Siberian Inst. Plant Physiol. Biochem.
Oligonucleotide DNA-probe for detecting linear mitochondrial plasmids
--E. L. Tauson, S. I. Belicov, J. M. Konstantinov, V. P. Kumarev
The molecules of the linear plasmid-like DNAs (S1, S2, 2.3kb plasmid or S3) found as individual elements of mitochondrial genome of maize are structurally unique in that they contain terminal inverted repeats significant for their biological functions. There are 16bp of perfect homology between terminal inverted repeats (TIRs) of these linear plasmids, which probably are protein binding sites (Fig. 1) (Bedinger et al., MGG 205:206-212, 1986). The S episomes of maize constitute a unique group of replicons with a common origin. At the present time it has become obvious that the genetic functions of these plasmids are not limited only by their role in the cytoplasmic male sterility trait.
Radiolabeled DNA-probes with specific sequences are a useful instrument for identification of small quantities of corresponding DNA without isolation and purification. The application of specialized DNA probes should be helpful in investigations of the role of some structural zones of these additional genetic elements of mitochondria of maize. In addition such probes can be used in experiments on recombination processes inside the mitochondrial genome and probable exchange of the genetic material between organelles. In the present work we have obtained such a DNA probe for S plasmids of maize mitochondria.
A 16bp DNA probe that has a perfect homology with three linear plasmids (2.3; S1 and S2) and free of mismatched nucleotides was synthesized according to a method of Frochler and Mattenci (Tetrahedron Lett. 27:469-472, 1986). The first and the second strand of the fragment was arranged with PstI sites (Fig. 2), phosphorylated with T4-polynucleotide kinase, annealed and inserted into pBR322 by a standard method (Maniatis et al., Molecular Cloning, NY, 1982).
The recombinant DNA (pmtZET16) was used to transform E. coli ÆM15. Cells were plated on agar containing 15µg tetracycline/ml. Colonies were transferred on Whatman 542 and hybridized with [32P] radiolabeled oligonucleotide probe. Colonies giving a strong signal were picked and transferred on other plates of LB-agar with tetracycline (15µg/ul) and LB-agar with tetracycline (15µg/ml) and LB-agar with tetracycline (15µg/ml) and ampicillin (50µg/ml). Colonies that were resistant to Tc, but sensitive to Ap, were selected. DNA was isolated from fresh cells of each clone, nick-translated and dot-hybridized with S2 plasmid-like DNA isolated and purified from mitochondria of etiolated seedlings of maize hybrid ìBekke LLOî (Fig. 3).
It can be concluded from the results of hybridization that recombinant plasmid pmtZET16 will be of use as a probe for recognizing corresponding DNAs. Moreover, it is very likely that localization of linear mitochondrial plasmids could facilitate the investigations of protein-binding sites in these DNAs.
The possibility that the same protein recognizes and binds the termini of all of the linear maize mitochondrial plasmids was proposed by Bedinger and coauthors (MGG 205:206-212, 1986). At present, however, it is unknown whether or not the 16bp fragment with a perfect homology within the TIRs of linear mitochondrial plasmids has functional significance in DNA-protein complex formation. The use of the cloned sequence in terms of the slight changes introduced into its synthesis, might be one of the ways to elucidate this problem.
Figure 1. Nucleotide sequence comparison of S plasmids and 2.3kb plasmid. Differences are indicated by the arrow.
Figure 2. 16bp DNA sequence of synthetic oligonucleotide, homologous to the linear plasmids of maize mitochondria. PstI sites marked with boxes.
3. Hybridization of radiolabeled DNA of several pmtZET16 clones to
S2 maize mitochondrial plasmid and pBR322; 1-5.7-clones with oligonucleotide
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