The site of initiation of chloroplast rRNA synthesis was determined by S1-mapping and by sequencing primary rRNA transcripts specifically labeled at their 5'-end. Transcription initiates at a single site 53 nucleotides upstream of the 5'-end of the mature 16S rRNA under all growth conditions examined. The initiation site is within a DNA sequence that is highly homologous to and probably derived from a tRNA gene-region located elsewhere in the chloroplast genome. A nearly identical sequence (102 of 103 nucleotides) is present near the replication origin. The near identity of the two sequences suggests a common mode for control of transcription of the rRNA genes and initiation of chloroplast DNA replication. The related sequence in the tRNA gene-region does not appear to serve as a transcript initiation site.

Fragments of Euglena chloroplast DNA generated by endonuclease R-Eco RI were separated by agarose-gel electrophoresis into 24 distinct bands. At least five fragments contain sequences complementary to chloroplast ribosomal RNA, Most of the Eco RI fragments have been cloned in a plasmid of Escherichia coli. Three of the cloned fragments were shown to contain chloroplast ribosomal RNA sequences by DNA-RNA hybridization.