Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes.
Trypanosomatid protozoan parasites cause several important tropical diseases and have been a fertile ground for the discovery of molecular paradigms such as trans-splicing and RNA editing. Transfection-based methods for the study of these organisms have recently been developed, and we have now designed an expression vector, pX, which contains only 2.3 kilobases of Leishmania DNA and can be stably transfected with high efficiency. Genes encoding Escherichia coli beta-galactosidase or a Leishmania amazonensis protective membrane glycoprotein (GP46A/M-2) were inserted into the pX expression site and transfected into Leishmania major, where they directed the synthesis of high levels of mRNAs formed by 5' and 3' processing events occurring predominantly at the sites used by the normal transcripts. Colony assays and immunoblot analysis showed that both proteins were produced; enzymatically active beta-galactosidase comprised approximately 1% of total protein. Sizes of the GP46A protein synthesized in transfected L. major or L. amazonensis were similar and differed from the predominant L. amazonensis GP46, suggesting that the GP46A gene may encode a variant GP46 family member. Because these vectors function efficiently in pathogenic species of Leishmania, pX will facilitate the genetic analyses of parasite proteins crucial for infectivity as well as the identification of cis-acting elements mediating transcription and replication.
LeBowitz
J H
JH
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Coburn
C M
CM
McMahon-Pratt
D
D
Beverley
S M
SM
D000818:Animals; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors; D007891:Leishmania / Q000235:genetics*; D007895:Leishmania tropica / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics*; D005810:Multigene Family; D010957:Plasmids; D015800:Protozoan Proteins / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D012333:RNA, Messenger; C066248:surface glycoprotein GP46-M-2, Leishmania; D013762:Tetrahydrofolate Dehydrogenase; D001616:beta-Galactosidase