Simultaneous transcription of duplicated var2csa gene copies in individual Plasmodium falciparum parasites.
BACKGROUND
Single nucleotide polymorphisms are common in duplicated genes, causing functional preservation, alteration or silencing. The Plasmodium falciparum genes var2csa and Pf332 are duplicated in the haploid genome of the HB3 parasite line. Whereas the molecular function of Pf332 remains to be elucidated, VAR2CSA is known to be the main adhesin in placental parasite sequestration. Sequence variations introduced upon duplication of these genes provide discriminative possibilities to analyze allele-specific transcription with a bearing towards understanding gene dosage impact on parasite biology.


RESULTS
We demonstrate an approach combining real-time PCR allelic discrimination and discriminative RNA-FISH to distinguish between highly similar gene copies in P. falciparum parasites. The duplicated var2csa variants are simultaneously transcribed, both on a population level and intriguingly also in individual cells, with nuclear co-localization of the active genes and corresponding transcripts. This indicates transcriptional functionality of duplicated genes, challenges the dogma of mutually exclusive var gene transcription and suggests mechanisms behind antigenic variation, at least in respect to the duplicated and highly similar var2csa genes.


CONCLUSIONS
Allelic discrimination assays have traditionally been applied to study zygosity in diploid genomes. The assays presented here are instead successfully applied to the identification and evaluation of transcriptional activity of duplicated genes in the haploid genome of the P. falciparum parasite. Allelic discrimination and gene or transcript localization by FISH not only provide insights into transcriptional regulation of genes such as the virulence associated var genes, but also suggest that this sensitive and precise approach could be used for further investigation of genome dynamics and gene regulation.
Department of Microbiology, Tumor and Cell Biology, Nobels Väg 16, Karolinska Institutet, SE-171 77 Stockholm, Sweden. kim.brolin@smi.se
Kim J M
KJ
Brolin
Ulf
U
Ribacke
Sandra
S
Nilsson
Johan
J
Ankarklev
Kirsten
K
Moll
Mats
M
Wahlgren
Qijun
Q
Chen
D000483:Alleles; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics*; D001483:Base Sequence; D001681:Biological Assay; D002467:Cell Nucleus / Q000378:metabolism; D018628:Gene Dosage / Q000235:genetics*; D020131:Genes, Duplicate / Q000235:genetics*; D017125:Genes, Protozoan / Q000235:genetics*; D008969:Molecular Sequence Data; D010271:Parasites / Q000166:cytology / Q000235:genetics*; D010963:Plasmodium falciparum / Q000166:cytology / Q000235:genetics*; D021381:Protein Transport; D015800:Protozoan Proteins / Q000235:genetics; D011786:Quality Control; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D053058:Trophozoites / Q000378:metabolism
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; D012333:RNA, Messenger; C512601:VAR2CSA protein, Plasmodium falciparum; C073415:antigen Pf332, Plasmodium