USE OF THE GREEN FLUORESCENT PROTEIN AS A MARKER IN TRANSFECTED LEISHMANIA

We have tested the suitability of the green fluorescent protein (GFP) of Aequorea victoria as a marker for studies of gene expression and pr otein targeting in the trypanosomatid parasite Leishmania. Leishmania promastigotes expressing GFP from episomal pXG vectors showed a bright green fluorescence distributed throughout the cell, readily distingui shable from control parasites. Transfection of a modified GFP gene con taining GC-rich synonymous codons and the S65T mutation (GFP+) yielded a much higher fluorescence. FACS analysis revealed a clear quantitati ve separation between GFP-transfected and control parasites, with pXG- GFP+ transfectants showing fluorescence signals more than 100-fold bac kground. Episomal DNAs could be recovered from small numbers of fixed cells, showing that GFP could be used as a convenient screenable marke r for FACS separations. GFP was fused to the C-terminus of the LPG1 pr otein, which retained its ability to restore LPG expression when expre ssed in the lpg(-) R2D2 mutant of L. donovani. The LPG1(GFP) fusion wa s localized to a region situated between the nucleus and kinetoplast; its pattern was similar to that of LPG2, which is known to be located in the Golgi apparatus. This is notable as LPG1 participates in the bi osynthesis of the glycan core of the LPG GPI anchor, whereas protein G PI anchor biosynthesis occurs in the endoplasmic reticulum. These stud ies suggest that the GFP will be a broadly useful marker in Leishmania .