Abstract
Extrachromosomal nucleoli were isolated from log phase cells of Tetrahymena pyriformis (amicronucleate strain) in a highly purified state. Nucleoli located at the periphery of the macronucleus were detached from the nucleoplasmic mass of isolated macronuclei with agitation and separated from macronuclei by filtration through a Nuclepore membrane filter (pore size 5 microm). The filtrate constitutes the crude nucleolar preparation, as judged by electron microscopy and DNA analysis. Further purification of the nucleoli was performed by isopycnic centrifugation of the filtrate in a Metrizamide density gradient. After this step, the purity of the nucleoli, as defined by rDNA content and measured by analytical CsCl centrifugation, was almost 100%. Electron microscopy of the purified nucleoli revealed structures that resemble those of an in situ nucleoli. Undegraded 35S pre-rRNA, together with 26S and 17S rRNA, could be isolated from purified nucleoli. In vitro RNA synthetic activity was associated with isolated nucleoli. This activity is insensitive to low and high concentrations of alpha-amanitin, indicating that the form I RNA polymerase is functioning.
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