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 protein targeting in the trypanosomatid parasite Leishmania. Leishmania promastigotes expressing GFP from episomal pXG vectors showed a bright green fluorescence distributed throughout the cell, readily distinguishable from control parasites. Transfection of a modified GFP gene containing GC-rich synonymous codons and the S65T mutation (GFP+) yielded a much higher fluorescence. FACS analysis revealed a clear quantitative separation between GFP-transfected and control parasites, with pXG-GFP+ transfectants showing fluorescence signals more than 100-fold background. Episomal DNAs could be recovered from small numbers of fixed cells, showing that GFP could be used as a convenient screenable marker for FACS separations. GFP was fused to the C-terminus of the LPG1 protein, which retained its ability to restore LPG expression when expressed in the lpg- R2D2 mutant of L. donovani. The LPG1(GFP) fusion was 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 biosynthesis of the glycan core of the LPG GPI anchor, whereas protein GPI anchor biosynthesis occurs in the endoplasmic reticulum. These studies suggest that the GFP will be a broadly useful marker in Leishmania.
Ha
D S
DS
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Schwarz
J K
JK
Turco
S J
SJ
Beverley
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005434:Flow Cytometry; D005819:Genetic Markers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D018320:Leishmania major / Q000166:cytology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D014162:Transfection
D005819:Genetic Markers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins