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pppAG-elongation on the correct antigenome 3′-end and on variants with an incorrect last nucleotide.

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posted on 19.02.2013, 23:26 by Barbara Selisko, Supanee Potisopon, Rym Agred, Stéphane Priet, Isabelle Varlet, Yann Thillier, Corinne Sallamand, Françoise Debart, Jean-Jacques Vasseur, Bruno Canard

pppAG-elongation by NS5PolDV on DV103′- templates (ACUAACAA-CN) varying the last nucleotide (correct -CU versus -CC, -CA and -CG). Control reactions were included without template. (A) pppAG elongation in the presence of Mn2+. pppAG (100 µM) and UTP (100 µM, containing αUTP) were used as substrates. Reaction mixtures were prepared as given in Materials and Methods plus 2 mM MnCl2, 500 nM NS5PolDV, and 1 µM template. Reactions were started by addition of MnCl2 and UTP. Samples were taken at given time points and analyzed by PAGE and autoradiography. OligoG marker is shown on the left, the identity of product bands is given on the right. (B) pppAG-elongation in the presence of Mg2+. pppAG (100 µM) and UTP (100 µM, containing αUTP) were used as substrates. Reaction mixtures were prepared as given in Materials and Methods plus 5 mM MgCl2, 5 µM NS5PolDV, and 1 µM template. Reactions were started by addition of MgCl2 and UTP. Samples were taken at given time points and analyzed by PAGE and autoradiography. OligoG marker is shown on the right; the identity of product bands is given on the left.

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