Two plasmid-like DNAs, S-1 and S-2 (formerly designated S-S and S-F respectively) of molecular weights 4 x 106 and 3.5 x 106, respectively are present in the mitochondria of members of the S group of cms in maize (PNAS 74:2904, 1977). Unlike cms-T and cms-C, the cms-S exhibits instability in the expression of sterility. An episomal mechanism was hypothesized to explain the high reversion rates of sterile to fertile plants (J. R. Laughnan and S. J. Gabay, 1975, Genetics and Biogenesis of Mitochondria and Chloroplasts, C. W. Birky, Jr., P. S. Perlman and T. J. Byers, ed.). Subsequently, the concomitant disappearance of S-1 and S-2 from mitochondria of spontaneous fertile revertants (Vg-revertants) was observed (MNL 53:83, 1979). Here, we report evidence that the plasmid-like DNAs or segments of them are integrated into mitochondrial chromosomal DNA (mtDNA) of Vg-revertants.
MtDNAs were isolated from each of several different cms-Vg and from Vg-revertants of them. The purified DNAs were digested with each of several restriction endonucleases and the resultant fragments electrophoretically resolved on agarose gels. Patterns (visualized by ethidium binding under UV) were compared between sterile parent and Vg-revertant progeny as well as among all sterile and fertile plants. While the restriction patterns of the several sterile cms-Vg were indistinguishable, the patterns of the revertant DNAs differed from those of the sterile cms-S and from one another. New restriction fragments, not present in mtDNA from sterile plants, were evident in the revertants and some of the new fragments were unique to a particular revertant. Analogous to prokaryotic genetic transpositions, the concomitant disappearance of the plasmid-like DNAs together with the appearance of new endonuclease restriction fragments in the Vg-revertants suggests the integration of the S-1 and S-2 DNAs upon the plant's reversion to fertility. To test this hypothesis we isolated and resolved the S-1 and S-2 DNAs and labeled them with (?-32P) by nick-translation for use as hybridization probes. Restriction digests of mtDNAs from the several cms-Vg and Vg-revertant plants were Southern blotted onto membranes and then hybridized with labeled fragments of either S-1 or S-2 DNAs.
Results obtained with one pair, i.e. a parental cms-Vg and a progeny fertile-revertant (296) are shown (diagrammatically) in Fig. 1. The electropherogram (compare lanes C and D) of Xho I digested mtDNAs shows that both cms-Vg and Vg-revertant DNAs yield almost identical restriction patterns. However, at least one "new" fragment (at arrow) is present in this Vg-revertant. The autoradiogram developed with S-2DNA as the hybridization probe (compare lanes E and F) shows: (1) this "new" fragment shares homologous sequences with S-2; (2) additional fragments of the Vg-revertant DNA share homology but differ in size from the Xho I digestion products of S-1 and S-2 detected in cms-Vg DNA. On the other hand, the autoradiogram developed with S-1 DNA as the hybridization probe (compare lanes A and B) yielded homologous patterns between cms-Vg and Vg-revertant DNAs except for the absence of the terminal Xho I fragments of both plasmid-like DNAs in the Vg-revertant pattern. The data from this and other cms-Vg and Vg-revertant analyses indicate that the mtDNA sequences have undergone rearrangement upon the apparent integration of portions of the plasmid-like DNAs.
We conclude that the S-1 and S-2 DNAs may be physical manifestations of the episomal fertility elements postulated by Laughnan and Gabay-Laughnan to account for high frequency cytoplasmic reversions to fertility. In any case, these observations provide a molecular approach to the mode of integration as well as the specific genetic information encoded in the integrated sequences and their relationship to cytoplasmic male sterility in maize.
B. D. Kim, R. J. Mans, D. R. Pring, M. F. Conde, C. S. Levings III, J. R. Laughnan and S. J. Gabay-Laughnan
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