at -70 C or extract the RNA immediately (see Protocol 6, step 3). 6 It might be necessary to increase the amount of RNase inhibitor. If so, the amount of solution A should be decreased to maintain the 25 pl reaction volume. As discussed in Section 4, mRNA is translated more efficiently in...

B

Time course of the formation of polyadenylation complexes and polyadenylated RNA. Polyadenylation complexes were formed according to Protocol 2 on an RNA (246 nt) containing the adenovirus L3 polyadenylation site. (A) Complexes were analysed on a non-denaturing 4 polyacrylamide gel. For an explanation of the complexes formed, see the text. Precursor RNA is indicated by 'pre'. A control lane with RNA in the absence of nuclear extract is labelled '-NXT'. (B) RNA was purified from...

B D Hames

Department of Biochemistry and Molecular Biology University of Leeds, Leeds LS2 9JT, UK Affinity Chromatography Anaerobic Microbiology Animal Cell Culture (2nd edition) Animal Virus Pathogenesis Antibodies I and II Behavioural Neuroscience Biochemical Toxicology Biological Data Analysis Biological Membranes Biomechanics Materials Biomechanics Structures and Systems Biosensors Carbohydrate Analysis Cell-Cell Interactions The Cell Cycle Cell Growth and Division Cellular Calcium Cellular...

A

This labelling procedure is described in Protocol 7. Protocol 7. Labelling of mRNA by reduction with 3H NaBH4 Capped mRNA (-100 pg ml) isolated as in Volume I, Chapter 1, Protocol 4 20mM sodium acetate buffer, pH 5.0 0.3 M sodium acetate buffer, pH 5.0 0.5 M sodium phosphate buffer, pH 7.0 3H NaBH4 (New England Nuclear 8.8 Ci mmol, 10mCi 10 jl in 50 mM NaOH) Elution buffer, Sephadex G-100 column, and 3.0 M potassium acetate, pH 4.5, as in Protocol 1 and metaperiodate reagent as in Protocol 5 1....

Methods for performing in vitro mRNA decay reactions

Three variables, the source of messenger RNase activity, the combination of subcellular fractions, and possible modifications of the basic system, need to (a) Reaction mixtures must contain either crude post-nuclear supernatant (S10), polysomes or RSW as a source of messenger RNase activity. Deproteinized, exogenous mRNA can be included with any of these, as desired. (b) Various combinations of fractions, for example, polysomes or RSW plus S130, with or without exogenous mRNA substrate, can be...

Preparation of undegraded mRNA and polysomes

When using an endogenous substrate, it is important that the mRNA and polysomes prepared are undegraded in order to obtain meaningful data. Contamination of glassware or reagents with RNases, which seem to be ubiquitous, can ruin a preparation. The following precautions, coupled with a large dose of common sense, will help maintain RNA integrity (a) Choose the proper cell or tissue. Some cells contain more non-specific RNases than others, so even the most careful investigator might have...

Analysis of rRNA processing in vitro 31 Introduction

In order to study the events of rRNA processing, it has proved extremely advantageous to have a system in which the processing can be re-created in vitro, using fractionated processing components and synthetic rRNA substrates. Processing extracts have been relatively easy to obtain from mouse and Xenopus for the 5' ETS cleavage reaction, and the reactions that excise the 18S region have also been amenable to in vitro analysis. The other metazoan rRNA processing events, however, have thus far...