Characterization of a Leishmania donovani gene encoding a protein that closely resembles a type IB topoisomerase

In order to clone the gene encoding a type I DNA topoisomerase from Leishma nia donovani, a PCR-amplified DNA fragment obtained with degenerate oligode oxyribonucleotides was used to screen a genomic library from this parasite. An open reading frame of 1905 bases encoding a putative protein of 635 ami no acid residues was isolated, A substantial part of the protein shares a s ignificant degree of homology with the sequence of other known members of t he IB topoisomerase family, in a highly conserved region of these enzymes t ermed the core domain. However, homology is completely lost after this cons erved central core. Moreover, no conventional active tyrosine site could be identified. In fact, the protein expressed in Escherichia coil did not sho w any relaxation activity in vitro and was unable to complement a mutant de ficient in topoisomerase I activity. The results of Southern blot experimen ts strongly suggested that the cloned gene was not a pseudogene, Northern a nalysis revealed that the gene was transcribed in its full length and also excluded the possibility that some form of splicing is necessary to produce a mature messenger. Furthermore, our results indicate that the gene is pre ferentially expressed in actively growing L.donovani promastigotes and that it is also expressed in other kinetoplastid parasites.