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Restriction mapping of mitochondrial DNA from cultured control and ethidium bromide resistant cells and new techniques for the analysis of DNA
Dissertation

Restriction mapping of mitochondrial DNA from cultured control and ethidium bromide resistant cells and new techniques for the analysis of DNA

Kathleen Pirollo
Doctor of Philosophy (Ph.D.), Hahnemann Medical College and Hospital of Philadelphia
Feb 1982
DOI:
https://doi.org/10.17918/00007593
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Pirollo_Kathleen_19824.96 MB
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Abstract

Biological Chemistry
Restriction endonucleases were used to analyze themitochondrial DNA (mt DNA) from the following cultured cells: (1) control BHK cells (BHK(,21)/C(,13)); (2) these same cells transformed by Rous Sarcoma Virus (RSV) (C(,13)/B(,4)) and each of these made resistant to the intercalating dye ethidium bromide (EB); (DIAGRAM, TABLE OR GRAPHIC OMITTED... PLEASE SEE DAI) respectively. The mt DNA from LVG outbred Syrian hamsters was also analyzed and mapped. Comparison of the patterns generated with fifteen different enzymes showed no differences between the mitochondrial genomes of the four cell lines or the parental hamster indicating that in these cells EB resistance is not due to any large alterations in the mitochondrial genome. During these investigations we observed that the restriction of native Syrian hamster mt DNA by various endonucleases was inhibited, to differing degrees, by the presence of increasing concentrations of EB or propidium iodide (PI), which led to the development of a procedure for the study of drug-DNA interactions with native DNA. Enzyme inhibition was found to be more sensitive to the presence of PI than EB. The degree of inhibition was also found to be independent of the type of cut made by the enzyme. We found that the sequences 5'-G-C-3' and 5'-A-T-3' within the enzyme recognition sites were the most sensitive to the presence of the dyes in accordance with published data on the ease of dye binding to dinucleotides. The observation that not all the restriction sites for an enzyme within a DNA molecule appear to have equal sensitivity to the dye facilitates mapping of the mt genome. A modification of the nick translation procedure was developed whereby already restricted DNA was adsorbed onto a nitrocellulose square and then subjected to the normal nick translation procedure. The labeled DNA was then electrophoretically transferred from the nitrocellulose into a 1% agarose slab gel and autoradiographed. The binding of the DNA to the nirtocellulose apparently protects it from degredation into small fragments as is usually the case during nick translation in solution. This procedure was found to be sensitive enough to detect nanogram quantities of DNA with a specific activity of 1 x 10('5) cpm/ug DNA.

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