Simultaneous transient expression assays of the trypanosomatid parasite Leishmania using beta-galactosidase and beta-glucuronidase as reporter enzymes.
We describe a transient transfection protocol for cultured Leishmania major promastigotes, utilizing Escherichia coli genes encoding beta-galactosidase and beta-glucuronidase inserted into an expression vector derived from the dihydrofolate reductase-thymidylate synthase locus. Less than 0.1 pg of either reporter enzyme can be detected with a simple fluorimetric assay, and transfection of 10 micrograms of either reporter construct yields activities at least 100-fold over background. Simultaneous introduction of both constructs showed that the activity of each reporter gene was unaffected by the presence of the other, allowing one reporter construct to serve as a control for experimental variability in test gene constructs containing the second reporter gene. These results show that it is feasible to apply transient expression assays to the identification of cis-acting elements of genes encoding nonabundant mRNAs in the genus Leishmania.
LeBowitz
J H
JH
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Coburn
C M
CM
Beverley
S M
SM
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics; D007700:Kinetics; D007895:Leishmania tropica / Q000235:genetics* / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D013762:Tetrahydrofolate Dehydrogenase; D001616:beta-Galactosidase; D005966:Glucuronidase