Cloning of the repertoire of individual Plasmodium falciparum var genes using transformation associated recombination (TAR)

Abstract

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.

Figure 1. Construction of the var TAR vectors.

(A) TAR cloning vectors pEB_upsA/B or C_ATS were constructed from the general TAR vector pEB2 (M. Becker and E.J. Louis, unpubl.). The 5′ targeting sites upsA (185 bp), upsB (263 bp) or upsC (203 bp) are indicated by black arrows. The 3′ targeting site is identical in all three TAR cloning vectors and contains part of the ATS region (287 bp) indicated with black squares. The yeast centromere and ARS are labelled as CEN and ARS, the positive selectable marker gene URA3 is indicated as light grey box (URA3), the negative selectable marker CYH2 with a white box (CYH2), and the Ampicillin resistance gene with a dark grey box (Amp-R). SalI and ClaI restriction sites are indicated. Homologous recombination between the targeting sites and P. falciparum genomic DNA results in the cloning of a var gene flanked by those sites as a circular molecule. (B) 5′ and 3′ targeting sequences used in the TAR cloning vectors pEB_upsA/B or C_ATS.

Figure 2. Southern blot analysis of var clones.

(A) Southern blot example of 41 samples of NotI-digested genomic DNA of yeast TAR clones probed with vector-backbone. One lane contains a negative control (ctrl). (B) Restriction fragment pattern of TAR clones upsAIII_10, upsBI_29 and upsCI_3 digested with NotI and NdeI. The vector backbone yielded a band of 1600 bp and is marked with an arrow. The molecular marker is indicated. The upper band of the upsAIII_10 clones represents a double band.