Sequence determination of the E̲u̲g̲ḻe̲ṉa̲ g̲ṟa̲c̲i̲ḻi̲s̲ chloroplast and cytoplasmic aspartic acid tRNas as a further investigation into the evolutionary origins of chloroplasts and E̲u̲g̲ḻe̲ṉa̲ g̲ṟa̲c̲i̲ḻi̲s̲

The comparison of tRNA and rRNA sequences has previously been used to probe the genetic relatedness between chloroplasts and organisms of prokaryotic and eukaryotic character. The sequences of the Euglena gracilis cytoplasmic and chloroplastic tRNAs-phe have previously been determined. Comparison of the Euglena tRNA-phe sequences to the tRNA-phe sequences from other pro- and eukaryotes suggests that the Euglena chloroplast tRNAs are more prokaryotic than eukaryotic in nature, and that the Euglena cytoplasmic tRNAs are more closely related to mammalian tRNAs than to tRNAs from lower eukaryotes such as yeast. As a further investigation into the evolutionary relationships suggested by these observations, the sequences of the Euglena cytoplasmic and chloroplastic aspartic acid tRNAs were determined.

The Euglena aspartic acid tRNAs were sequenced by combining the benefits of several tRNA sequencing technologies. The sequence of 5’-³²p-labeled tRNA fragments, obtained by either RNase T1 or Pancreatic RNase digestion and 5'-terminal labeled by using T4 polynucleotide kinase, were determined by Snake Venom Phosphodiesterase and Nuclease Pl partial digestion and analysis by two-dimensional homochromatography. Sequence information was also obtained by in vitro 5’-³²P-labeling tRNA fragments obtained by thermal digestion of an intact tRNA molecule. The family of 5'-terminal ³²P-labeled fragments obtained in a thermal digest is composed of all possible tRNA fragments extending from some point in the interior of the tRNA molecule to its 3'-CCA end. The family of labeled tRNA fragments may be resolved into successive units, each, one nucleotide longer by polacrylamide gel electrophoresis. When each successive unit is recovered from the polyacrylamide gel, the identity of the 5'-terminal nucleotide is determined by digestion with RNase T2 and PEI-cellulose thin-layer chromatography in two different solvent systems. Selected 5’-³²P-labeled tRNA fragments were also purified and their sequences determined by Nuclease Pl partial digestion and analysis by two-dimensional homochromatography.

The sequence of the Euglena gracilis cytoplasmic aspartic acid tRNA is: pU-C-U-U-C-G-G-U-A-G-U-A-γ-A-G-D-Gm-G-D-C-A-G-U-A-U-G*-ψ-C-C-G-C-U-G-U-C-A-C*-G-C-G-G-A-A-G-A-C*-C*-C-G-G-G-T-ψ-C-A-m¹A-U-U-C-C-C-G-G-C-C-G-G-A-G-A-G-C-C-A. The sequence of the Euglena gracilis chloroplast aspartic acid tRNA is: pG-G-G-A-U-U-G-U-A-G-U-U-U-A-A-D-G-G-D-D-A-G-A-G-C-A-C-C-G-C-C-C-U-G-U-C-A-C-G-G-C-G-G-A-A-G-m⁷G-U-C*-G-C-G-G-G-T-ψ-C-G-A-G-U-C-C-C-G-U-C-A-G-U-C-C-C-G-C-C-A.

These results further support the observation that the Euglena chloroplast tRNAs are more prokaryotic than eukaryotic in nature, and that the tRNAs found in the Euglena cytoplasm more closely resemble mammalian tRNAs than the tRNAs from lower eukaryotes.