Isolation of the kinetoplast DNA of Leishmania tarentolae in the form of a network.

Abstract

SYNOPSISThe major kinetoplast DNA complement of Leishmania tarentolae promastigotes has been isolated as a single sheet of interconnected molecules on the basis of its relative stability to shear forces and its high sedimentation coefficient. Two successive differential centrifugations were sufficient to recover 50 ± 10% of the total kinetoplast DNA free of nuclear DNA contamination. We use the term “network” to describe this unusual type of DNA configuration.Leishmania networks have a molecular weight of ∼1010 daltons and an S20,W in neutral sucrose gradients of 1729 7plusmn; 189 [n = 19] and exhibit an extremely low specific viscosity due to the compactness of packing of the DNA. The networks were visualized in the electron microscope, and in the light microscope either by fluorescence in solution after staining with acridine orange or in dried smears after staining with Giemsa.Purified networks from stationary phase cells banded in the position characteristic of closed monomeric minicircles in ethidum bromide‐CsCl equilibrium gradients, and were stable in alkaline sucrose. Treatment of the closed networks with RNase and pronase had no effect on the ethidium bromide‐CsCl banding pattern. However, treatment of closed networks with DNase I or II, X‐irradiation or γ‐irradiation changed the banding pattern by introducing single strand and double strand breaks, yielding an upper band and in some cases a intermediate band.