At levels of organization between the Watson-Crick model of DNA on the one hand, and the microscopically visible mitotic or meiotic chromosome on the other, very little is known about the structure or function of chromosomes in eukaryotic organisms. The studies reported in this thesis were an attempt to learn more about the arrangementof certain DNA sequences in mammalian chromosomes, about the size of the DNA molecules in such chromosomes, and about the replication of these DNA molecules.
Part I contains the results of experiments designed to determinethe distribution of the hundreds of genes (DNA sequences) for ribosomal RNA among the chromosomes of HeLa cells. In the course of these experiments, methods were developed for isolating metaphase chromosomes ona large scale from HeLa cells and for fractionating them on the basis of sedimentation velocity. Hybridization experiments between ribosomal RNA and DNA from the various fractions of isolated chromosomes showed that the genes for ribosomal RNA are confined entirely to small HeLa cell chromosomes.
In Part II are reported the results of autoradiographic experiments intended to help determine the size and manner of replication of the DNA in mammalian chromosomes. All the experiments described in Part II are the result of collaboration with Dr. Arthur D. Riggs. Weused a modification (by Dr. Riggs) of the technique for autoradiography of individual DNA molecules which had been developed by Cairns (J. Mol.Biol. 6, 208 (1963)). Our application of this technique to the DNAof Chinese hamster cells demonstrated the presence in Chinese hamster cell chromosomes of DNA fibers up to 1,800 µ long. Subsequent pulse labeling studies showed that such long fibers are divided into many shorter replication units, and that DNA replication probably startsin the interior of each unit and then proceeds outward in both directions, at fork-like growing points, to the ends of the unit.
