Abstract
Two different methods have been devised for the analysis and purification of spliceosomes formed in a yeast in vitro splicing system. The first method relies on the electrophoretic separation of ribonucleoprotein particles in composite acrylamide-agarose gels. A large fraction of added substrate is located in spliceosomes, the formation of which can be shown to be dependent on the presence of both a yeast 5′ splice junction and a TACTAAC box on the RNA substrate. The second method relies on oligo(dT)-cellulose chromatography of spliceosomes formed with a polyadenylated substrate. Purification of spliceosomes by either method indicates that at least three small nuclear RNAs, approximately 160, 185, and 215 nucleotides in length, are specifically associated with yeast spliceosomes.
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