Gene disruption of the DNA topoisomerase IB small subunit induces a non-viable phenotype in the hemoflagellate Leishmania major
The unusual heterodimeric leishmanial DNA topoisomerase IB consists of a large subunit containing the phylogenetically conserved "core" domain, and a small subunit harboring the C-terminal region with the characteristic tyrosine residue in the active site. RNAi silencing of any of both protomers induces a non-viable phenotype in the hemoflagelate Trypanosoma brucei (Bakshi and Shapiro, Mol.
Biochem. Parasitol.136: 249-255; 2004).
Unfortunately, this approach is not suitable is Leishmania where gene replacement with an antibiotic marker is the only approach to generate lack-of-function mutants. We have successfully replaced both topS loci with two selection markers each conferring resistance to hygromycin B and puromycin, respectively.
However, to achieve the second transfection round, we have had to rescue the null-homozygous with an episomal vector carrying the Leishmania major topS gene. This result evidences the essentiality of this gene in L.
major. Phenotypic characterization of the L.
major rescued strain and a L. major strain, which co-overexpresses both subunits, shows few differences in DNA relaxation and CPT cytotoxicity when it was compared to the WT strain.
Studies on phosphatidylserine externalization show a poor incidence of CPT-induced programmed cell death in L. major, but an effective cell-cycle arrest occurs within the first 24 h.
S-Phase delay and G2/M reversible arrest was the main outcome at lower concentrations, but irreversible G2 arrest was detected at higher CPT pressure. Reversibility of the CPT effect points to the existence of effective checkpoint mechanisms in Leishmania parasites.
Author: Rafael Balana-Fouce, Carlos Garcia-Estrada, Yolanda Perez-Pertejo and Rosa M Reguera
Credits/Source: BMC Microbiology 2008, 8:113
Published on: 2008-07-08
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