Chloroplast genomes are generally believed to encode all of the transfer RNAs (tRNAs) involved in chloroplast protein synthesis. Since chloroplast genomes are of small size, readily isolated in intact form, and easily cloned in fragments of desired size as recombinant DNAs, it is a feasible goal to locate and characterize every chloroplast tRNA gene, and to determine the anticodon and cognate amino acid for each. We have been involved in a characterization of the complete set of tRNA genes and flanking sequences in chloroplasts of Euglena gracilis. This project is focused on completing the DNA sequence analysis of tRNA coding loci of the Euglena chloroplast genome, determining how tRNA genes are organized into transcription units and transcribed into precursor tRNAs, and on understanding mechanisms involved in regulation of tRNA gene expression during chloroplast biogenesis. DNA sequence of tRNA coding loci will be carried out with either the chemical cleavage or primed, dideoxy method. Characterization of primary tRNA transcription and processing reactions will be based on studies with homologous in vitro transcription systems from both Euglena gracilis and Spinacia oleracea choloroplasts. DNA template modification experiments will be used to determine the role of DNA sequences in regulation, initiation and termination of transcription. Finally, synthetic deoxyoligonucleotides will be used as hybridization probes to study the synthesis and maturation of tRNAs in vivo during chloroplast development.
