Graduate Thesis Or Dissertation
 

The association of DNA with other cellular materials in Escherichia coli lysates

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  • The association of other materials with DNA has been studied extensively in higher organisms and more recently in bacteria. However, investigation of these complexes in bacteria have for the most part been restricted to the study of a specific function this association may serve, or a specific kind of association. The degree of protein, RNA and membrane association of DNA within a given species has not been compared at one time under the same experimental conditions. These studies were undertaken in order to compare DNA associated with protein, RNA and cell membrane fractions in a single bacterial system. Escherichia coli strain CR34 (requiring thymine, leucine, threonine, and B₁) or strain TAU bar (requiring thymine, arginine, uracil, methionine, proline and tryptophan) were grown 5-5.5 hours in media containing ³H thymidine, ¹⁴C uracil, ³H tryptophan or ¹⁴C arginine, harvested, washed three times with buffer and lysed with 1 percent sodium lauryl sulfate (SLS) or sarkosyl. Untreated, sonicated, RNAse and pronase treated lysates were then subjected to starch block electrophoresis or CsC1 density gradient analysis. After electrophoresis of a ³H thymidine labeled SLS lysate, about 30 percent of the DNA was found at the origin. This association with materials of low electrophoretic mobility was not found when phenol-extracted, ³H labeled DNA was added to cells prior to lysis and electrophoresis. In electrophoresis of sonicated lysates virtually none of the DNA was found at the origin, while over 80 percent of the DNA appeared 20 or more cm from the origin, showing a distribution very similar to that of unsonicated, phenol-extracted DNA. These results indicated that DNA association with materials of low electrophoretic mobility occurs in the cell prior to lysis and that sonication will remove DNA from this complex. Treatment of unsonicated SLS lysates with pronase prior to electrophoresis decreased by about one fifth the amount of DNA retained at the origin. Similar treatment with RNAse reduced the amount of DNA remaining at the origin by about one half. Electrophoretic profiles for ³H tryptophan and ¹⁴C arginine labeled lysates were similar, with most of the radioactivity at or near the origin. Sonication did not release radioactivity to faster migrating fractions. Electrophoresis of cell lysates labeled with ¹⁴C uracil showed that only 4 percent of the ¹⁴C label was found at the origin. From these findings it seemed that about 30 percent of the DNA was bound to materials of low electrophoretic mobility, in conjunction with protein and a minor amount of the cells' RNA. Enzymatic destruction of protein or RNA affected the amount of DNA bound to this complex and thus either may function as a linker of DNA to substances of low electrophoretic mobility, possibly the cell membrane. CsC1 density gradient analysis of ³H thymidine-labeled lysates showed that most of the DNA was associated with a mucous film of light density floating on top of the gradient. From this appearance and the reports of others, this pellet was identified as the cell membrane fraction. If the lysate was sonicated before centrifugation, most of the DNA was not found in the floating pellet, but was distributed in a broad band throughout the center of the gradient. Pooling and recentrifugation of widely separated portions of this broad density band resulted in a single peak, indicating all portions to be of similar density. Treatment of a ³H thymidine- labeled SLS lysate with pronase before centrifugation reduced the amount of label in the floating pellet by 10 percent. Similar treatment with RNAse reduced the total radioactivity in the floating pellet by 4 percent. After centrifugation of a ³H tryptophan or ¹⁴C arginine-labeled lysate, virtually all of the radioactivity was found in the top few fractions and sonication of a lysate before centrifugation did not change this. After centrifugation of a ¹⁴C uracil-labeled lysate, ¹⁴C label was found in all portions of the tube, with about 50 percent of the label in the floating pellet. Sonication of a lysate prior to centrifugation reduced by 10 percent the ¹⁴C in the floating pellet. Paper chromatography of hydrolysed top, intermediate and bottom fractions from a density gradient of a sonicated, ¹⁴C uracil labeled lysate revealed the ¹⁴C label to be mostly in RNA in the top and bottom fractions, and to be largely in DNA, by the conversion of ¹⁴C uracil to ¹⁴C cytosine, in the intermediate fractions. A small amount of ¹⁴C uracil was found in fractions of intermediate density, probably indicating RNA-DNA hybrids. Similar studies showed the floating pellet to contain a greater percent of RNA which may be released by sonication to the positions of intermediate density than does the lysate as a whole. Thus the membrane fraction may be a preferential location of RNA-DNA hybrids.
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