The in vivo synthesis of deoxyribonucleic acid from labeled precursors was studied in the rat intestinal mucosa in an attempt to elucidate the complex process of DNA replication. In one set of experiments, the rats were injected with ³H-thymidine and then starved for 24 hours, in which time the stable DNA became labelled with tritium. (14)C-thymidine was then administered and the animals were sacrificed 5 minutes later. By this procedure
the newly synthesized DNA was labelled with (14)C.
The DNA, was fractionated by chromatography on a methylated-albumin kieselguhr column. Only one main peak of DNA was eluted with a sodium chloride solution ranging in concentration from 0.5-0.6 M. The thermal denaturation temperature for the DNA in each.fraction from this peak was determined and the G + C content was calculated:, Within the DNA peak obtained from MAK chromatography, the G + C content of the DNA decreased with increasing fraction number.
In addition to these differences in base composition, there were differences in metabolic activity between the fractions, which were indicated by their ³H/ (14)C ratios. The ³H/ (14)C ratio of the DNA fractions from MAK chromatography increased with
fraction number to a maximum at fraction 4 or 5 and then decreased. It was found that the ³H/O.D. ratio of the fractions was not constant, thus suggesting that the tritium might be unevenly distributed throughout the fractions. If the time interval between the ³H and (14)C-thymidine injections was reduced to 3 ½ hours, the ³H/O.D. ratio became constant while the pattern of the ³H/14C ratios remained unchanged. If (14)C-thymidine was administered 20 minutes before the animals were sacrificed, the ³H/(14)C ratio of the DNA fractions from MAK chromatography increased with increasing fraction number. From these results it was concluded that small molecular weight, newly synthesized DNA, which was highly labelled with (14)C, was being incorporated with time into the high molecular weight, stable DNA fraction, which is labelled with ³H.
During these experiments it was observed that the pattern of ³H/(14)C ratio versus fraction number varied according to the treatment given to the DNA sample prior to the preparation for radioactive counting. If the sample was denatured by heating to obtain its T(M) value, and then dialyzed against distilled water, small molecular weight nucleotides passed into the dialysate.
The denatured DNA sample also gave different results from the native DNA sample on digestion with snake venom phosphodiesterase. On the denatured sample, the pattern of release of ³H and (14)C labelled material into the acid soluble material, indicated that both these labels were uniformly distributed along the DNA chain. On the other hand, with the native 5 min. DNA samples, the release of (14)C labelled material into the acid
soluble fraction was that expected for DNA which had incorporated (14)C-preferentially into the 3’ terminal positions.
The separation of the pyrimidine clusters of DNA indicated that those were not uniformly labelled with (14)C and ³H. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/34687 |
| Date | January 1969 |
| Creators | Flanagan, Mary Louise |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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