DNA polymerase beta mRNA determination by relative quantitative RT-PCR from Leishmania infantum intracellular amastigotes

Abstract.

Gene expression level is extremely difficult to assess in the intracellular form of Leishmania infantum, the amastigote, due to host mRNA contamination, low supply of parasites and stress degradation problems. We obtained and analyzed L. infantum DNA polymerase β (Li Pol β) , a suitable enzyme for differential expression studies. The amount of Li Pol β mRNA was determined in different forms of the parasite (extracellular promastigote, intracellular amastigote) by Northern blot and by relative quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Amastigote transcript levels were determined from both gradient-purified parasites and directly from a population of infected macrophages. The mRNA, undetectable when obtained from amastigotes by the classic gradient centrifugation method and subsequent Northern analysis, was clearly and specifically detectable by this quantitative RT-PCR method from the mixed macrophage/parasite cell population. Li Pol β displays a different pattern of expression in the distinct forms of the parasite cycle that correlate with the infectivity of the protozoon. Thus, Li Pol β mRNA expression is developmentally regulated, being clearly higher in the more infective forms of the parasite.