A. The left panel shows a Western analysis of proteins derived from procyclic cells expressing ectopic TbZFP1, detected using the BB2 antibody to the Ty1 tag engineered into the protein. Cells were either induced (+) or uninduced (−) with tetracycline and the parental (Pc449; ‘WT’) strain is shown as a control. The right panel shows the same protein samples probed with an anti-peptide antibody to TbZFP1 and shows both the native molecule (lower band – all samples) and the transgenic protein (upper band).
B. Western analysis of wild-type bloodstream form cells (‘Wt BSF’) or bloodstream form cells transfected with a gene encoding a copy of TbZFP1 comprising a C-terminal Ty1 tag (‘TbZFP1-C’). No tagged protein is detectable using the anti-tag antibody BB2. Procyclic cells containing the same expression construct are shown (‘PC’) as a control. Cells were grown in the presence of 1 μg ml⁻¹ tetracycline to induce transgene expression.
C. Northern analysis of RNA samples taken from bloodstream form TbZFP1 expression cell lines induced (+) or uninduced (−) with tetracycline. Two clonal lines and the parental (Bs449 ‘WT’) were probed with the TbZFP1 coding region. The asterisk indicates endogenous TbZFP1 mRNA, the arrowhead indicates the transgene-derived mRNA. Cross hybridizing rRNAs are also detected (labelled ‘rRNA’).

A. Schematic diagram showing the strategy used to create TbZFP1 null mutants in bloodstream forms. The tetracycline repressor and neomycin cassette were used for the first knockout, and a set of targeting sequences internal to the first knockout targeting sequences were used for the hygromycin cassette for the second knockout. This ensured that the first integration could not be re-targeted during the second knockout. Note that each drug resistance cassette has also been engineered to contain an EP1 promoter (arrowhead), which is active in bloodstream forms, to ensure sufficient transcription of the drug resistance cassettes.
B. Northern analysis of bloodstream form cells deleted for TbZFP1. RNA samples from bloodstream forms were probed with a TbZFP1 specific riboprobe. Wild-type (+/+), two single gene knockout cell lines (+/−) and a double knockout (−/−) mutant are shown. The blot has been overexposed to reveal the low level endogenous TbZFP1 mRNA in the wild-type and single knockout lines and to confirm its absence in the null mutant line. The ethidium bromide image of the RNAs is shown as a loading control.
C. Verification of the deletion of TbZFP1 in the null mutant cells. In each case genomic DNA from either wild-type cells or the null mutant lines (Null A, Null B) was subjected to PCR amplification using primers specific for either the TbZFP1 coding region, or the coding region of TbZFP2 (Hendriks et al., 2001), as a control. Each amplification reaction was run simultaneously.

A. Graph showing VSG loss for two TbZFP1 null mutants (null A and null B) and the wild-type (WT) cell line 6 h and 72 h after exposure to differentiation conditions.
B. Graph showing EP procyclin expression for the same cells as in A.

A. Kinetoplast repositioning of wild-type cells (WT, ZFP+/+), two independently derived TbZFP1 null mutants (ZFP1 −/−; null A, B), and the null B mutant expressing a rescue copy of TbZFP1 (ZFP1 −/−/+). For the left hand panel, individual cell measurements are shown for the kinetoplast to posterior dimension 72 h after incubation in differentiation conditions. The right hand panel is a separate experiment in which the cell samples were taken 96 h after the initiation of differentiation. This time point was chosen to match the percentage differentiated cells (as assessed by expression of EP procyclin) seen in the experiment presented in the left panel, thereby allowing the relatively kinetoplast positioning in the respective populations to be more easily compared. Cells undergoing differentiation were identified by their expression of EP procyclin. Each open circle represents an individual cell measurement and 100 cells were measured for each time point. B. Images of the relative extent of kinetoplast repositioning in wild-type cells, the null B mutant and the null B mutant re-expressing a rescue copy of TbZFP1. In each case a phase-contrast-DAPI image is shown. The arrowhead marks the position of the kinetoplast. Scale bar = 8 μm.

Absence of posterior microtubule extension on the TbZFP1 null mutant. Representative images are shown of the TbZFP1 null mutant cells and null mutants expressing an ectopic rescue copy of TbZFP1 at 96 h after the initiation of differentiation to procyclic forms. Procyclic form cells are also shown, as a control. In each case phase contrast images of cells are shown (‘Phase’) as are the same cells counterstained with DAPI to reveal the position of the kinetoplast (‘Phase DAPI’). The lower panels show the same fields stained with Yl1/2, which detects tyrosinated microtubules within the cell, this being diagnostic for posterior extension of the cytoskeleton. Example cells in which there is active posterior microtubule extension are highlighted with arrowheads. Bar = 15 μm.

Cells progress into the cell cycle despite a failure in kinetoplast repositioning. Cells from the null B population are shown 96 h after being induced to differentiate to procyclic forms. In each case the left panel shows cells stained with DAPI to indicate that the kinetoplasts have segregated, despite being located at the extreme posterior of the cell. Kinetoplasts are labelled with an arrowhead; nuclei are labelled with an arrow. The right panel shows the same cells stained with EP procyclin, confirming that each cell is undergoing differentiation and is not merely a proliferating bloodstream form parasite.
A and B. Showing a cell where kinetoplast repositioning is initiating.
C and D. Showing a cell where the kinetoplasts are well segregated, but where one kinetoplast remains close to the posterior of the cell. This cell may also be undergoing an aberrant attempt at cytokinesis.
E and F. Show a cell where kinetoplast segregation is large and mitosis has occurred. Nonetheless one kinetoplast remains close to the posterior end of the cell. Bar = 10 μm.

Schematic representation of the phenotypic consequences of TbZFP1 knockout in the context of the pathway of events during bloodstream stumpy-procyclic form differentiation. The expression profile of TbZFP1 is shown in the wild-type cells as a dumbbell.