Quantitative tritium autoradiography of mammalian chromosomes. I. The basic method

Abstract

A technique has been developed that allows repeated autoradiographs to be made of the isotope distribution in the chromosomes of a single cell. A series of 10 separate autoradiographs were made of a Chinese hamster diploid male metaphase cell which had been labeled with tritiated thymidine during the first 15 minutes of its DNA synthesis period in the previous interphase. Each autoradiograph had low grain densities above the chromosomes so that quantitation was feasible. The separate autoradiographs were photographically combined into a single composite in which grain images were converted to lines oriented at right angles to the chromosome axis. The line densities were then measured with a recording microdensitometer to yield graphs reflecting the isotope distribution along each chromosome. The area under each graph was directly proportional to the total number of grains counted above the corresponding chromosome in the 10 separate autoradiographs. The distribution of isotope along the chromosomes was different for each chromosome, and in some cases homologs also differed in their early labeling patterns.