Figure 4

Cell cycle dependent expression of CAT is regulated at the translational level. (A) Northern-blot analysis of total RNA samples for promastigotes transfected with pXCAT5'III/3'I treated with 5 mM HU either for 12 h (lane 0) or at the indicated time (in h) after removal of the drug. The blot was sequentially probed with an oligonucleotide reverse and complementary to the 3' end of the CAT coding region and with a cDNA coding for L. infantum H4. Ethidium bromide staining of the corresponding gel is also shown (rRNA panel). (B) Graphic showing the ratio between the densitometric values of the blots hybridized with CAT probe (black columns) or H4 probe (white columns) and the rRNA bands revealed by ethidium bromide staining of the gel. Also the percentage of cells in the S phase of the cell cycle determined by flow cytometric analysis (black line) is shown. (C) Cytoplasmic extracts from L. infantum promastigotes transfected with pXCAT5'III/3'I and treated with 5 mM HU either for 12 h or 3 h after drug removal were separated on 15–40% sucrose linear gradients and RNA was purified and resolved in a 1% agarose-formaldehyde gel. The ethidium bromide staining of the gel is shown. Migration of 18 S, 24 S-α and 24 S-β are indicated. A graphic showing the densitometric values of the gel obtained 3 h after drug removal is included. The migration of the 80S particles has been marked in the gradients by an asterisk. (D) RNA from these gels were transferred on to nylon membranes and sequentially probed with the CAT and H4 probes. The autoradiographic exposure of the blot and the densitometric analysis is shown. Results are plotted as percentages of the total signal to allow direct comparison of the polysomal profiles. Data correspond to one representative experiment of three independent assays with similar results.