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Specific detection of kinetoplast DNA in cytological preparations of trypanosomes by hybridization with complementary RNA

par Steinert, Maurice ;Van Assel, Suzanne ;Borst, Piet;Mol, J. N M;Kleisen, C.M.;Newton, B.A.
Référence Experimental cell research, 76, 1, page (175-185)
Publication Publié, 1973

Article révisé par les pairs

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Résumé :

We have used the method of cytological hybridization with complementary RNA to study the base sequence homology between kinetoplast DNAs of different hemoflagellates. 3H-labelled complementary RNA (spec. act. 20 × 106 dpm/μg) was synthesized on purified kinetoplast DNA with RNA polymerase of Escherichia coli. Smears of hemoflagellates were fixed with a formaldehyde-containing fixative, the DNA was heat-denatured in situ in the presence of 50% formamide and hybridization with complementary RNA was carried out with 20 μl RNA at 0.6 μg/ml. Unspecifically bound RNA was removed by treatment with ribonucleases and hybridization was quantitatively evaluated on autoradiograms with a microphotometer. In intraspecific hybridization reactions with Crithidia luciliae and Trypanosoma mega, RNA was bound only to the kinetoplast and not to the nucleus. In the interspecific hybridization reaction no cross-hybridization was found at all, either on cytological preparations or with kinetoplast DNA fixed to nitrocellulose filters. This shows that the evolution of a major part of kinetoplast DNA is not highly restricted. Cytological hybridization may, therefore, provide a useful method to differentiate closely related species of pathogenic hemoflagellates. © 1973.