UBC Theses and Dissertations
The biosynthesis of deoxyribonucleic acid in vivo in the intestinal mucosa of rat Mezei, Catherine
The in vivo biosynthesis of deoxyribonucleic acid (DNA) from labelled thymidine has been investigated in rat intestinal mucosa. The DNA preparations were fractionated by column chromatography and the fractions were assayed for radioactivity by liquid scintillation counting methods. In the first experiments the DNA was isolated from the intestinal mucosa of rats which had received H³-thymidine 5, 10 or 20 minutes or 24 hours before sacrifice. When the macromolecules were fractionated on ECTEOLA-cellulose the results obtained were inconclusive because no definits pattern of incorporation of radioactivity was observed in the fractions. Chromatography on ECTEOLA-cellulose was considered unsatisfactory, because of the variations in the elution patterns of DNA preparations from experiment to experiment and evidence indicating degradation of DNA during the fractionation procedure. In subsequent experiments fractionation on methylated albumin-kieselguhr (MAK) columns was employed and double labelling experiments were carried out. The animals were injected intravenously with H³-thymidine and 24 hours later with C¹⁴-thymidine. The rats were killed 20 or 40 minutes after the second injection and the double labelled DNA was isolated from the intestinal mucosa. On fractionation by MAK columns reproducible elution patterns were obtained even after storage of the DNA solutions. The main DNA peak was always eluted at the same range of sodium chloride concentration and 95-97 percent of the radioactivity was eluted in this peak. Each subfraction comprising the main peak was examined for H³ and C¹⁴ activity. By studying the H³/C¹⁴ ratios of the fractions newly synthesized material could be compared with older, presumably stabilized DNA. When the animals were exposed to the C¹⁴-labelled thymidine for 40 minutes the H³/C¹⁴ ratios of the subfractions were constant, indicating no metabolic differences between the newly synthesized DNA (C¹⁴-labelled and the "old" (H³-labelled) DNA. However, when the time of exposure to the C¹⁴-labelled precursor in vivo was 20 minutes, the H³ /C¹⁴ ratios of subtractions increased as the sodium chloride concentration of the eluant increased. These results indicated some metabolic differences amongst these fractions. Stepwise enzymatic degradation by snake venom phosphodiesterase of the double labelled DNA preparations, and the main peak obtained after MAK chromatography, indicated the incorporation of thymidine into newly synthesized and "old" DNA occurred well within the chain.
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