Mutually Exclusive Expression of Virulence Genes by Malaria Parasites Is Regulated Independently of Antigen Production
Figure 5
Integration of a bsd Expression Cassette into an Internal Chromosomal var Locus
(A) Schematic diagram showing integration of the construct pVcBB/IDH into the var PFL1960w locus through a single homologous recombination event within the var upstream region.
(B) Southern analysis was performed using BamHI and StyI digested gDNA hybridized with probes to bsd and pUC18. The 8.3-kb bsd band and the 13.2-kb pUC18 band correspond to the fragments flanking the integrated concatamer. The doublet at ~7.1 kb corresponds to fragments from within the integrated concatamer.
(C) Analysis of the level of transcription of each var gene within the parasite genome shows that selection with blasticidin results in silencing of all members of the var gene family. The only var locus expressed by parasites grown under drug pressure (top panel) was the gene that contained the integrated bsd expressing plasmid. 10 wk after drug removal, the culture became transcriptionally heterogenous (bottom panel).