Gary L. Stiegler

Organization and Expression of the Chloroplast Genome of Euglena gracilis

We have been involved in a characterization of the chloroplast (ct) genes of the unicellular photosynthetic eucaryote Euglena gracitis. Previous work from several different laboratories on physical mapping and cloning of this DNA, and on the characterization of the ribosomal RNA (rRNA) operons has been recently reviewed.1 In the present article we describe our recent studies on the organization and expression of transfer RNA (tRNA) genes. Nucleotide sequence data is now available for 18 different species. We also describe a new Euglena chloroplast extract that catalyzes DNA-dependent, selective in vitro transcription of Euglena ct tRNA genes.

CHLOROPLAST DNA OF EUGLENA GRACILIS GENE MAPPING AND SELECTIVE IN VITRO TRANSCRIPTION OF THE RIBOSOMAL RNA REGION

ABSTRACT Purified chloroplast 5S, 16S, and 23S rRNAs and tRNAs were labeled in vitro and hybridized by the Southern method to ctDNA restriction fragments and cloned ctDNA fragments. Transfer RNA coding loci were found scattered throughout the genome, as well as in the rRNA coding region. 5S, 16S and 23S rRNAs and tRNA(s) all hybridized to the same strand of a 5.6 kbp DNA, repeated three times in tandem on the chloroplast genome. The RNA product transcribed in vitro by Euglena chloroplast RNA polymerase in a purified, transcriptionally active complex with ctDNA has been characterized as largely a selective transcript of the rRNA coding region. In preliminary experiments, sequence homology between Euglena chloroplast DNA and a fragment coding for the large subunit of RuDP carboxylase in C. reinhardii has been mapped.