Cloning of the repertoire of individual Plasmodium falciparum var genes using Transformation Associated Recombination (TAR)

Article English OPEN
Gaida, Annette ; Becker, Marion M. ; Schmid, Christoph D. ; Bühlmann, Tobias ; Louis, Edward J. ; Beck, Hans-Peter (2011)
  • Publisher: Public Library of Science
  • Journal: PLoS ONE, volume 6, issue 3 (eissn: 1932-6203)
  • Related identifiers: pmc: PMC3049791, doi: 10.1371/journal.pone.0017782
  • Subject: Research Article | Biology | Plasmodium Falciparum | Infectious Diseases | Genome Analysis Tools | Genomics | Genome Sequencing | Parasitic Diseases | Medicine | Malaria | Comparative Genomics

One of the major virulence factors of the malaria causing parasite is the Plasmodium falciparum encoded erythrocyte membrane protein 1 (PfEMP1). It is translocated to It the membrane of infected erythrocytes and expressed from approximately 60 var genes in a mutually exclusive manner. Switching of var genes allows the parasite to alter functional and antigenic properties of infected erythrocytes, to escape the immune defense and to establish chronic infections. We have developed an efficient method for isolating VAR genes from telomeric and other genome locations by adapting transformation-associated recombination (TAR) cloning, which can then be analyzed and sequenced. For this purpose, three plasmids each containing a homologous sequence representing the upstream regions of the group A, B, and C var genes and a sequence homologous to the conserved acidic terminal segment (ATS) of var genes were generated. Co-transfection with P. falciparum strain ITG2F6 genomic DNA in yeast cells yielded 200 TAR clones. The relative frequencies of clones from each group were not biased. Clones were screened by PCR, as well as Southern blotting, which revealed clones missed by PCR due to sequence mismatches with the primers. Selected clones were transformed into E. coli and further analyzed by RFLP and end sequencing. Physical analysis of 36 clones revealed 27 distinct types potentially representing 50% of the var gene repertoire. Three clones were selected for sequencing and assembled into single var gene containing contigs. This study demonstrates that it is possible to rapidly obtain the repertoire of var genes from P. falciparum within a single set of cloning experiments. This technique can be applied to individual isolates which will provide a detailed picture of the diversity of var genes in the field. This is a powerful tool to overcome the obstacles with cloning and assembly of multi-gene families by simultaneously cloning each member.
  • References (41)
    41 references, page 1 of 5

    1. World Health Organization (2008) The world health report 2008: primary health care now and more than ever. Available at: http://www.who.int/whr/ 2008.

    2. Baruch DI, Gormely JA, Ma C, Howard RJ, Pasloske BL (1996) Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1. Proc Natl Acad Sci U.S.A 93: 3497-3502.

    3. Jensen ATR, Magistrado P, Sharp S, Joergensen L, Lavstsen T, et al. (2004) Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes. J Exp Med 199: 1179-1190.

    4. Chookajorn T, Ponsuwanna P, Cui L (2008) Mutually exclusive var gene expression in the malaria parasite: multiple layers of regulation. Trends Parasitol 24: 455-461.

    5. Freitas-Junior LH, Hernandez-Rivas R, Ralph SA, Montiel-Condado D, Ruvalcaba-Salazar OK, et al. (2005) Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites. Cell 121: 25-36.

    6. Bull PC, Lowe BS, Kortok M, Molyneux CS, Newbold CI, et al. (1998) Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat Med 4: 358-360.

    7. Bull PC, Lowe BS, Kortok M, Marsh K (1999) Antibody recognition of Plasmodium falciparum erythrocyte surface antigens in Kenya: evidence for rare and prevalent variants. Infect Immun 67: 733-739.

    8. Kyes SA, Kraemer SM, Smith JD (2007) Antigenic variation in Plasmodium falciparum: gene organization and regulation of the var multigene family. Eukaryotic Cell 6: 1511-1520.

    9. Kraemer SM, Smith JD (2006) A family affair: var genes, PfEMP1 binding, and malaria disease. Curr Opin Microbiol 9: 374-380.

    10. Voss TS, Kaestli M, Vogel D, Bopp S, Beck H (2003) Identification of nuclear proteins that interact differentially with Plasmodium falciparum var gene promoters. Mol Microbiol 48: 1593-1607.

  • Related Research Results (3)
  • Metrics
    No metrics available