Characterization of Bleomycin-Resistant DNA

After reaction of DNA with high concentrations of bleomycin, approximately 80% of the DNA becomes trichloroacetic acid (TCA) soluble. The remaining 20% of the DNA remains TCA insoluble. Upon further treatment of this TCA-insoluble material with high concentrations of the drug, no further drug action can be detected. Drug action is defined as fragmentation of DNA to smaller molecular size, release of free bases, and TCA solubilization. This material which is not attacked by bleomycin has been termed bleomycin-resistant DNA. This bleomycin-resistant DNA does not compete with native DNA in the bleomycin reaction indicating that there is no binding or inactivation of the drug by the resistant DNA. The resistant DNA shows very little hyperchromicity when heated through the melting temperature for the corresponding native DNA, indicating a single-stranded structure. Results of sedimentation and equilibrium analyses yield a molecular weight of about 4,000 daltons. This value is the same regardless of the source of the native DNA. Finally, the bleomycin-resistant DNA exhibits a base composition similar to that of the native DNA from which it was derived.     

Automatic collection and processing of data from the ultracentrifuge using a programmable desk calculator.

Previous investigators [Trautman, R., Spragg, S. P., and Halsall, H. B. (1969) Anal. Biochem.28, 396–415] have published a detailed protocol for the analysis of sedimentation velocity measurements which is adaptable to data generated by an ultraviolet scanning system. The advent of programmable desk calculators capable of sampling the output of digital measuring devices has made it possible to develop inexpensive and highly convenient systems for collecting and processing scanner data. Basing our approach on the referenced protocol, we have developed algorithms for dealing with real data, that is, data characterized by a relatively high level of noise. The techniques are applieable to both sedimentation equilibrium and sedimentation velocity measurements using the seanning system and multicell rotors. With known concentration dependence, valid estimates of weight-average sedimentation coefficients, diffusion coefficients, and heterogeneity parameters have been obtained for both simulated and actual sedimenting anddiffusing macromolecular solutes. We find, however, that concentration dependence derived internally from a single sedimentation velocity measurement is unreliable.