In vitro splicing of SV40 late mRNA in isolated nuclei from CV-1 cells.
An in vitro splicing system utilizing isolated nuclei of SV40 infected cells has been developed. Nuclei were isolated from CV-1 cells at a late stage of SV40 infection after a pulse-labeling with 3H-uridine. In nuclei prepared under mild isotonic conditions, 19S viral coded RNA synthesized in vivo was converted in vitro into 16S mRNA. In contrast, the nuclei prepared with RSB, a hypotonic medium, showed a very low splicing activity only. Addition of a "nuclear extract" to these nuclei restored the activity almost to the original level. These results indicate that 1) 19S RNA is indeed a precursor to 16S mRNA 2) the splicing of 19S RNA into 16S RNA takes place in the nucleus, and 3) at least a part of the enzyme system required for splicing could be extracted from the nucleus. This in vitro system may be useful for the assay of the splicing enzyme(s).
D001483:Base Sequence; D002467:Cell Nucleus / Q000648:ultrastructure*; D007038:Hypotonic Solutions; D007552:Isotonic Solutions; D012333:RNA, Messenger / Q000235:genetics*; D011995:Recombination, Genetic; D013539:Simian virus 40 / Q000378:metabolism*
D007038:Hypotonic Solutions; D007552:Isotonic Solutions; D012333:RNA, Messenger