Integration of exogenous DNA into the trypanosome genome occurs by homologous recombination only. To test whether a high degree of homology between donor and target DNA is required, we have inserted marker genes for drug resistance into the promoter area of variant surface glycoprotein (VSG) gene expression sites of Trypanosoma brucei, using targeting fragments from two expression sites that are 92% identical. We observed integrations into expression sites that are known to be perfectly matched to the donor flanks, and into subsets of uncharacterized expression sites that are specific for each type of targeting fragment, and that could be similar or identical to the donor flanks. This requirement for very high homology was found in both procyclic and bloodstream-form trypanosomes. We speculate that trypanosomes have a mismatch repair system that suppresses recombination between divergent DNA sequences, and we discuss ways in which the trypanosome might circumvent the requirement for perfect DNA homology in the duplicative transposition of a VSG gene into a VSG gene expression site.

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PubMedDate not supplied

Drug resistance and P-glycoprotein gene amplification in the protozoan parasite Leishmania.

Amplification of the H circle is often associated with methotrexate (MTX) selection in Leishmania species. We have shown that the H circle of Leishmania tarentolae contains an open reading frame, ItpgpA, that has the attributes of P-glycoproteins (large plasma membrane proteins known to extrude lipophilic drugs from mammalian cells). H region amplification was also noted in some mutants selected for resistance to arsenite and vinblastine. Mutants having the complete 68-kb circles were cross-resistant to MTX, but two arsenite mutants having only part of the H region amplified, but including ItpgpA, were not cross-resistant to MTX. These results suggest that the putative determinant for MTX resistance present on the H circle is not ItpgpA. We have also determined how ItpgpA-containing plasmids were generated from the chromosomal copy. The H circle contains a 30-kb inverted duplication separated by two unique DNA segments. The corresponding H region of chromosomal DNA has only one copy of the duplicated DNA. We have shown that the two unique segments in chromosomal DNA are flanked by inverted repeats suggesting that H circles could be formed by a foldback mechanism (see fig. 2). Unexpectedly, a plasmid present in cells selected for arsenite resistance lacked part of the H region and the long inverted repeats. It appears to have been formed by intrachromosomal recombination between two P-glycoprotein genes, ItpgpA and ItpgpB, located adjacent to the H region. Our results show that under drug pressure, the same P-glycoprotein-encoding region in Leishmania may be amplified by very different mechanisms and yield different amplicons.

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