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|Title:||Chromatin Structure of Schizosaccharomyces Pombe: From the Lowest to the Highest Orders of Folding|
|Author(s):||Godde, James Scott|
|Doctoral Committee Chair(s):||Widom, Jonathan|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||I have used new methods for chromatin isolation, together with conventional methods for measuring the nucleosome repeat length, to determine the repeat length of Schizosaccharomyces pombe chromatin. I obtained a result of 156 $\pm$ 2 bp. Equivalent results are obtained using a psoralen crosslinking method for measuring the repeat length in viable spheroplasts. That result, together with other control experiments, rules out many possible artifacts. The measured value of 156 $\pm$ 2 bp is smaller than the length of DNA found in the chromatosome. Thus, the chromatosome cannot be the fundamental unit of chromatin structure in all eukaryotes. The cross-linker model of chromatin higher-order structure is incompatible with a nucleosome repeat length of 156 bp, and thus cannot apply to all eukaryotes. The solenoid model of higher order structure is compatible with this repeat length only if the solenoid is right-handed. I note two other properties of this chromatin. (1) Early in digestion, the DNA length of mononucleosomes from S. pombe and A. nidulans exceeds the nucleosome repeat length. (2) Many methods for isolating chromatin from S. pombe yield an apparent nucleosome repeat length of $\sim$140 bp; this result is found to be an artifactual consequence of nucleosome sliding.
I attempted to characterize histone H1 in S. pombe using a biochemical approach; this met with little succes. Sequences obtained from purified proteins which share many characteristics with H1 revealed homology to a known ribosomal protein and to an unknown protein. The latter bears no resemblance to H1 at the level of primary sequence but has limited homologies to various nucleic acid binding proteins.
I have also isolated mitotic chromosomes from S. pombe. These chromosomes were studied using both transmission and scanning electron microscopy and appear as condensed structures in the size range of 0.7-2.5 $\mu$m wide by 1.4-3.9 $\mu$m in length often surrounded by a periphery of filamentous material. I have verified that these chromosomes are made up of nucleosomal subunits by dispersing the samples with a low ionic strength buffer. Immunoelectron microscopy reveals the presence of histone H2B and DNA in the periphery of these isolated chromosomes. Chromosomes dispersed to intermediate extents enabled the visualization of higher levels of structure, namely fibers about 23-26 nm in diameter. Thin sectioning of these chromosomes after embedding in resin enables more fibers in this size range to be visualized. SEM reveals surface structure consisting of protrusions in various size ranges, including 25-35 nm and 110-133 nm.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.
|Date Available in IDEALS:||2014-12-17|