Nucleic Acids Research, 1982, Vol. 10, No. 6 2145-2161
© 1982
MOLECULAR BIOLOGY |
Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3
Department of Biology, University of Rochester Rochester, NY 14627, USA
Received January 6, 1982. Revised February 16, 1982. Accepted February 16, 1982.
The fraction of ribosomes loaded on polysomes is about 95% in logarithmically growing Tetrahymena thermophila, and about 4% in starved cells. Cytoplasmic extracts from cells in these two physiological states were used to develop column chromatographic methods for the purification of polysomes. Bio-Gel A 1.5 m was found to separate total cytoplasmic ribosomes from many soluble proteins, including RNAse, with no detectable change in the polysome size distribution. Polysomes can be separated from monosomes and non-polysomal mRNA by chromatography on Bio-Gel A 15 m without size selection. These methods can easily be adapted to large scale preparations of polysomes, even from cells where a small fraction of the ribosomes is on polysomes. A method is described for reversible precipitation of polysomes and monosomes from dilute solutions at pH 5.3 which greatly facilitates polysome isolation. Hybridization of 3H-labeled polyU to RNA isolated from column fractions has been used to demonstrate that purification of EDTA released polysomal mRNA can be performed using the column chromatography procedures described here. These methods have been employed to demonstrate that most of the cytoplasmic mRNA in log-phase Tetrahymena is loaded onto polyribosomes while most of the mRNA in starved cells exists in a non-polysomal form.
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