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 mitochondrion would benefit greatly from the ability to insert and express exogenous DNA in the organelle. However, even with a method to introduce DNA, the current lack of knowledge about mitochondrial transcription would hinder efforts to obtain expression. To circumvent this problem, Leishmania tarentolae promastigotes and Trypanosoma brucei procyclic cells have been transfected with bacteriophage T7 RNA polymerase targeted to the mitochondrion. Mitochondria isolated from the transfectants contained active T7 RNA polymerase, as shown by a comigration in density gradients of mitochondrial marker enzymes and T7 polymerase activity. A DNA cassette under T7 control was introduced into isolated mitochondria from the transfectants by electroporation and the DNA was shown to be transcribed. This system should allow the transcription of foreign genes of choice within the mitochondrial matrix either in a transient assay using electroporation of DNA into isolated mitochondria, or in a stable assay using cells transfected with DNA by the biolistic gun method.
Estévez
A M
AM
Howard Hughes Medical Institute, UCLA School of Medicine, Los Angeles, CA 90095-1662, USA.
Thiemann
O H
OH
Alfonzo
J D
JD
Simpson
L
L
D000818:Animals; D017123:Bacteriophage T7 / Q000201:enzymology; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000235:genetics; D018274:Electroporation; D018390:Gene Targeting / Q000379:methods*; D007891:Leishmania / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D003576:Electron Transport Complex IV; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases