Figure 8.

Preferential translation induced in the S phase by the H2A4 5'UTR is maintained when a Histidine tagged H2A was employed as a reporter. (A) Schematic representation of the pXhisH2A5'H2A construct. The position of the 6 × Histidine tag coding region is indicated. (B) Immunoprecipitation of the His-H2A of nuclear protein extracts from ³⁵S-labelled L. infantum promastigotes transfected with pXhisH2A5'UTRH2A and treated with 5 mM HU either for 12 h (lane 0) or at the indicated time period (in h) after removal of the drug. The autoradiography (De novo panel) and western blot performed with the anti-His monoclonal antibody (TP panel) is shown. Data correspond to one representative experiment of two independent assays with similar results. (C) Densitometric analysis showing the ratio between de novo synthesis of His-H2A and total amount of His-H2A (columns). The percentage of cells in the S phase is also included (black line). (D) Northern blots prepared with RNA samples separated by sucrose gradients from promastigotes transfected with pXhisH2A5'H2A and treated 12 h with HU (lane 0) or 3 h after drug removal were sequentially hybridized with the 6 × His and the H4 probe. The autoradiographic exposure of the blots and the densitometric analysis is shown. Data correspond to one representative experiment of two independent assays with similar results. (E) Graphic showing the percentages of His-H2A and H4 mRNAs on polysomes at G1 and at the S phase obtained from two independent assays.