Escape from translational repression during differentiation from bloodstream to procyclic forms.

<p>A. Mean scintillation counts ± standard error of [³⁵S]-methionine incorporation during 1 hour windows throughout the differentiation from stumpy forms to procyclic forms (n = 3). Samples were taken at various time-points, shown in hours (h), post-addition of <i>cis</i>-aconitate (6mM); 0 h represents stumpy form cells. Established procyclic form cells (427 449 strain; PCF) and slender bloodstream monomorphic cells (single marker strain; BSF) were also analysed for comparison. Shown on the right <i>y</i>-axis is the mean count as a percentage of the mean count for established procyclic form cells (100%), these values also being shown above each data point. The experiment was performed twice and the numbers in parentheses are those obtained from the second experiment. B. Visualization of protein synthesis throughout differentiation by [³⁵S]-methionine labelling. 6mM <i>cis</i>-aconitate was added to stumpy form <i>T. b. brucei</i> AnTat1.1 cells; samples were then labelled with [³⁵S]-methionine for one hour at the time indicated post-addition of cis-aconitate (hr). The 0 hr time-point represents stumpy form cells. Monomorphic slender bloodstream form (BSF) and established procyclic form (PCF) cultures were also analysed. Protein samples were separated by SDS-PAGE (10%) and stained with Coomassie (left hand panel) to show loading or assayed by fluorography (right hand panel) to detect [³⁵S]-labelled proteins. The major labelled bands are tubulin and VSG (for Slender forms), which closely migrate. Stumpy forms show less obvious VSG since MSP-B and GPI-PLC activity during parasite isolation and pre-incubation before labelling resulted in a significant loss of cell-associated VSG.