Chromosomal Protein-DNA Interactions

Author: Murphy, Robert Francis

Year: 1980

Degree: Dissertation (Ph.D.)

Advisor: Bonner, James Frederick

Committee Members: Bonner, James Frederick; Mitchell, Herschel K.; Lazarides, Elias; Strauss, James H.; Richards, John H.

Option: Biochemistry

DOI: 10.7907/3ecg-z764

Abstract

The nature and function of some of the protein-DNA interactions in eukaryotic chromatin were investigated. The nucleosome structure of isolated template-active chromatin was determined. In vitro chemical acetylation of chromatin was shown to result in a structure similar to that of deproteinized DNA, which represents a shift towards the properties of isolated template active chromatin, and chromatin containing specific transcribed genes.

The process of chromatin replication was shown to include a shortening of the internucleosomal spacer, resulting in decreased nuclease sensitivity. Newly-replicated chromatin was separated from bulk chromatin in shallow metrizamide density gradients. Newly-synthesized histone and newly-acetylated protein were shown to be present predominantly in the unreplicated chromatin fraction.

The accuracy and reproducibility of non-linear least squares determinations of the thermal denaturation transitions of DNA and chromatin were determined using computer programs designed for ease of use and adaptability to mini-computer configurations. Direct fitting of melt data to a normalized error function gave results very similar to those obtained by fitting Gaussian curves to derivatized data. This approach avoids errors introduced by the derivatization method, and requires fewer data points.

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