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A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomycin D (7-amino-AMD), a fluorescent analogue of antibiotic actinomycin has been investigated, and a particular staining procedure has been developed. The procedure includes short fixation in 70% ethanol and staining for 20 min in 10W5M solution of 7-amino-AMD at pH7. The results of DNA content measurements are very reproducible. The histograms obtained have a coefficient of variation less than 3%.The absorption maximum of the complex of 7-amino-AMD with DNA is situated in the green spectrum region, making this stain particularly suitable for argon laser flow cytornetry.

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Interaction of DNA with the analogs of the antibiotic distamycin A having different numbers of pyrrolcarboxamide groups and labeled with dansyl was studied. The binding isoterms of the analogs to synthetic polydeoxyribonucleotides were obtained. Analysis of the experimental data leads to the following conclusions: (1) the free energy of binding of the analogs to poly(dA).poly(dT) depends linearly on the number of amide groups in the molecule of the analog whereas attachment of each pyrrolcarboxamide group produces changes of 2 kcal/mole in the free energy; (2) attachment of a pyrrolcarboxamide unit to the GC pair results in the free energy change of 0.95 kcal/mole; (3) the binding of analogs to poly(dA).poly(dT) is a cooperative process, presumbly, dependent on conformational changes induced by the binding of analogs to DNA.

It is shown here that distamycin A and actinomycin D can protect the recognition sites of endo R.EcoRI, EcoRII, HindII, HindIII, HpaI and HpaII from the attack of these restriction endonucleases. At proper distamycin concentrations only two endo R.EcoRI sites of phage lambda DNA are available for the restriction enzyme--sRI1 and sRI4. This phenomenon results in the appearance of larger DNA fragments comprising several consecutive fragments of endo R.EcoRI complete cleavage. The distamycin fragments isolated from the agarose gels can be subsequently cleaved by endo R.EcoRI with the yield of the fragments of complete digestion. We have compared the effect of distamycin A and actinomycin D on a number of restriction endonucleases having different nucleotide sequences in the recognition sites and established that antibiotic action depends on the nucleotide sequences of the recognition sites and their closest environment

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Optical methods, such as fluorescence, circular dichroism and linear flow dichroism, were used to study the binding to DNA of four symmetrical cyanine dyes, each consisting of two identical quinoline, benzthiazole, indole, or benzoxazole fragments connected by a trimethine bridge. The ligands were shown to form a monomer type complex into the DNA minor groove. The complex of quinoline-containing ligand with calf thymus DNA appeared to be the most resistant to ionic strength, and it did not dissociate completely even in 1 M NaCl. Binding of cyanine dyes to DNA could also be characterized by possibility to form ligand dimers into the DNA minor groove, by slight preference of binding to AT pairs, as well as by possible intercalation between base pairs of poly(dG)-poly(dC). The correlation found between the binding constants to DNA and the extent of cyanine dyes hydrophobicity estimated as the n-octanol/water partition coefficient is indicative of a significant role of hydrophobic interactions for the ligand binding into the DNA minor groove.

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