Several major mtDNA rearrangements characterize the genomic configurations that distinguish N cytoplasms from T cytoplasms. One rearrangement is the duplication of ca. 5 kb of a single copy region of N cytoplasm mtDNA which is 5' to the gene atp6 (Dewey et al., Cell 44:439, 1986; Wise et al., Proc. Natl. Acad. Sci., submitted). The duplicated region in T places the atp6 transcription start signals 5' to URF13-T and ORF25. To identify the presumed progenitor region in Wf9(N) mtDNA contiguous to the repeated region of Wf9M, a 1.7 kb BamHI clone, representing the left junction fragment of the repeat in T, was used as probe on SstII, SmaI, and XhoI digests of Wf9(N) and A188(N) mtDNAs, and mapped by the coordinate map of Lonsdale et al. (MNL 60:170). A 14 kb XhoI fragment left of the repeat in T was also used as a probe. Sequences of the two clones mapped to approximate coordinates 231-244. Thus the unique configuration of contiguous T sequences includes regions which are 240 kb apart on the Wf9(N) map. Interestingly, the 5 kb repeat and the adjacent sequences in T are very close to the recombinationally active 5.2 kb repeats of N cytoplasm; the 244 coordinate is within 2 kb of the N repeat, and the right edge of the 5 kb repeat of T is within 8 kb of the second copy of the N repeat. The 14 kb clone contained sequences which are represented in two regions of N cytoplasm. A188(N) and Wf9(N) mtDNAs were distinguishable with both the 1.7 and 14 kb clones.
Sequences adjacent to ORF25 vary within N cytoplasms, and we have found that A188(N) is more similar to T than is Wf9(N) in this region. Fifteen probes, covering ca. 11 kb from T cytoplasm, were hybridized to digests of Wf9(T), Wf9(T), A188M, and A188(N) mtDNAs, using at least three endonucleases. A188(N) is colinear with T from within ORF25 to more than 3.5 kb 3' to the gene. This region appears to be repeated in T cytoplasm. In contrast, at least 3.5 kb of sequences adjacent to ORF25 were not present in Wf9(N) mtDNA. At least part of this sequence is transcribed in T and A188(N). The colinear region of T and A188(N) diverged within a 1.16 kb HindIII-XhoI fragment at the right hand end of the 6.7 kb XhoI fragment. It is apparent that A188(N) and T cytoplasm mtDNAs share genomic configuration through this region, and that Wf9(N) is clearly divergent. The data do not imply that A188(N) can be considered as a progenitor to T, but it is evident that Wf9(N) does not carry sequences which are represented and transcribed in A188(N) and T. It is interesting to note that A188(N) and T also share the 2.1 kb minilinear DNA, while Wf9(N) has a larger 2.3 kb counterpart.
John C. Kennell, Torbert R. Rocheford, Roger P Wise and Daryl R. Pring
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