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Nucleic Acids Research, 1980, Vol. 8, No. 3 587-599
© 1980

Articles

In vitro splicing of SV4O late mRNA in isolated nuclei from CV-1 cells

Hiroshi Hamada, Tersuya Igarashi and Masami Muramatsu

Department of Biochemistry, Cancer Institute, Japanese Foundation for Cancer Research Kami-Ikebukuro, Toshima-ku, Tokyo, 170, Japan

Received December 11, 1979. An in vitro splicing system utilizing isolated nuclei of SV4O infected cells has been developed.

Nuclei were isolated from CV-1 cells at a late stage of SV4O 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) l9S 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).


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