By using immobilized trypsin, I have been able to
prepare well-defined, stable trypsinized nucleosomes. The
difficulties of lacking of control in the extent of
trypsinization, which were encountered in previous studies
with the use of free trypsin, have been eliminated. The
nucleosomes and oligonucleosomes prepared by immobilized
trypsin are suitable for biochemical and biophysical
studies to analyze the function of the histone N-terminal
regions ("tails"), which are removed by trypsin treatment,
on chromatin structure and stability.
Studies were first conducted using the trypsinized
nucleosome core particles to examine the role of the
histone tails in the stabilization of the nucleosome core
particle. While it was found that these tails have little
effect on either the nucleosome dissociation or the
conformational transition in salt, they play a very
important role in determining thermal stability of the
nucleosome. The differential effects of selective removal
of these tails also provided more insight about histone-DNA
interactions in the nucleosome core particle.
Experiments have also been carried out to investigate
the change in structure and hydration of nucleosome core
particles which may be associated with the salt-dependent
conformational transition. Changes in the tertiary
structures are suggested to be responsible for the salt-dependent
transition.
Roles of the histone tails in determination of
nucleosome positions along specific DNA sequences were
examined by analysis of nucleosome positioning on a
specific eukaryotic gene sequence (Lytechinus Variegatus 5S
rRNA gene) after in vitro nucleosome reconstitution with
native and trypsinized histone octamers. Data obtained
indicate that the histone tails are not required for
nucleosome positioning. Results also seem to restrict the
portions of histones which are responsible for determining
nucleosome positions to the globular regions of (H3/H4)₂
tetramer, and possibly H2B. Studies with different DNA
templates strongly suggest that the most important
determinants of nucleosome positioning are the mechanical
properties (such as bending and flexibility) of the DNA
molecule.
Taking together, it seems that the N-terminal tails of
the histones may play roles in stabilizing both nucleosome
structure and the higher-order structure of chromatin. / Graduation date: 1991
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/37327 |
| Date | 13 July 1990 |
| Creators | Dong, Feng |
| Contributors | van Holde, Kensal E. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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