UBC Theses and Dissertations
Studies on histone modification and chromatin structure in developing trout testi Honda, Barry M.
During spermatogenesis in rainbow trout, a synchronous development of stem cells → spermatocytes → spermatids → mature sperm occurs, with replacement of the histones in chromatin by protamines. PART A: Histone methylation. Along with histone acetylation and phosphorylation, methylation of specific lysyl residues of histones H3 and H4 can be observed. This histone methylation occurs predominantly in the large diploid stem cells and primary spermatocytes, which actively synthesize DNA and histones. In spermatids, histone methylation is minimal and so probably has no role in the replacement of histones by protamine. Other labelling experiments suggest that histone H4 methylation is a late event in the cell cycle, occurring after the synthesis, acetylation and deacetylation of histone H4. This methylation may be necessary for histone phosphorylation or chromatin condensation prior to cell division. PART B: Chromatin subunit structure. When a sample of trout testis nuclei is digested with micrococcal nuclease, the DNA is cleaved almost entirely to discrete fragments approximately 200 base pairs long and multiples thereof. The same DNA fragments can be obtained when isolated chromatin, as opposed to intact nuclei, is nuclease digested. These DNA fragments can also be found in discrete chromatin "subunits" isolated from nuclease-digested nuclei. Sedimentation through sucrose gradients, or velocity sedimentation in an analytical ultracentrifuge separates these chromatin subunits into 11S (monomer), 16S (dimer), 22S (trimer) etc. species. Subunits can also be fractionated on a Sepharose 2B column equilibrated and run in low salt. High salt (>40 mM NaCl) or divalent cations (≃5 mM) cause subunit precipitation. Chromatin subunits have a protein:DNA ratio of approximately 1.2 and contain all the histones, including the trout-specific histone H6. There are however no detectable nonhistone chromosomal proteins. Mg⁺⁺ precipitates of the 11S chromatin monomers, when pelleted, are thin and clear, while oligomer Mg⁺⁺ pellets are thick and white. This could reflect a more symmetrical or ordered packing of 11S monomers, which are deficient in histone Hi. This histone may crosslink the larger oligomers, resulting in a disordered Mg⁺⁺ complex. These results are consistent with the subunit model of chromatin structure, based on 200 base pair long regions of DNA associated with histones. These subunits would be separated by nuclease-sensitive DNA spacer regions, and crosslinked by histone Hi. Testis consisting predominantly of early spermatids (meiotic tissue, containing mainly nucleohistone) gives similar yields of DNA fragments and 11S subunits. Later stage testis (protamine has replaced the histones) however, gives no DNA fragments or 11S subunits. This presumably reflects large differences in structure between nucleoprotamine and nucleohistone.
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