Cloning and characterization of the linear 2.3 Kbp mitochondrial plasmid of maize

The mitochondria of maize, as well as many other higher plants, contain small circular and linear DNA molecules known as mitochondrial plasmids. The mitochondria of all maize lines examined to date contain a linear 2.3 kilobase pair (or related 2.1 Kbp) linear plasmid. Since this is the only mitochondrial plasmid found consistently in all maize lines, it is the best candidate for encoding an essential mitochondrial function. It has been shown that this plasmid has protein(s) tightly associated with its 5' termini that would interfere with standard molecular cloning techniques. In order to clone this mitochondrial plasmid into a bacterial vector, we first separated the 2.3 Kbp plasmid from the high molecular weight (HMW) main mitochondrial genome on CsCl gradients. Next, complementary homopolymer "tails" were added to the 2.3 Kbp plasmid and the linearized bacterial vector pUC8, and the annealed DNAs were transformed into bacterial cells. By using this technique we were able to bypass the terminally bound 5' protein and obtain full length DNA clones of the 2.3 Kbp plasmid, which we have named pZm2.3. Nucleotide sequence analysis of one end of this clone reveals that 15 out of 17 base pairs are homologous with the termini of the linear S plasmids, which are found in plasmid form only in cms-S type mitochondria. The S plasmids also have protein(s) tightly associated with their 5' termini. Further analysis using pZm2.3 as a hybridization probe has shown that there is an integrated form of the plasmid in the HMW DNA of N(fertile), cms-T, cms-C and cms-S mitochondria. It has been previously shown that S plasmid related sequences exist in an integrated form in N and cms-S HMW mitochondrial DNA. Small amounts of dimers of the 2.3 Kbp plasmid can be detected, and may represent replicative forms. The 2.1 Kbp linear plasmid found in cms-T and some fertile inbred lines is very homologous to the 2.3 Kbp plasmid, and apparently is deleted near one terminus. We are currently sequencing the cloned plasmid and are especially interested in investigating the possibility that the 2.3 Kbp plasmid may encode the protein that is associated with the 5' termini of this plasmid.

P. Bedinger, E. de Hostas and V. Walbot

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