Detection of hybrid phenotypes in African trypanosomes by high resolution two-dimensional gel electrophoresis.

Abstract

High resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and autoradiography were used to analyze the protein phenotypes of Trypanosoma brucei (T.b. brucei and T.b. gambiense) clones suspected of being hybrids. Procyclic culture forms of parental and suspected hybrid trypanosomes were biosynthetically labeled with [35S]methionine and labeled proteins were resolved by multiple 2D-PAGE (the ISO-DALT system) to allow accurate inter-gel comparisons. Autoradiography of the gels showed that the parental clones had qualitative differences in at least seven sets of spots. Five of these sets represented charge differences and one represented proteins of altered relative molecular mass (Mr) and charge. Autoradiographs of the gels of the putative hybrid trypanosomes showed both forms of the proteins found separately in the parental clones indicating that new, nonparental phenotypes had been generated by transmission of mixed trypanosome clones through tsetse flies. The 2D-PAGE patterns from parasites cultivated for extended periods were identical, showing that the individual cloned parasites were phenotypically stable. The results indicate that analytical 2-D gels can be used to study the phenotypes of "parental" or "hybrid" African trypanosomes without having any previous knowledge of the molecular characteristics of the parasites. In addition, the technique allows an extension of phenotypic analysis to hundreds of different proteins in populations of cloned parasites.