Isolation of Mitochondria and Mitochondrial RNA from Crithidia fasciculata

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Abstract
Two methods were used to isolate mitochondria from C. fasciculata. In the 1st method, cells were weakened by exposure to hypotonic conditions and then disrupted by blending; mitochondria were subsequently isolated using disodium 3,5-diacetoamido-2,4,6-triiodobenzoate gradients. In the 2nd, cells were treated with digitonin before disruption; mitochondria were purified by differential centrifugation. Both preparations were examined by EM and also possess several characteristic biochemical properties of mitochondria. Kinetoplast DNA was present in the mitochondria, uncontaminated by nuclear DNA. Analysis by polyacrylamide gel electrophoresis showed 2 RNA components of MW 0.47 .times. 106 and 0.22 .times. 106, in addition to cytoplasmic RNA contamination. Four mitochondrial components with sedimentation coefficients of 14.6S, 11.4S, 10.1S and 6.9S were identified on sucrose density gradients. Ethidium bromide abolished the incorporation of [5-3H]uridine into the presumed mitochondrial RNA.