A fine restriction map of the linear mitochondrial DNA of Tetrahymena pyriformis: genome size, map locations of rRNA and tRNA genes, terminal inversion repeat, and restriction site polymorphism

Abstract

A fine restriction map of the linear mitochondrial DNA of Tetrahymena pyriformis strain ST is presented. 1. Based on agarose gel electrophoresis data together with limited nucleotide sequences available on some restriction fragments, we estimate the actual size of this genome to be about 55,000 base pairs. 2. Seven tRNA gene locations have been assigned, which are scattered along the genome length. Six of these locations encode the genes for tRNA(phe), tRNA(his), tRNA(trp), and tRNA(glu), and the duplicate tRNA(tyr) genes which are located at the inverted terminal repeat segments. The tRNA gene(s) encoded in one location has not been identified. We have not yet found the tRNA(leu) and tRNA(met) genes, which were previously shown to be encoded in the genome (Chiu et al. 1974; Suyama 1982). 3. We have mapped the 14S rRNA gene by sequencing the 170 bp segment of EcoRI fragment 8 and by aligning its sequence with E. coli 16S rRNA. From our recent complete sequence data the gene size was found to be about 1,650 bp, which is unexpectedly large for the 14S rRNA which has an estimated size of 1,300 bp. The 14S rRNA is probably a cleavage product of the larger primary transcript of which 200-300 bases of the 5' end are missing. 4. The duplicate copies of the 21S rRNA gene at the terminal duplication inversion segments were analyzed. ClaI fragment 7 (1,500 bp) corresponds in sequence from base position 850 to 2,390 of the 20S rRNA gene of Paramecium mitochondrial DNA (Seilhamer et al. 1984b). The 21S gene is approximately 2,500 bp long. The presence of some restriction site polymorphism is apparent in this segment. 5. Each of the 21S gene copies precedes the tRNA(tyr) gene, but the space flanking one tRNA(tyr) gene differs in size and restriction sites from the space flanking another tRNA(tyr) gene. Thus, this space corresponds to the segment of an imperfect match in the terminal duplication inversion of Goldbach et al. (1978a). 6. Saccharomyces cerevisiae mitochondrial probes including Cob, ATPase VI and IX, and cytochrome oxidase I gene sequences, 21S and 15S rRNAs, and mouse mitochondrial DNA showed no significant hybridization with any restriction fragments of Tetrahymena mitochondrial DNA. The results are in accordance with an extensive sequence divergence previously found in the Tetrahymena mitochondrial genome (Goldbach et al. 1977).