Effect of <i>AQP1</i> 3’-UTR from different species of <i>Leishmania</i> on levels of <i>LUC</i> mRNA, protein expression and activity when transfected in promastigotes of <i>L</i>. <i>donovani</i>.

<p><b>A</b>. Chimeric constructs with the luciferase (LUC) reporter gene placed under the control of the 3’-UTR of the AQP1 mRNA from different <i>Leishmania</i> species were prepared to assess the role of these sequences in the species-specific regulation process. The neomycin resistance gene (NEO) and <i>LUC</i> transcripts in pSPYNEOαLUC vector were processed either by a 92 synthetic polypyrimidine stretch (Y90AG) (YNEO) or by the intergenic region of the α-tubulin gene (α-IR), respectively. The 3’-UTR (approximately 1.8 kb) followed by a 200 bp intergenic sequence of the <i>AQP1</i> 3’-UTR (IR) from different species were PCR amplified from the genome of different species of <i>Leishmania</i> and introduced at the 3’ end of the <i>LUC</i> gene. The LUC-expressing constructs were transfected into different species of <i>Leishmania</i>, and the effects of the AQP1 3’-UTR on <i>LUC</i> mRNA and protein expression and activity were measured. <b>B</b>. <i>LUC</i> mRNA levels: Total RNA was isolated from promastigotes of <i>L</i>. <i>donovani</i> expressing different chimeric constructs of <i>LUC</i>, and <i>LUC</i> mRNA expression levels were estimated using qPCR. Relative (with respect to <i>LUC</i>) <i>LUC</i> mRNA expression levels were calculated using 2^-ΔΔCt method. Data were expressed as mean ± SD of three independent experiments in triplicate. <b>C</b>. LUC activity and expression: Estimation of LUC activity (□) was carried out using whole cell lysates. Percent LUC activity was calculated keeping vector control at 100%. Data were expressed as mean ± SE of three independent experiments in triplicate. <b>D</b>. Representative Western blot analysis of transfected promastigotes: Whole cell (1 x 10⁶/lane) lysates of different transfectants were fractionated on SDS-PAGE and blotted onto nitrocellulose membrane. Levels of LUC expressions were detected using an anti-luciferase antibody. α-tubuline was used as loading control. Lanes: 1. pLUC, 2. pLUC-Ld, 3. pLUC-Li, 4. pLUC-Lm, 5. pLUC-Lt, 6. pLUC-Lb, and 7. pLUC-Lp. Amount of luciferase expression (■) relative to cells transfected with pSPYNEOαLUC was estimated by densitometric analysis using ImageJ software followed by normalization against the amount of α-tubulin of the respective cells. Error bars were calculated from the mean ± SE of two independent experiments. <i>Ld- L</i>. <i>donovani</i>, <i>Li- L</i>. <i>infantum</i>, <i>Lm- L</i>. <i>major</i>, <i>Lt- L</i>. <i>tropica</i>, <i>Lb- L</i>. <i>braziliensis</i>, <i>Lp- L</i>. <i>panamensis</i>.