A study of var gene transcription in vitro using universal var gene primers.
The polymorphic multigene family, var, encodes the variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), present on the surface of erythrocytes infected with the human malaria parasite, P. falciparum. PfEMP1 has been implicated in the pathology of malaria through its ability to bind to host endothelial receptors and uninfected erythrocytes. Understanding the relationship between host pathology, immune response and parasite variation is crucial, but requires a method of reliably detecting and differentiating all possible var genes. Several primer pairs used to date are biased and limited in their detection capacity. Here we describe a set of PCR primers that amplify the majority of var genes in the laboratory isolates 3D7 and A4, and appear to work equally well on all isolates tested. We use these universal primers to examine the relationship between var gene transcription as assessed by reverse transcriptase-PCR (RT-PCR) with that measured by Northern analysis of parasite RNA. Phenotypically selected young parasites have multiple transcripts detected by RT-PCR, but the full-length transcript appears to be homogeneous. In addition, we demonstrate that the choice of primers used for RT-PCR is crucial in data interpretation.
Taylor
H M
HM
Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford, UK. htaylor@nimr.mrc.ac.uk
Kyes
S A
SA
Harris
D
D
Kriek
N
N
Newbold
C I
CI
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D017931:DNA Primers; D017125:Genes, Protozoan; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology; D008969:Molecular Sequence Data; D005810:Multigene Family; D010963:Plasmodium falciparum / Q000235:genetics* / Q000254:growth & development; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D017931:DNA Primers; D015800:Protozoan Proteins; D016053:RNA, Protozoan; C085731:erythrocyte membrane protein 1, Plasmodium falciparum