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Studies on DNA and DNA polymerases from the intestinal mucosa of rat

University of British Columbia

PART I - The base compositions of deoxyribonucleic acid, isolated from whole cells, nuclei, and mitochondria of rat intestinal mucosa were compared. DNA from whole cells or nuclei was fractionated by column chromatography on methylated albumin kieselguhr, MAK. The guanine plus cytosine content of these DNA fractions was determined mainly by the method of heat denaturation, although the methods of acid hydrolysis and equilibrium centrifugation in cesium chloride solutions were also used. The mole % G+C for the DNA fractions from whole cellular extracts ranged between 34.39 and 52.92, while the nuclear DNA fractions showed a range between 37.31 and 50.97. Although the main DNA band which was eluted with 0.6 M NaCl solution had separated into two or three peaks, the detection of a major base compositional class of DNA was not evident. Unfractionated DNA from whole cells or nuclei has a G+C content of 42.2 which corresponds to a midpoint of thermal denaturation, Tm of 86.6° C. DNA isolated from the mitochondria of intestinal mucosa was observed to have a Tm of approximately 85.0° C and a density of 1.702 g/cm³. This density corresponded to the value determined for unfractionated DNA from the whole mucosa cells. PART II - In initial experiments, DNA polymerase from Escherichia coli was isolated and partially purified by treatment with streptomycin, ammonium sulfate, and by chromatography on DEAE-cellulose. After these introductory experiments, DNA polymerases from the small intestinal mucosa of the rat were studied. Using suitable assay systems with ¹⁴C-2-dTTP or ¹⁴C-8-dATP, both a replicative and a terminal DNA nucleotidyl-transferase were detected in extracts of nuclei. The replicative enzyme incorporated a labeled precursor into a native or heat denatured DNA primer in the presence of all four complementary triphosphates. The terminal enzyme preferentially incorporated single deoxyribonucleoside triphosphates onto the terminal position of heat denatured DNA primers. Treatment of the DNA products formed in the terminal addition reaction with snake venom phosphodiesterase indicated that the labeled precursors were added to 3'-hydroxy terminal positions of the chains. A heterogeneous nature of the DNA polymerases from rat intestinal mucosa was indicated by the appearance of three fractions of enzyme activity following DEAE-cellulose chromatography. A distinct peak of terminal-addition enzyme activity was detected by rechromatography on DEAE-cellulose. Gel filtration through Sephadex G-150 or G-200 and sucrose density gradient centrifugation showed that these DNA polymerases varied in molecular sizes. The molecular weights of the DNA polymerase fractions were estimated to be between 2.5 x 10⁴ and 3 x 10⁵ by comparisons with marker proteins.

Text

2011-08-19

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http://hdl.handle.net/2429/36776

Biochemistry and Molecular Biology

University of British Columbia

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