3′-UTRs can mediate TR in P. berghei gametocytes. (A) Cartoons of the transcripts produced by a set of reporter constructs with the gfp ORF flanked by the 5′-UTR of ccp2 and the 3′-UTR of a series of mapped gene transcripts. The predicted lengths (in nt) of the 3′-UTRs were determined by 3′ RACE on gametocyte cDNA (dhfr/ts, pb25, pb47, ccp2, pb-245- and pb-633-) or have been published before (pb28, pg28) (37,38). The positions of the predicted 47nt TR motif (black box) or U-rich regions (grey shaded box in pg28) are indicated. (B) Western analysis of GFP production in gametocytes of transgenic P. berghei lines expressing gfp under control of different 3′-UTRs using anti-GFP antibodies. As a loading control an antiserum against the female gametocyte-specific protein Pb47 is used. (C) GFP fluorescence intensity in purified gametocytes of lines expressing GFP reporter transcripts with different 3′-UTRs as determined by FACS analysis of 30 000 cells. The left hand peak with fluorescent values between 0 and 10² are non-fluorescent cells such as male gametocytes and residual asexual stages that co-purify. The second, right hand peak represents the population of GFP-positive female gametocytes. The mean fluorescence intensity of the female gametocyte populations is indicated. Right panels: Representative GFP fluorescence images of gametocytes (g) and ookinetes (o) of the respective parasite lines. (D) Northern analysis of steady state levels of mRNA from purified transgenic gametocytes expressing gfp transcripts as shown in (A). The ccp2 5′ region was used as a probe which hybridizes to the gfp reporter transcripts as well as to endogenous ccp2 mRNA that serves as a loading control. (E) Relative GFP mRNA and protein expression levels in gametocytes expressing gfp transcripts as shown in (A). The GFP protein production is defined as the mean GFP-fluorescence intensity as determined by FACS analysis. GFP mRNA production of the same samples is determined by quantification of the intensity of the hybridization signal on northern blots relative to the internal control (ccp2). The GFP protein, mRNA and protein/mRNA level in each parasite line is normalized relative to the level obtained with the control reporter strain expressing GFP under control of the dhfr/ts 3′-UTR.

The 47nt 3′-UTR motif is involved in the mediation of TR. (A) Cartoons of the 3′-UTRs of gfp transcripts produced by a set of reporter constructs with gfp under the control of the ccp2 promoter and mutated/truncated pb28 3′-UTR regions. Regions of the pb28 3′-UTR are defined as follows: I; 5′ part of the pb28 3′-UTR (bases 1–375), II; 47nt element (bases 376–422), III; 3′ part of the pb28 3′-UTR (bases 423–477). Fusions were made with the complete dhfr/ts 3′-UTR (dhfr). Various combinations of the four regions were tested (i–v) in addition to 3′-UTR regions in which region II of pb28 was replaced with either the reversed sequence of the 47nt element (a) or with the putative 47nt elements of the 3′-UTR of pb-245- (b) and pb-633- (c) (vi–viii). (B) FACS analysis of GFP fluorescence intensity of gametocytes (as described for Figure 2C) expressing transcripts as shown in (A). (C) Northern analysis of steady state levels of mRNA from purified gametocytes expressing transgene transcripts as shown in (A). The ccp2 5′ region was used as a probe as described for Figure 2D. (D) Relative GFP mRNA and protein expression levels in gametocytes expressing gfp transcripts with 3′-UTRs as shown in (A). GFP mRNA and protein levels were determined as described for Figure 2E. Upper panel: Protein, mRNA and protein/mRNA levels displayed relative to the reporter parasite line expressing gfp with the pb28 3′-UTR. Lower panel: Protein, mRNA and protein/mRNA levels displayed relative to the dhfr/ts 3′-UTR control.

