Euglena gracilis chloroplast transfer RNA transcription units. I. Physical map of the transfer RNA gene loci

Abstract

The locations of transfer RNA genes with respect to the restriction endonuclease cleavage map of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA have been determined. Purified chloroplast tRNAs were treated with snake venom phosphodiesterase to remove the 3'-CCA terminus, and radioactively labeled by the action of Escherichia coli tRNA nucleotidyltransferase in the presence of [alpha-32P]CTP. Chloroplast DNA was treated individually and with combinations of the enzymes Bal I, Bam HI, Eco RI, Pst I, Pvu II, Sal I, and Xho I. The location of tRNA genes with respect to the cleavage sites for these enzymes was determined by hybridization of the 32P-labeled tRNAs to membrane filter blots of the chloroplast DNA restriction nuclease fragments following gel electrophoresis. The 145-kilobase pair genome was resolved into nine areas of strong tRNA hybridization, separated by areas of weak or no tRNA hybridization. The loci of tRNA genes are within the Eco RI fragments Eco A, B, G, H, I, J', P, Q, and V.