H3-Uridine Autoradiography of Human Chromosomes

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Abstract
Human lymphocyte and fibroblast metaphase chromosomes were labeled immediately prior to mitosis with tritiated uridine to study the feasibility of utilizing autoradiography to localize areas of genetic activity. Specifically, two questions were investigated. 1. Are consistent patterns of RNA labeling detectable by H3-uridine autoradiography of metaphase chromosomes? 2. Does H3-uridine autoradiography of metaphase chromosomes detect the decreased rate of RNA synthesis by the inactive X chromosome? H3-uridine autoradiography of XX, XY, X isochromosome X, and XXXXY cells demonstrated localization of grains to metaphase chromosomes. There were, however, no consistent patterns of RNA labeling, and no decrease in the number of grains over the inactive X chromosomes. Incubation of cells with various compounds to stimulate or suppress RNA synthesis, or treatment of metaphase preparations with varying concentrations of ribonuclease, failed to accentuate any patterns of labeling. Female cells labeled with H3–5-uridine for five minutes showed a marked decrease in grains over the sex chromatin body. Cells pulse labeled with H3-5-uridine and chased with cold uridine for up to 48 hours, also showed localization of grains to metaphase chromosomes but no consistent patterns of labeling. These results indicate that in humans sufficient amounts of newly synthesized RNA do not remain on the metaphase chromosomes to allow autoradiographic localization of their sites of synthesis. What labeling does occur is the result of random, non-specific adherance of labeled RNA to metaphase chromosomes.