Detection of hDHFR-GFP and GAPDH (loading handle) was performed on entire mobile lysates of parasites harvested at sixty four hpi. Primary antibody dilutions had been: mouse anti-GFP (Roche Diagnostics, 11814460001), 1:five hundred monoclonal mouse antiGAPDH 1-10B [eighty], one:20,000. Pre-synchronised parasites cultures were synchronised two times 16 hours aside to obtain an eight-hour development window. Whole RNA digested with EcoRV and NcoI and ligated into EcoRV/NcoIdigested pET-forty one (EMD Biosciences). To amplify EcoRV/NcoI restriction fragments containing the hdhfr coding sequence, a main PCR response was executed making use of T7 and hdhfr_R1 (R1) as ahead and reverse primers, respectively. The primary PCR solution was diluted one:two hundred and utilised as template for a semi-nested PCR reaction using T7 and hdhfr_R2 (R2) as ahead and reverse primers, respectively. This amplicon was then sequenced employing primer hdhfr_R3 (R3). The nucleotide sequence is proven at the base (reversed sequence). It commences with an EcoRV site in the intron of var gene PF3D7_0400100 on chromosome four (orange letters) and proceeds into the 39 stop of exon one (purple box). The environmentally friendly letters highlight the 10 bp sequence concerned in the recombination event in between the var and hdhfr-gfp loci. The grey box signifies the commence of the hdhfr-gfp coding sequence. A in depth schematic check out of the recombination event is depicted over the nucleotide sequence. A single-crossover happened among the ten bp sequence (environmentally friendly letters) immediately upstream of the hdhfr-gfp reporter (gray box) on chromosome two, and an equivalent sequence (eco-friendly letters) at the quite 39 end of exon 1 of var gene PF3D7_0400100 (purple box) on chromosome four. As a consequence, the hdhfr-gfp reporter (grey box) was put under handle of the var gene intron promoter (orange line) on the reverse strand via gene conversion.
Determine S2 Further verification of the gene conversion function among chromosomes two and four in WR-chosen 3D7/pBKminC parasites. (A) The map schematically depicts the end of chromosome two like the integrated plasmid concatamer (blue box) in 3D7/pBKminC parasites. kahrp promoter sequences are depicted by thick black lines. The upsC 59 UTR sequence is depicted in purple.The gene accession 349085-82-1 biological activity variety refers to the most telomereproximal upsB var gene PF3D7_0200100. The decrease map shows a zoom-in view of the built-in concatamer (blue box). Restriction web sites used in Southern analysis are revealed by vertical dashed arrows, and envisioned fragment lengths are indicated and colourcoded. The hdhfr probe utilized for hybridisation15215179 is proven below the hdhfr-gfp coding sequence 
(gray box). EcoRI web sites are absent from the plasmid sequence. That’s why, the EcoRI web sites up- and downstream of the built-in concatamer launch a restriction fragment in the measurement of 6228 bps (chromosomal DNA) in addition n times 9475 bps (complete plasmid size) in accordance to the amount of copies in the concatamer. (B) The map schematically depicts the conclude of wildtype chromosome four including var gene PF3D7_0400100 (orange box) in unselected 3D7/pBKminC parasites. The PF3D7_0400100 exon one probe utilized for hybridisation is shown underneath the coding sequence. The placement of the EcoRI restriction site downstream of the var locus and the envisioned fragment duration are indicated. (C) The map schematically depicts the conclude of chromosome 4 soon after the gene conversion celebration among chromosomes two and 4 in WRselected 3D7/pBKminC parasites (“chromosome 4/2 end”). The border between the eco-friendly and blue boxes identifies the website of one-crossover recombination. The green and blue packing containers depict sequences of the acceptor (chromosome four) and donor (chromosome 2), respectively, of the gene conversion celebration.
