Characterization of trypanosomosis resistance QTL, Tir1, in the mouse.

In order to characterise the D17MIT16 BAC DNA clone which is part of a 1200 kilobase DNA insert believed to contain mouse trypanotolerance genes, restriction endonucleases were used to digest the insert for restriction mapping, identification of DNA markers, sub cloning and sequencing. The D17MIT16 BAC DNA was digested with five rare-cutting restriction endonucleases and separated by agarose gel electrophoresis. Southern blot analysis of the resulting gel revealed that there are 3, 3, 3 and 2 restriction sites for the restriction endonucleases, CIa I, Eag I, Mlu I and Sal I respectively, in the 116 kb DNA insert. Fragments obtained from digestion of the BAC DNA with the frequent-cutting restriction endonuclease, EcoR I, were also separated by agarose gel electrophoresis. Southern blot analysis using a (GT)lO ((CA)lO on the complementary strand) microsatellite probe showed the presence of nine (CA)n DNA markers. The EcoR I-digested fragments were also sub cloned in a pUC19 cloning vector. Positive clones, as determined by histochemical identification, were selected for futher analysis. DNA isolated from these positive clones were sequenced using the ABI Prism© Sequencing Kit. Sequence analyses of positive clones using the ABI Prism© 377 Sequence Analysis software revealed the presence of a (GT)16 microsatellite that is probably polymorphic. The Basic Local Alignment Search Tool (BLAST) programme was used to determine the homology of each of the clones to known nucleotide aad.protein sequences. Five of the clones showed >65% homology at the nucleotide level. Four of these showed high homology to known genes. Because of the high homology of clones A7, €12.and C60 to the genes JAK3 and GAG/POL2, the reported involvement of these genes in certain immunological pathways and the fact that they are found in the Tir1 :region of interest suggests that these could be possible candidate trypanotolerance genes