Integrated sequences of the S1 and S2 episomal DNAs in Wf9(N) mitochondrial DNA (mtDNA) have been mapped through cosmid cloning (D. M. Lonsdale, R. D. Thompson and T. P. Hodge, Nucl. Acids Res. 9:3657, 1981). Since extensive deletion of these sequences is a characteristic of the T and C male-sterile cytoplasms, we surveyed 12 common North American normal cytoplasm lines to assess the frequency and conservation of these sequences in male-fertile cytoplasms. Lines examined were W64A, Mo17, A619, A632, F44, F6, B37, NC7, Wf9, W182BN, A188, and Black Mexican Sweet. MtDNAs were prepared from each line, restricted by BamHI, electrophoresed, and transferred to nitro-cellulose. Nick-translated S1 and S2, as well as cosmids 2c11 and 2c44, were used as probes. Cosmid 2c11 spans the S1 region, while cosmid 2c44 spans the S2 region; these cosmids were cloned from Wf9.

The mtDNA from each line displayed major homology to S1 and S2. Homology to S1 occurred in a BamHI fragment of 6.9 kb in 8 of 12 lines: Wf9, W64A, Mo17, NC7, A619, A632, F44, and B37, consistent with the Wf9 model structure. Homology in F6, W182BN, and A188 occurred in a BamHI fragment of 6.6 kb, while Black Mexican Sweet displayed major homology at 8.1 kb. Homology to S2 was conserved among all entries, with major homology at 4.4, 0.95, and 2.1 kb, consistent with the structure of the model Wf9 mtDNA.

Probing with cosmid 2c11 verified that mtDNA structure of the eight lines which carried homology to S1 at 6.9 kb was conserved through a 40 kb region spanning the S1 site. F6, W182BN, and A188 were altered in fragments adjacent to the S1 site, and by rearrangement of sequences ca. 16 kb from the S1 site. Black Mexican Sweet displayed singular rearrangements, unlike all other lines examined. Hybridization with cosmid 2c44 demonstrated that the S2 region was conserved among all entries.

As was demonstrated with Wf9, none of the normal cytoplasms carried complete integrated copies of the S1 DNA; the 1400 bp repeat, characteristic of isolated S1 and S2 DNAs, was absent in the integrated form of S1.

Associated with the rearrangements which characterize F6, W182BN, and A188, we detected an alteration in the size of a linear, free DNA, occurring at 2.3 kb in most lines. In these three lines, no 2.3 kb DNA was apparent, but a 2.1 kb DNA was detected, co-migrating with a similar DNA in T cytoplasm mtDNA.

Although this survey included only defined male-fertile cytoplasms, and not exotics nor teosintes, integrated copies of S1 and S2 sequences would appear to be a constituent of these normal cytoplasms. Whether or not integrated copies are an obligate component of a "normal" mtDNA cannot be determined by these data. MtDNA from S cytoplasm also carries extensive homology to the episomes, which clearly distinguishes S from C and T cytoplasms. The role(s) which sequences of these DNAs may play in differentiating normal cytoplasms from the S male-sterile cytoplasm is thus obscure. MtDNA structure of the S cytoplasm through the S1 and S2 regions is different from all normal cytoplasms examined to date, and our current efforts are directed toward constructing restriction maps of these regions in S cytoplasm. (Supported in part by NATO Research Grant 283.81 to DML and DRP).

J. W. McNay, D. R. Pring and D. M. Lonsdale

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