Sequence Elements Required for the Insertion of Base J into DNA in Leishmania

Flagellar kinetoplastid parasites such as trypanosomes and Leishmania cause diseases that include Chagas, African sleeping sickness, and leishmaniasis. These deadly parasites have an unusual DNA modification known as base J (glycosylated thymine). The absence of base J causes RNA polymerase readthrough and produces anti-sense RNA. The cells cannot live with large loss of J. The DNA sequence elements needed for the insertion of J are yet to be determined. The strategy for finding the sequences requirements for the insertion of J was creating plasmids, growing the plasmids in Leishmania, determining if J was added, and then shortening and/or mutating the sequences of the insert. Plasmid constructs were made using InFusion cloning followed by transformation into E. coli. The plasmids will be transfected into Leishmania cells and then the plasmids will be isolated. DNA immunoblot and SMRT sequencing will be used to determine the location of J. In this project, the vector fragment and insert fragment were prepared for InFusion cloning. In addition, plasmids were isolated from Leishmania cells.