A study of nucleoprotein interactions in mammalian systems

Barclay, Alan B (1972) A study of nucleoprotein interactions in mammalian systems. PhD thesis, University of Glasgow.

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As a first approach to the study of the interactions between the constituent macromolecules of mammalian chromatin, some physicochemical properties of two components of chromatin, GAR histone and DNA, have been characterized. Studies of the self-association of GAR histone by ultracentrifugal techniques have indicated that the histone probably exists in a monomer-dimer equilibrium in 0.075M NaCl/0.01M HCl and in a monomer-dimer-tetramer equilibrium in 0.15M NaCl/0.01M HCl. Gel filtration studies confirmed these results, also indicating that the tetramer formed is of compact shape, similar to a tetrahedron. Preliminary ultracentrifugal studies indicated that the mode of association of GAR histone is independent of pH in the range 2.5 - 7.0. ORD studies of GAR histone in the above conditions suggested that the amount of helix present in the protein was low and was not altered by the self-association of the histone. The heterogeneity of the C. T, DNA also used in these studies was quantitated using the g(s) distribution of sedimentation coefficients. Ultracentrifugal analysis of complexes of GAR histone-C. T. DNA prepared by salt-gradient dialysis and by direct addition of the two components, revealed considerable heterogeneity of the complex with respect to sedimentation coefficient. Thermal denaturation studies did not detect any significant differences in the absorbance melting profile of complexes formed by the direct addition of GAR histone to DNA in conditions in which GAR existed as a monomer and in conditions where GAR probably existed as a monomer-dimer equilibrium. To lessen the difficulties in interpretation of the properties of complexes of GAR histone with heterogeneous C. T. DNA, homogeneous preparations of relaxed circular SV40 DNA and supercoiled SV40 DXA were used. The number of histone molecules bound per molecule of each form of DNA was calculated as a function of the amount of histone added to the DNA preparations and the results were interpreted as indicating co-operative binding. Sedimentation coefficients and sedimentation coefficient distributions of the complexes of GAR with each form of SV40 DNA in-various salt conditions were measured. Comparison of the frictional coefficient and diffusion coefficients of the complexes with those of free SV40 DNA and SV40 virus suggested that the complexes were not as compact as the spherical virus particle.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: R Eason
Keywords: Biochemistry
Date of Award: 1972
Depositing User: Enlighten Team
Unique ID: glathesis:1972-73792
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56
URI: https://theses.gla.ac.uk/id/eprint/73792

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