Electron microscopic studies of mitotic chromosomes in yeast
Sakane, Holly Masae
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Permalink
https://hdl.handle.net/2142/19285
Description
Title
Electron microscopic studies of mitotic chromosomes in yeast
Author(s)
Sakane, Holly Masae
Issue Date
1995
Doctoral Committee Chair(s)
Widom, Jonathan
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Cell
Biology, Microbiology
Chemistry, Biochemistry
Language
eng
Abstract
Yeast are highly genetically manipulatable organisms which have been used successfully as model systems to determine many of the components involved in cell processes such as cell cycle control and secretion. One of the unresolved problems in yeast cell biology is whether or not the chromosomes of the yeast, Saccharomyces cerevisiae, condense during mitosis and, if they do, to what extent they are condensed.
In this thesis, I demonstrate the presence of electron dense objects in various preparations of S. cerevisiae cells for electron microscopy. These electron dense objects appear to be the condensed mitotic chromosomes of this yeast. They are very similar in appearance to chromosomes visualized in Schizosaccharomyces pombe. Additionally similar objects have been visualized in lightly-fixed, low-temperature embedded cells. These objects have been labeled with antibodies against histone H2B and DNA presenting evidence that these are the condensed mitotic chromosomes of this yeast. This is the first direct visualization of the condensed mitotic chromosomes of S. cerevisiae.
To further study these chromosomes, they have been reconstructed using serial thin sections in the electron microscope to obtain a three-dimensional view of these objects. The structures observed vary in width from 30 nm to 160 nm (typically with widths of 80 nm) and can be traced within and between sections as they form objects which look similar to condensed chromosomes seen in higher eukaryotic cells. This is the first analysis of the condensed mitotic chromosomes of S. cerevisiae in three dimensions. In the study, there are hints that the chromosomes may form a metaphase plate and that filaments found within the nucleus are involved in the positioning of chromosomes at mitosis.
Finally, methods were developed for visualizing and analyzing isolated mitotic chromosomes in the yeasts, S. pombe and S. cerevisiae. Chromosomes were visualized by adsorbing them to grids and expanding them gently. Both yeast contain chromatin fibers which are on the order of 25 nm. In addition, fibers containing nucleosomes could be observed. Mitotic chromosomes from both yeast were shown to label with antibodies directed against histone H2B and DNA This demonstrates that condensed mitotic chromosomes from both yeast can be isolated. In addition, the similarity of their substructure to those of higher eukaryotic cells indicates that the chromatin folding which they undergo is similar to that which occurs in higher eukaryotic cells at the early levels of folding.
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