T7 RNA polymerase-driven transcription in mitochondria of Leishmania tarentolae and Trypanosoma brucei

The study of RNA editing and other molecular processes in the trypanosome m itochondrion would benefit greatly from the ability to insert and express e xogenous DNA in the organelle. However, even with a method to introduce DNA , the current lack of knowledge about mitochondrial transcription would hin der efforts to obtain expression. To circumvent this problem, Leishmania ta rentolae promastigotes and Trypanosoma brucei procyclic cells have been tra nsfected with bacteriophage T7 RNA polymerase targeted to the mitochondrion . Mitochondria isolated from the transfectants contained active T7 RNA poly merase, as shown by a comigration in density gradients of mitochondrial mar ker enzymes and T7 polymerase activity. A DNA cassette under T7 control was introduced into isolated mitochondria from the transfectants by electropor ation and the DNA was shown to be transcribed. This system should allow the transcription of foreign genes of choice within the mitochondrial matrix e ither in a transient assay using electroporation of DNA into isolated mitoc hondria, or in a stable assay using cells transfected with DNA by the bioli stic gun method. (C) 1999 Elsevier Science B.V. All rights reserved.