Part I. Studies on Mycoplasm gallisepticum. Part II. Autoradiography of Chromosomal DNA Fibers from Chinese Hamster Cells

Citation

Riggs, Arthur Dale (1966) Part I. Studies on Mycoplasm gallisepticum. Part II. Autoradiography of Chromosomal DNA Fibers from Chinese Hamster Cells. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/P60E-AW11. https://resolver.caltech.edu/CaltechETD:etd-08252008-095724

Abstract

Part I:

The cell size, DNA content, and chromosome size and structure of Mycoplasma gallisepticum A5969 was studied. After calibrating a phase microscope with polystyrene latex spheres of known diameter, it was determined that the average cell volume in the living state is only 36 ± 9 x 10^-15 cm³, which is equivalent to the volume of a sphere 0.40 ± 0.04 μ in diameter.

By a DNA determination method based on analysis for thymine, the DNA content of M. gallisepticum was found to be 4.0 ± 0.6% of the dry weight. The DNA per colony forming unit was found to be 300 x 10⁶ daltons. For these experiments, the cells were harvested by centrifugation. The results of later experiments, in which the cells were directly precipitated from a broth culture by the addition of trichloroacetic acid (TCA), suggested that there had been DNA loss during the centrifugation steps of the earlier experiments and that the DNA per clone forming unit is 1,200 ± 200 x 10⁶ daltons.

The Cairns autoradiographic technique was used to visualize the chromosome of M. gallisepticum. The DNA autoradiograms seen indicated that its chromosome is 630 ± 150 μ long. This corresponds to 1,200 ± 300 x 1010⁶ daltons of DNA. The DNA autoradiograms often showed "replication forks" and were sometimes circular.

The Kleinschmidt technique was also used to provide evidence on the size and structure of the chromosome of M. gallisepticum. Tangles of DNA were found that were very similar to those obtained from bacteria. The total length of DNA in the tangles indicated that the chromosome of M. gallisepticum is 500 ± 200 μ long, a value that supports the estimate obtained by autoradiography.

Part II:

Linear DNA autoradiograms were found when the Cairns technique was applied to Chinese hamster cells. At least 6% of these autoradiograms are more than 0.8 mm long - roughly the size of the E. coli chromosome and considerably longer than previously reported DNA fibers from higher organisms. Some rare autoradiograms are as long as 1.6 - 1.8 mm. The implications of these results in terms of models of chromosome structure are discussed.

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