In maize there are several basic types of cytoplasmic male sterility which can be distinguished on the basis of nuclear genes that restore fertility. Restriction endonuclease fragment analysis also separates the various cytoplasms by distinctions in the banding patterns of their mtDNA. By the hybridization experiments described below, differences have been demonstrated among the mtDNAs from normal (fertile) and the T and S male-sterile cytoplasms. Sequence similarities, duplications, possible rearrangements and deletions of portions of the mitochondrial genome were evident.
MtDNA isolated from the male-fertile hybrid, NC7 x T204, was digested with Bam H1 and the resulting fragments cloned with the plasmid PBR 322 in E. coli, strain LE 392. Fragments which had been taken up by the plasmid and replicated were screened to insure that each insert contained only one restriction fragment. Cloned inserts were isolated and 32p-labelled by nick translation for use as hybridization probes.
MtDNAs from the normal and cms-T strains were restricted with Bam H1 and the resulting fragments fractionated by gel electrophoresis on a .8% agarose gel. Unrestricted cms-S mtDNA and the insert, which would later be used as the probe in the hybridization experiments, were also loaded in adjacent slots on the gel. In this case, the insert served as a marker while homology with S-1 and S-2 DNA species could be assayed on the slot containing the unrestricted cms-S mtDNA.
DNA digests were transferred from the electrophoretic gels to nitrocellulose membranes according to the Southern blot technique. Labelled probes were heat denatured and incubated with the nitrocellulose membranes for 20-40 hours at 650 C. Autoradiography was used to detect hybridization.
At the present time, fourteen different cloned fragments have been studied by the hybridization techniques. Of these, eight demonstrated homology with bands of the same electrophoretic mobility in the normal and T restriction patterns (Fig. 1a). Earlier restriction enzyme analysis showed that when mtDNA from normal and male-sterile cytoplasms were digested with a common enzyme, 75-80% of all the fragments appeared to be of the same molecular weight. Therefore, it was expected that most of the autoradiographs would show identical hybridization patterns between N and T. It can now be added that not only are the majority of the fragments similar in size but they contain sequence homologies as evidenced by hybridization with the labelled probes.
The next most frequent type of autoradiographic pattern (four of the fourteen) showed an "extra" band present in T that was not detected in N (Fig. 1b). One of the two bands in T was analogous to the band in N while the additional band appeared smaller in molecular weight. This suggests that recombinational events which either duplicated or rearranged DNA sequences may have occurred in T. Interestingly, none of the labelled clones has displayed homology with more than one band in the normal pattern.
One autoradiograph exhibited a pattern where the band in T was displaced or did not line up exactly with the band in N (Fig. 1c). This result could have arisen by sequence rearrangements in the mtDNA or perhaps even a deletion event.
Evidence has been obtained for additional sequences present in normal mtDNA that is missing in male sterile mtDNA. One of the cloned fragments of normal mtDNA showed no homology with any of the T restriction bands (Fig. 1d).
Another cloned fragment is particularly noteworthy. The autoradiographic pattern indicated homology with corresponding bands in N and T and also with S-1, one of the plasmid-like DNAs found associated with the mtDNA in cms-S (Fig. 1e). This demonstrates that sequences in common with the plasmid-like DNA, S-1, are also found in the mitochondrial chromosomal DNA.
In summary, hybridization with cloned fragments has revealed: sequence homology between a majority of the restriction fragments from normal and cms-T mtDNA, a single case of sequence differences between normal and cms-T mtDNA, evidence for recombinational and/or deletional events among the mtDNAs, and evidence that DNA sequences in common with the plasmid-like DNAs are found in the mitochondrial chromosomal DNA.
W. M. Spruill Jr., R. R. Sederoff and C. S. Levings III
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