STABLE TRANSFORMATION OF TRYPANOSOMA-BRUCEI

We have further analyzed parameters affecting stable transformation of Trypanosoma brucei. Linear DNA was much more efficient than circular DNA and in the vast majority of transformants analyzed the plasmid DNA had inserted into the chromosomes by homologous recombination. The pr esence of non-homologous (vector) DNA at one or both ends of linear co nstructs inhibited transformation efficiency. Less than 1 kb of homolo gous flanking sequence was sufficient for efficient targeting of a mar ker gene into the tubulin gene array. When transformants with a single neomycin phosphotransferase (neo(r)) gene replacing a beta-tubulin ge ne were selected for higher levels of G418 resistance, the neo(r) gene was amplified and spread through the tubulin gene cluster. The additi onal neo(r) gene copies were adjacent in the tubulin gene array and we re added to the array rather than replacing beta-tubulin genes. These results are compatible with asymmetric post-replication recombination (unequal sister chromatid exchange) as the mechanism for neo(r) gene a mplification. Starting with a circular construct containing the neo(r) gene between tubulin intergenic regions, we obtained a single transfo rmant that maintained the neo(r) genes as an extrachromosomal plasmid. We show this plasmid to consist of a circular pentamer of the input c onstruct. All other attempts to derive a shuttle vector that replicate s extrachromosomally in T. brucei were unsuccessful. Our experiments e xtend previous observations suggesting that T. brucei has a strong pre ference for chromosomal insertion of exogenous DNA by homologous recom bination.