IRKUTSK, RUSSIA
Institute of Plant Physiology and Biochemistry
Russian Academy of Sciences

NOVOSIBIRSK, RUSSIA
Institute of Cytology and Genetics
Russian Academy of Sciences

Protein factor binds specifically to putative origin of replication of the mitochondrial 1.9 kb plasmid

— Tarasenko, VI; Subota, I; Kobzev, VF; Konstantinov, YM

It is well known that plant mitochondria have more complex genome organization than the mitochondria of animals or fungi. In addition to large mtDNA molecule, the maize mitochondrial genome also composed of smaller autonomously replicated plasmid-like DNA molecules (Fauron et al., Trends Genet. 11:228–235, 1995). There are complete sequence data on these plasmids. Based on analysis of these data some predictions were made concerning potential regulatory regions in the molecules. In particular, the 1.9 kb extrachromosomal double-stranded circular DNA plasmid was found to contain regions which in other systems are known to be part of origin of DNA replication (Ludwig et al., Gene 38:131–138, 1985). One of two AT-rich regions found in this molecule contains in triplicate the 11-bp sequence ATTTATATCTA. In yeast, similar sequences function as autonomously replicated elements (Kearsey, Cell 37:299–307, 1984). This suggests that they may function as ori in mitochondria. Neverthless, no experimental studies were made to verify this hypothesis. In this investigation we tried to search for protein factors able to bind specifically to given DNA region. The finding of such a protein would suggest that this region actually plays some regulatory role in the metabolism of plasmid molecule.

The mitochondria were isolated from 4-day-old etiolated seedlings of hybrid VIR46 MV and lysed by high salt and Triton X-100. The lysate was applied on DEAE Toyopearl chromatography column. The proteins were eluted by discontinious gradient of KCl concentration (0.05 – 0.75 M). EMSA was carried out as described (Kagoshima et al., J. Biol. Chem. 271:33074–33082, 1996) with minor modifications. We used PCR-generated 230 bp double-stranded DNA probe named ATR (AT-rich Region). This DNA fragment contained potential origin of replication and flanking regions (base pairs 1120–1350).

Some fractions eluted from DEAE Toyopearl contained activity which formed single DNA-protein complex in EMSA when ATR was used as a probe (Fig. 1). The main part of activity was detected in the fractions eluted at 0.2–0.25 M KCl concentration. The retardation complex was stable in high salt and in the presence of non-specific competitor DNA poly[dIdC-dIdC] demonstrating the specificity of the interaction.

To test further DNA binding specificity of protein factor under investigation we performed mobility shift competition studies. The principle of these experiments was to add an excess of unlabelled specific DNA to a reaction containing the radiolabelled ATR probe and protein fractions obtained by DEAE Toyopearl chromatography. Figure 1 shows the results of such an experiment. When competitor DNA added to the reaction was ATR fragment the formation of retardation complex was greatly reduced due to the sequestration of DNA binding factor by these DNA. However we did not detect a decrease in retardation complex intensity upon addition of competitor DNA contained rps13 gene sequence or upstream region of cox1 gene. These results clearly indicate that hypothetical protein factor specifically binds to region of the 1.9 kb plasmid containing putative origin of replication. It is possible to speculate that this protein indeed involved in realization of genetic functions of plasmid molecule.

It is unclear where this protein is encoded. There are a few putative orfs in sequence of the 1.9 kb plasmid but in our opinion they are not long enough to encode functional protein. The largest one would encode a protein comprised of 82 amino acid residues. So, we suppose that protein could be encoded in mitochondrial chromosome or nuclear genome. Additional experiments are needed to reveal the nature of this protein factor. Further purification and characterization of the protein is on the run in our laboratory.

 

Fig. 1. DNA binding activity exhibited by protein fractions isolated by chromatography on DEAE-Toyopearl (DF, eluted with 0.25 M KCl). Competition study using non-specific DNA competitor and probes contained mitochondrial sequences. rps13 — coding region of rps13 gene (470 bp); cox1 promoter — upstream region of cox1 gene (230 bp). Unlabelled competitors were present in the reactions in a 30-fold excess relative to the labelled probe. Numbers indicate the positions of DNA-protein complex and the free radiolabelled ATR probe.


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