Knockout of the glutamate dehydrogenase gene in bloodstream Trypanosoma brucei in culture has no effect on editing of mitochondrial mRNAs

Glutamate dehydrogenase (GDH) was shown previously to bind the 3' oligo[U] tail of the mitochondrial guide RNAs (gRNAs) of Leishmania tarentolae, appa rently in the dinucleotide pocket (Bringaud F, Stripecke R, Frech GC, Freed land S, Turck C, Byrne EM, Simpson L. Mel. Cell. Biol. 1997;17:3915-3923). Bloodstream Trypanosoma brucei cells in culture represent a good system to investigate the genetic effects of knocking out kinetoplastid nuclear genes to test a role in RNA editing, since editing of several mitochondrial gene s occurs but is dispensable for viability (Corell RA, Myler P, Stuart K. Me l. Biochem. Parasitol. 1994;64:65-74 and Stuart K. In: Benne R, editor. RNA editing-the alteration of protein coding sequences of RNA. New York: Ellis Horwood, 1993:25-52). Both GDH alleles of bloodstream T. brucei in culture were replaced by drug resistant markers without any effect on viability. T he ratios of edited to unedited mRNAs for several cryptogenes were assayed by primer extension analysis. The steady state abundances of these edited R NAs were unaffected by the double knockout. This evidence suggests that GDH may not play a role in the editing reaction in bloodstream trypanosomes in culture, but this conclusion is tentative since there could be redundant g enes for any biological function. We employed a double allelic replacement technique to generate a tetracycline inducible conditional expression of an ectopic copy of the deleted gene in bloodstream trypanosomes in culture. W e used this strategy for genes encoding mitochondrial proteins which are no t required during this stage of the life cycle, but as a general strategy i t should be appropriate for generation of conditional null mutants for esse ntial genes as well. (C) 1999 Elsevier Science B.V. All rights reserved.