Title

Isolation of Euglena chloroplastic tRNA and purification of chloroplasticPhe

Abstract

The chapter discusses the isolation of Euglena chloroplastic tRNA and purification of chloroplastic tRNAPhe. Total mitochondrial and chloroplast tRNAs are isolated using zonal rotors, essentially free from contaminating cytoplasmic components for purposes of identification and characterization and use in hybridization studies. Individual species of organelle tRNA are purified for use in structural and hybridization studies. The various purification procedures, particularly those involving large-scale organelle isolations, are the subject of the chapter. The isolated chloroplasts are suspended in breaking buffer (10 mM Tris.HCl, pH 7.5, 0.1 M sodium chloride, 10 mM magnesium acetate, 10 mM 2-mercaptoethanol, and 3 mM sodium azide) plus 1% sodium dodecyl sulfate, and the total tRNA is isolated. After phenol and chloroform extractions, the deproteinized nucleic acids are precipitated with ethanol (2.5 volumes of ethanol) and resuspended in breaking buffer. RNA obtained from DEAE-cellulose are fractionated using benzoylated DEAE-cellulose (BD-cellulose) column chromatography to separate individual isoacceptors, tRNA is first deacylated in 0.5 M Tris HCl buffer, pH 8, for 1-2 hr at room temperature. Chloroplastic tRNAs can easily be identified in whole-cell preparations of Euglena tRNA assayed for amino acid acceptor after chromatography on BD cellulose columns. © 1979, Elsevier Inc. All rights reserved.

Publication Title

Methods in Enzymology

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