Detection of P-4 and GP-46 expression in Leishmania amazonensis amastigotes and promastigotes by RT-PCR

OBJECTIVE: To detect the expression level of stage-specific genes in Leishmania promastigotes and amastigotes. METHODS: Total RNAs were isolated from Leishmania amazonensis stationary promastigotes and three sources of amastigotes: freshly obtained from mouse skin lesions, infected J774.G8 macrophages, and transformed from the cultured promastigotes. mRNAs were conversely transcribed into cDNA with SuperScripII reverse transcriptase and oligo dT primers. The polymerase chain reaction (PCR) was used to amplify the specific fragments of amastigote-specific nuclease (P-4) and promastigote-specific membrane glycoprotein (GP-46). PCR products were analyzed in 1.5% agarose gel. RESULTS: A P-4-specific band (273 bp) was observed in all three types of amastigotes with similar density, but it was almost undetectable in promastigotes. In contract, a GP-46-specific band (325 bp) was expressed at a higher level in promastigotes than in all three types of amastigotes. CONCLUSION: Promastigote-derived amastigotes express high level of P-4-specific gene and can be used as a source of amastigotes for biochemical and immunological studies.