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Nucleic Acids Research, 1980, Vol. 8, No. 23 5551-5566
© 1980

MOLECULAR BIOLOGY

In vitro splicing of the ribosomal RNA precursor in isolated nucleoli from Tetrahymena

M. Carin, B.F. Jensen, K.D. Jentsch, J.C. Leer, O.F. Nielsen and O. Westergaard

Department of Molecular Biology and Plant Physiology, University of Aarhus DK-8000 Arhus C, Denmark

Received October 20, 1980. The macronuclear rRNA genes of Tetrahymena thermophila contain an 0.4 kb intervening sequence in the 26S rRNA coding region. The sequence is represented within the primary transcription product. We demonstrate in this paper that the enzyme activities necessary for the endonucleolytic cleavage as well as for the ligation of the transcript are associated with the isolated nucleoli. Both of these processes occur posttranscriptionally. The intervening sequence is excised as an unique molecule, which is stable in vitro. About 50% of the in vitro synthesized RNA is processed. Faithful in vitro transcription occurs in the presence of the divalent ions Mg2+, Mn2+ and Co2+ while processing takes place only in the presence of Mg2+. The absolute requirement for Mg2+ in the excision reaction enables us to synthesize labelled pre-rRNA in the presence of Mn2+ or Co2+. The synthesized RNA can be used as a substrate in studies of the processing enzymes in vitro.


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