Doctoral Dissertations

Date of Award

8-1986

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

Donald E. Olins

Committee Members

Francis T. Kenney, Audrey Stevens, Howard Adler

Abstract

Macronuclear soluble chromatin of the hypotrich Euplotes eurystomus was characterized employing a variety of biochemical and biophysical techniques and fractionated to enrich for individual genes as intact chromatin molecules. Macronuclear chromatin is nucleosomal and contains a normal complement of inner his tones, an Hl-like 17 kDa protein which is very sensitive to proteolysis, and a considerable number of non— his tone proteins. The nucleosome repeat length of Euplotes macronuclear chromatin was found to be 186 base pairs. Thermal denaturation and circular dichroism studies revealed that the biophysical properties of macronuclear chromatin resemble those of whole chicken erythrocyte soluble chromatin, with some small differences. These differences were attributed to the increased amounts of nonhistone proteins in macro nuclear chromatin, compared to their paucity in chicken erythocyte chromatin. Electron microscope studies of spread macronuclear chromatin molecules demonstrated a linear relationship between molecule length and number of nucleosomes per chromatin molecule. Two dimensional chromatin/DNA agarose gel electrophoresis established a strict correlation between DNA and chromatin electrophoretic mobility. Hl-depleted macronuclear chromatin resembled "core" chicken erythocyte chromatin in its biophysical properties and lacked the 17 kDa protein band. This 17 kDa band was also absent in mononucleosomes derived from microccocal nuclease digested macronuclear chromatin which was fractionated by two consecutive sucrose density gradient ultracentrifugations. Oligonucleosome fractions from these gradients were very similar in their biophysical properties to whole chicken erythrocyte polynucleosomes.

Macronuclear chromatin, fragments fractionated on isokinetic sucrose density gradients on the basis of their sedimentation properties, were found to be enriched for specific genes in individual fractions. The individual fractions contained intact gene-sized chromatin molecules as evidenced by Southern blot hybridization.

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