Fig 1. Generation of mismatch repair null mutants in T. cruzi and T. brucei.

(A) Agarose gel electrophoresis of reverse transcriptase (RT) PCR products to verify the absence of MSH2 or MLH1 mRNA in T. brucei procyclic form mutants. cDNA derived from wild type (WT) cells, blasticidin (BSD) or puromycin (PUR) resistant clones of Tbmsh2 single allele knockouts (+/-) and Tbmsh2 double allele knockouts (-/-) were PCR-amplified with MSH2 or MLH1- specific primers; the size of the PCR product is indicated and a control reaction without cDNA is indicated by C. The bottom panel shows, as positive controls, cDNAs from the same cells PCR-amplified (RT+) with primers specific for the RAD51 gene; a control for genomic DNA contamination, in which reverse transcriptase was excluded from the cDNA synthesis reaction (indicated by RT-) is also shown. (B) Total RNA extracted from T. cruzi epimastigote form wild type (WT) cells, a Tcmsh2 single allele knockout (+/-) mutant and three independent clones of Tcmsh2 double allele knockouts (-/-) were transferred to a nylon membrane and hybridized with [α-³²P]-labeled probe specific for the T. cruzi MSH2 gene. The bottom panel shows hybridization with a probe for 24Sα rRNA, used as loading control.