The major objectives of this project are: to examine the intragenomic distribution of specific and total adducts induced by the aromatric amines (N-2-acetylaminofluorene (AAF) and N-2-acetylaminophenanthrene (AAP), and to locate and identify the damaged nucleotides in DNA sequences. The adducts will be analyzed by a previously described 32p-fingerprinting method which requires 1-2 Mug of DNA for replicate analyses with a sensitivity of detection of 1 adduct in 10-100 million total nucleotides; all experiments, therefore, can and will be conducted in whole animals (rats) to study the adduct formation and removal rates. The addduct distribution will be examined in (i) low and high-salt soluble chromatin DNA and matrix-bound DNA, isolated after endogenous digestion of nuclei; (ii) 145 bp core DNA, 165 bp chromatosomal DNA, 50 bp nucleosomal linkers, and 30 bp chromatosomal linkers, which will be isolated by gel electrophoresis of the mononucleosomal DNA released by micrococcal nuclease digestion of nuclei; (iii) 100 bp central core DNA, isolated by 3 feet - and 5 feet- exonucleolytic """"""""trimming"""""""" of the core DNA, followed by gel electrophoresis; and (iv) repetitive DNA sequences, isolated by restriction of total nuclear DNA with Hind III or EcoRl endonuclease and gel electrophoresis. Adducts in in vitro modified DNA sequences will be identified directly as (32p)-nucleotides by a procedure currently under development. The use of a well known hepatocarcinogen (AAF) and a nonhepatocarcinogen (AAP) will address the question whether the fine chromatin structural features show significant differences as to the distribution of adducts induced by the two compounds, whixh may have relevance to the process if carcinogenesis. The proposed studies will enable us and others to investigate the actions of carcinogens, at molecular level, in a much more sensitive and precise way than has been hitherto possible. These studies will be extended to structural genes, viz., transfer and ribosomal RNA genes, which are the major long-term objectives of this proposal.
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