Date of Award
Doctor of Philosophy (PhD)
The Indian deer, (Muntiacus muntjak), has the lowest known diploid chromosome number (seven in the male) for a mammal. Mitotic chromosomes can be removed from cells either by extraction with microneedles or by inducing chromosome expulsion with a drop of silicone oil applied to the cell. The entire diploid complement is removed by virtue of interconnecting fibers among the chromosomes. The chromosomes are brought to the surface of the cover slip for analysis, where each can be identified morphologically with phase contrast optics. The chromosomes are sticky and extensible, and parts of chromosomes can be dissected by cutting with microneedles. Within muntjac metaphase cells, chromosomes are arranged radially on the mitotic spindle, with centromeres facing the center and arms pointing outwards. The position of individual chromosomes on the spindle is random however. There is no homologous pairing or tendency towards association of particular chromosomes. The radial array is preserved when chromosomes are microsurgically removej from cells. Immunofluorescent staining with tubulin antiserum reveals that the spindle is at the center of the extracted array and holds it together. When cells are treated with spindle poisons, the mitotic chromosomes are not radially arranged and chromosomes can be removed from the cells individually. The spindle is therefore responsible for maintaining the configuration of mitoitic chromosomes. Interconnecting fibers among microsurgically extracted chromosomes are of several types. Some are adhesions of sticky chromosome fibers accidentally brought into contact with one another during micromanipulation. Cognizance of this possibility allows such artifacts to be avoided. Centromeres are connected to the spindle, which appears as a fibrous network at the center of the radial array with phase contrast and scanning electron microscopy. When chromosomes are displaced from the spindle during micromanipulation, their connections to the spindle stain for DNA and histone and are sensitive to DNase. These connections may represent stretched chromosome regions which are still attached to the spindle. Further studies may elucidate the mode of connection of chromosomes to the spindle. Examination of intact mitotic cells with fluorescent stains for DNA confirm that chromosomes are not connected to one another by DNA during mitosis. Scanning electron microscopy, immunofluorescent staining for histone, and the effects of enzyme treatments on microsurgically-isolated chromosomes provide insights into chromosome structure. Chromosome arms are labile to DNase but not protease. This suggests that chromosomes do not contain protein cores which run from one end to the other. Centromere regions are resistant to nuclease and protease treatments, and consist of tightly packed fibers. The remaining surface of the chromosome consists of looping fibers, except for the nucleolus organizers, where fibers are loosely packed and longitudinally oriented. The latter regions are especially prone to breakage by DNase. Staining for histones H1 and H2B reveals that these are present throughout the lengths of the chromosomes and are distributed more or less uniformly. These studies exploit the ability of micromanipulation to obtain chromosome preparations from cells without chemical treatments or destruction of three-dimensional relations. Production of artifacts can be monitored and often avoided. Micromanipulation can therefore be a valuable adjunct to other forms of analysis in studying chromosome structure and function.
Korf, Bruce R., "Probing Mitotic Chromosome Structure and Arrangement Using Micromanipulation" (1979). Student Theses and Dissertations. 476.