FIG 1.

RNA expression analysis of LABCG2 in L. major lines. (a, top) LABCG2 gene expression determined by reverse transcription-PCR, as indicated by the amplified 82-bp ABCG2 fragment. (Bottom) GAPDH gene expression as the internal loading control showing the amplified 227-bp GAPDH fragment. Total RNA was extracted from the pUCNEO line (control), LABCG2 lines, and LABCG2 parasites grown for 15, 30, 60, and 90 days in the absence of G-418 pressure (LABCG2rev 15D, LABCG2rev 30D, LABCG2rev 60D, and LABCG2rev 90D lines, respectively) and then reverse transcribed to single-stranded cDNA by specific priming as described in Materials and Methods. PCR products were electrophoresed on a 4% agarose gel, stained with ethidium bromide, and viewed under a UV illuminator, and the relative intensity was measured against that of GAPDH by using a densitometer. The positions of molecular markers (base pairs) are indicated on the left. (b) Relationship between LABCG2 expression (arbitrary units [a.u.]) and Sb^III susceptibility (EC₅₀ values ± standard deviations from three independent experiments) in L. major promastigote lines. Data from reverse transcription-PCR assays, representative of at least three independent experiments, are shown.