This work presents the development of a new highly sensitive and selective chemical assay for poly(ADP-ribose) which is routinely useful for the determination of polymer levels in vivo. This method was used to carefully measure poly(ADP-ribose) levels in normal and in DNA-damaged cells. The results of these studies strongly suggest that synthesis of poly(ADP-ribose) is involved in some aspect of DNA repair. A review of the literature is presented in the
introduction of this work. Poly(ADP-ribose) synthesis has
been implicated in aspects of transcription, in DNA syn
thesis, and in DNA repair largely based on evidence from in
vitro studies. It is apparent that current methodology has
not allowed the routine quantification of poly(ADP-ribose) in vivo, hence the lack of i^n vivo data concerning the function(s) of the polymer. The body of this work presents the development of two chemical methods for the quantification of poly(ADP-ribose) and the application of one of these methods to the measurement of polymer levels in normal and DNA-damaged cells. Preliminary studies are presented on the utilization of combined gas chromatography/mass spectroscopy for the selective quantification of nucleoside derivatives. A second method makes use of the unique chemistry of the polymer for quantification. The polymer was selectively adsorbed to dihydroxyboryl-sepharose which allowed the removal of most RNA, DNA, and protein from the samples. The polymer was hydrolyzed to the unique nucleoside 2'—^-l*'-ribosyladenosine by digestion with venom phosphodiesterase and bacterial alkaline phosphatase. The 1-N^-etheno derivative of ribosyladenosine was formed by reaction with chloroacetaldehyde and this derivative was seperated from other fluorescent species by reversed phase high pressure liquid chromatography.
| Identifer | oai:union.ndltd.org:unt.edu/info:ark/67531/metadc330896 |
| Date | 12 1900 |
| Creators | Sims, James L. |
| Contributors | Jacobson, Myron, Gracy, Robert W., Norton, S. J., Jones, Paul R. |
| Publisher | North Texas State University |
| Source Sets | University of North Texas |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation |
| Format | vii, 145 leaves : ill., Text |
| Rights | Public, Sims, James L., Copyright, Copyright is held by the author, unless otherwise noted. All rights reserved. |
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