Model genes and reporter system to study TR in P. berghei gametocytes. (A) Expression of P. berghei pb28 and pb25. Upper panel: northern analysis of mRNA of P. berghei asexual blood stages (asex), purified gametocytes (gct) and ookinetes (ook) hybridized with probes of the open reading frames of pb28 and pb25 and with the 87R probe (specific for a fragment of the LSU rRNA) as a loading control. RNase H digestion of gametocyte and ookinete RNA was performed in the absence or presence of oligo(dT). Bottom panel: western analysis of gametocyte and ookinete preparations using Pb28 and Pb25 antibodies. Antiserum against the gametocyte-specific protein Pb47 was used as a control for loading of the blot. (B) Northern analysis of mRNA of P. berghei pb000633.00.0 (pb-633-) and pb000245.02.0 (pb-245-). (C) Reporter constructs used to transfect P. berghei to generate expression of GFP under control of the female gametocyte-specific ccp2 promoter with different 3′ regions. The constructs contain the tgdhfr/ts as a positive selectable marker and sequences of the ssu gene of the d-rrna locus for targeted integration into the genome. amp, ampicillin resistance gene; R, EcoRV; B, BamHI; X, XbaI; A, Asp718; K, KspI.

Factors responsible for pb28 3′-UTR-mediated TR are exclusively present in female gametocytes. (A) Each panel shows: Left, a cartoon of the transgene introduced into P. berghei, centre representation of the FACS analysis of Hoechst and GFP fluorescence in red blood cells infected with transgenic P. berghei, right, representative bright field (top) and GFP fluorescent (bottom) images of the transgenic parasites. Upper panel: Blood stage parasites expressing GFP under control of the constitutive ef-1αa promoter and the pb28 3′-UTR. Infected blood with a parasitemia of 1.5% was stained with Hoechst-33258 to be able to distinguish between infected and non-infected erythrocyte population. 57% of the Hoechst-stained cells are GFP-positive (region II) demonstrating that asexual parasites express GFP. The non-GFP fluorescent parasites (region I) are mainly young ring forms that do not express GFP (25) Middle panel: Blood stage parasites expressing GFP under control of the female specific ccp2 promoter and the pb28 3′-UTR. Both asexual parasites and gametocytes are GFP-negative. Lower panel: Infected red blood cells expressing GFP under control of the female specific ccp2 promoter and the dhfr/ts 3′-UTR. Asexual parasites are GFP-negative and only gametocytes are GFP-positive (region II). (B) The two panels each show: cartoons of the transgene introduced into P. berghei and representative GFP fluorescence images of exflagellating male gametocytes. Gfp is expressed under control of the pb28 3′-UTR and the male specific promoter regions of either α-tubulin II (α-tubII) (top) or pb000791.03.0 (bottom) showing the lack of TR in male gametocytes.

TR regulatory sequences exist in the 5′-UTR of pb25. (A) Cartoons of the GFP expression cassettes in reporter constructs for the expression of GFP under control of the promoter/5′-UTRs of ccp2, pb28 and pb25 and the 3′-UTR region of dhfr/ts. (B) GFP expression in purified gametocytes transfected with the expression cassettes as shown in (A). Upper panel: northern analysis using a gfp probe to detect the reporter transcripts and the female gametocyte specific ccp2 probe as an internal control. Bottom panel: western analysis using GFP antibodies. Antiserum against the gametocyte-specific protein Pb47 was used as a control. (C) Relative GFP mRNA and protein expression levels in gametocytes transfected with the expression cassettes as shown in (A). GFP mRNA and protein levels were determined as described for Figure 2E. The GFP protein, mRNA and protein/mRNA level in each parasite line is presented relative to the level obtained with the control reporter strain expressing GFP under control of the ccp2 5′ region and the dhfr/ts 3′-UTR. (D) The two panels each show: Top, cartoons of the transgene introduced into P. berghei and bottom, FACS analysis of purified gametocytes transfected with reporter constructs expressing GFP under control of a 3′-UTR comprising 350 bp of the 5′-UTR of pb25 in the right (5′pb25) and reversed (5′pb25rev) orientation. GFP fluorescence intensity of gametocytes was measured as described in Figure 2C. (E) Northern analysis of steady state levels of mRNA from purified gametocytes transfected with reporter constructs expressing gfp under control of the ccp2 5′ region and the 3′-UTRs of pb28, ccp2 and ccp2 with insertions of the 5′-UTR of pb25. The ccp2 5′ region was used as a probe as described for Figure 2D. (F) Conceptually transcribed sequence of the pb25 upstream region that was inserted into the 3′-UTR of the reporter constructs as described in Figure 5D. The 40nt U-rich TR-associated elements identified by MEME are underlined.