AUTORADIOGRAPHIC AND IMMUNOFLUORESCENT DETECTION OF LOW CONCENTRATIONS OF ACTINOMYCIN D BOUND TO HUMAN METAPHASE CHROMOSOMES.
AuthorBROTHMAN, ARTHUR RICHARD.
Binding sites (Biochemistry)
AdvisorLindell, Thomas J.
Committee ChairHarris, Robert M.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractThe binding of low concentrations of actinomycin D (Act D) to fixed human metaphase chromosomes was studied using both autoradiographic and immunofluorescent techniques. At the concentration range of 0.001 - 0.1 μg/ml Act D is known to selectively inhibit rRNA synthesis. Although it was previously suggested that at these low concentrations Act D would selectively bind to the ribosomal cistrons, evidence also exists that the drug binds to non-ribosomal DNA, and inhibits rRNA transcription in an indirect fashion. Because of the conflicting data on Act D binding and a lack of focus on biologically relevant concentrations of drug, it was decided to systematically investigate the distribution of the drug binding in low concentrations to chromosomes from 72-hr human lymphocyte cultures. Autoradiographic detection of [³H]Act D bound to chromosomes showed no selective binding of the drug at concentrations that maximally inhibit rRNA synthesis. A new technique was employed using Formvar and potassium chromium sulfate as a pretreatment to autoradiography. This technique permitted simultaneous detection of silver grains and chromosome identification by G-banding. With autoradiographic exposure times of 1 and 7 days, there was a positive correlation of autoradiographic grains with chromosome length. To increase sensitivity in detection of Act D bound to chromosomes, a specific anti-Act D antibody was generated in rabbits. Antibody avidity was evaluated on the basis of a rapid charcoal assay. This charcoal assay was then used in development of a radioimmunoassay for Act D which is sensitive in quantitating the drug down to 0.005 μg/ml. The anti-Act D antibody was characterized to be IgG, and was shown to be specific for the pentapeptide lactone portion of the Act D molecule. Indirect immunofluorescence of Protein A-purified IgG containing anti-Act D was used to detect drug bound to fixed human chromosomes. The antibody was shown to be specific for drug bound to chromatin. When 0.1 μg/ml Act D was bound to chromosomes, the drug was observed bound throughout the genome, with no selective binding at the ribosomal cistrons. This confirms the autoradiographic data and supports the model of extranucleolar regulation of rRNA synthesis. Preliminary results suggest that Act D binds to GC-rich DNA, since an R-banding pattern was observed in 5% of the immunofluorescent metaphases examined.