Date of Award

Summer 1986

Degree Type

Thesis - Restricted

Degree Name

Master of Science (MS)

Department

Biology

Abstract

Regulation of gene expression occurs at several levels, including chromatin structure, transcription, RNA processing, translation and post translational modification. The roles of these various forms of regulation in particular physiological changes is not well understood in eukaryotes. The aim of these research projects was to explore certain of these levels of regulation and their dependence on circadian rhythms. The first studies investigated the sensitivity of chromatin to DNase 1 by use of "in situ nick translation," an enzymatic procedure that theoretically yields radioactive labelling of actively expressed DNA sequences. In an attempt to purify these DNA sequences the chromatin was nick translated with biotin labelled nucleoside and these labelled sequences were isolated by affinity chromatography. This method yielded an enrichment for sequences in "active" conformation, i.e., those sequences preferentially nicked by DNase 1; however, the purification was not complete, nor was it adequate for further studies. The second set of experiments studied the regulation of a2~globulin at the RNA level. Alpha 2-microglobulin is a protein synthesized in the liver of adult male rats and secreted into the plasma. At various phases of the circadian cycle, total and polysomal RNA was isolated from adult rat liver, transferred to nitrocellulose and assayed using a cloned eDNA probe. These data demonstrated that a2~-globulin regulation is dependent on a circadian rhythm. Polysome bound mRNA (translationally active), but not total cellular mRNA, underwent a marked circadia11 variation at the translational level. Alpha 2-microglobulin is one of the few proteins for which the circadian regulatory mechanisms have been studied at the nucleic acid level. These data demonstrated an exciting and relatively new technique for studying circadian dependence of many proteins at the molecular level in an effort to understand the various mechanisms ·responsible for regulation of gene expression.

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