A var Gene Upstream Element Controls Protein Synthesis at the Level of Translation Initiation in Plasmodium falciparum
Figure 1
Integration of the upsC 5′ upstream sequence into a heterologous context at the kahrp locus.
(A) Schematic map of the transfection construct pBKminC. Single-crossover integration was guided by kahrp 5′ homology. The position of the kahrp TSS is indicated [81]. Numbers refer to the nucleotide positions relative to the ATG start codon. The bsd resistance cassette selects for stably transfected parasites. The var intron is indicated by a bold dashed line. hsp86 5′, hsp86 promoter; Pb DT 3′, P. berghei dhfr-thymidylate synthase terminator; rep20, 0.5 kb TARE6 repeat element; hrp2 3′; histidine-rich protein 2 terminator. MEE, location of the 101 bp mutual exclusion element MEE [54]. (B) Genomic situation after integration of the pBKminC concatamer into the endogenous kahrp locus. Restriction sites used in Southern analysis and fragment lengths are indicated and colour-coded. S, StuI; B, BglII. The Southern blot on BglII/StuI-digested gDNA shows integration of pBKminC into the endogenous locus of kahrp. The membrane was hybridised with hdhfr (top) and kahrp (bottom). Fragments are colour-coded according to the integration map. wt, size of the kahrp fragment in 3D7 wild-type parasites. i, integration event; p, plasmid fragment. (C) The upsC 5′ UTR sequence represses kahrp promoter activity. The bars represent the ratio of relative hdhfr-gfp and msp8 transcript levels in 3D7/pBKminC parasites (open bars) compared to the 3D7/pBKmin control (black bars) cultured in absence of WR. Results are the mean +/− s.d. of three independent experiments. Values are normalised for PF3D7_1331700 transcripts.