Molecular analysis of the NCS3 mitochondrial mutant

--M. D. Hunt and K. J. Newton

NCS3, one of the nonchromosomal stripe mutations, arose in a WF9 line carrying cms-T mitochondria. It has a restriction enzyme profile which distinguishes it from other NCS mutants and from "normal" cms-T mitochondria. This mutation has been removed from the WF9 background, and thus stabilized, by repeated outcrossings with pollen from other inbred lines. We would like to determine what sort of DNA rearrangement has taken place, and this, in turn, should shed some light upon the mechanism by which NCS mutations occur. A potential use for these mutants lies in defining functional genes which have not yet been identified in the plant mitochondrial genome.

We have found that a new 20 kb XhoI fragment which appears in NCS3 (Newton and Coe, PNAS 82:6879) is the result of a rare recombination event between two regions of the cms-T genome, one contained within a 14kb and the other within a 16kb XhoI fragment. These two progenitor fragments have been cloned, and hybridization studies have indicated that the two regions share no apparent homology. By sequencing through the junction point of both progenitor fragments and the mutant fragment, however, we have found a 12-base reiterated sequence at the point of recombination. This situation is very similar to several described in Oenothera mitochondria (Manna and Brennicke, Mol. Gen. Genet. 203:377) and it may indicate that, under some circumstances, a very limited region of homology may be sufficient to initiate a recombination event in plant mtDNA. Because we have found no evidence of a reciprocal recombination product in the mutant and because part of one of the progenitor fragments seems to be missing from the mutant genome, we suspect that this event has given rise to a deletion involving at least one functional mitochondrial gene. The extent of the deletion is not yet known.

Using probes taken from the recombination junction of the 14 and 16kb XhoI fragments of cms-T DNA, we have identified two transcripts which are missing or severely reduced in NCS3. These RNAs are not homologous to any previously identified plant mitochondrial genes. We are currently analyzing these regions of the genome in an effort to identify a function for the affected transcripts.

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