Stable and unstable DNA- 3H in Entamoeba histolytica grown with Bacteroides sp.

Abstract

Entamoeba histolytica strain K9, grown with non-multiplying Bacteroides sp. was incubated with thymidine-methyl- 3H for 72 hours. The distribution of tritium in the amebae was then followed by liquid-scintillation spectrometry, after resuspension in unlabeled medium over a 72-hour period. An approximate 40% reduction in total acid-insoluble tritium (DNA) occurred within 24 hours; a further reduction in DNA- 3H over the next 48 hours was small. This initial drop in high molecular weight tritium was probably a reflection of the unstable cytoplasmic DNA fraction in amebae (much of which is of bacterial origin) detected previously by autoradiography. Mitomycin C (MC), at a concentration which partially inhibits growth of amebae (0.0002%), was without significant effect on the loss of such acid-insoluble tritium over a 72 hour period. This indicates that this concentration of MC does not degrade DNA. Further evidence that MC does not degrade DNA was revealed by the presence of a low activity, comparable with that of control cells, in the low molecular weight (acid-soluble) fraction of cells treated with MC. This same concentration of MC does result in a considerable increase in loss of tritium in thymidine-methyl- 3H-prelabeled Bacteroides, the associate cell. However, since this is a nonpropagating population, to begin with, it is not clear whether this is the result of the effect of MC directly on DNA, or a secondary result, due to disintegrating cells.