Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

A humoral factor elaborated by tumor tissue has been suggested as the etiological agent which causes the systemic effects accompanying malignant disease. Because of the important role of the nucleic acids in the metabolism of the cell, it was postulated that the tumor factor might produce the systemic effects by altering the nucleic acid metabolism of the host tissues. With these considerations in mind, a study has been made of the effect of several transplantable tumors on the incorporation of formate-C¹⁴ and tritiated thymidine into the nucleic acids of the host tissues. Studies on the incorporation of thymidine into the host tissues have been emphasized since this compound is a specific precursor of DNA thymine. Liquid scintillation counting methods were developed in order to assay the radioactivity of the tritium-labelled thymine. Methods for the liquid scintillation counting of carbon-¹⁴ labelled purines and pyrimidines were also established. The presence of the ascitic and subcutaneous forms of the Ehrlich tumor was found to have little effect on the incorporation of formate-C¹⁴ into the nucleic acids of the host tissues. In contrast an increased uptake of tritiated thymidine by the DNA of the host tissues was observed in animals bearing the Ehrlich ascites tumor, Novikoff hepatoma and the Walker 256 carcinosarcoma. This effect was particularly striking in the case of the liver and spleen of animals bearing the Walker 256 tumor. Other investigators have isolated a substance known as toxohormone from tumor tissue which has been shown to produce certain systemic effects similar to those of tumor tissue. It was postulated that the increased incorporation of thymidine into the DNA of the host tissues might be the result of the action of toxohormone. In order to test this hypothesis, the effect of toxohormone on the incorporation of tritiated thymidine into the DNA of rat liver and spleen was studied. Crude toxohormone caused an increased uptake of thymidine by the DNA of spleen, but the results obtained for liver were equivocal. A highly purified fraction of toxohormone was prepared by ion-exchange chromatography and gel filtration. However this fraction had no effect on the incorporation of thymidine into the DNA of rat liver and spleen. These results suggested that tumor tissue might contain a factor which stimulates DNA synthesis and which could be isolated in company with crude toxohormone. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Thesis (Ph. D.)--University of Wisconsin, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The work in this thesis consisted of initial experiments designed to elucidate the role of hormones in a hormonal dependent tumor. Various aspects of nucleic acid synthesis in a hormone dependent tumor in the presence (growing) and absence (regressing) of the hormone were studied. The rates of nucleic acid synthesis were studied in whole animals by injecting radioactive formate and allowing the animal to incorporate radioactivity for various periods of time. Nucleic acids were extracted by PAS, phenol procedure and separated on a MAK column. Labelling of all species of nucleic acid was decreased in regressing tumors. In order to determine whether estrogen is acting directly on cells or at some indirect physiological level; the ability of cells from growing and regressing tumor to synthesize nucleic acids in vitro was determined. Results of experiments with these cell suspensions demonstrate that cells from the regressing tumor had a decreased ability to synthesize nucleic acids relative to growing tumor. The rate of DNA synthesis was decreased somewhat more than RNA. In preliminary experiments the activity of DNA dependent DNA polymerase and RNA polymerase from regressing tumor was compared with the same enzyme in growing tumor. The specific activity of both RNA and DNA polymerase was decreased in the regressing tumor. In target tissue like uterus stimulation with estradiol results in an increased rate of synthesis of several species of RNA. In the tumor system used in these preliminary experiments, stimulation with estrogens has a greater effect on the synthesis of DNA than RNA. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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