Fig. 1.

Gene fragmentation approaches to improve P. falciparum yeast two-hybrid screens. A. Gene fragments generated by partial DNAse I digestion (bottom) and PCR (top). Black bar represents PfMyb2 (PF10_0327). Bars below PfMYB2 represent fragments generated by partial DNAse I digestion of PfMYB2 that were expressed in yeast. Tick marks above the bar indicate the positions of PCR primers, which were spaced at ~150-bp intervals. Short bars above PfMyb2 represent 300-, 450-, and 600-bp fragments; dashed lines separate fragments by size. All fragments were cloned into pOBD.111 by homologous recombination in the yeast strain R2HMet and tested for expression of the fragment by growth on synthetic dropout (SD) medium lacking methionine; the MET2 gene is fused to the 3’ end of the PfMYB2 fragment and is expressed only if the PfMYB2 fragment is also expressed. White bars represent gene fragments that were expressed, whereas gray bars represent those that were not. Numbers and letters indicate fragments that were retested for interaction with PFC0365w and PFL1705w in part B; letters designate 150-bp fragments. The twelve DNAse I-generated fragments and all 600-bp defined fragments were screened against a P. falciparum yeast two-hybrid library. Striped bars indicate fragments that interacted with both PFC0365w and PFL1705w in the yeast two-hybrid assay. B. Confirmation of yeast two-hybrid interactions with PfMYB2 and mapping of the PfMYB2 protein interaction domain. PfMYB2 150-bp fragments K-N, 300-bp fragments #10-13, and 600-bp fragments #41-43 were cloned into pOBD.111 in the yeast strain R2HMet. Similarly, the PFC0365w and PFL1705w fragments that interacted with PfMYB2 fragment #42 were PCR-amplified and inserted into pOAD.102 by homologous recombination in the yeast strain BK100. Yeast harboring DNA-binding domain and activation domain plasmids were allowed to mate and then grown on SD medium lacking tryptophan and leucine to select diploid cells containing both plasmids. Yeast were grown to mid log phase, diluted to an OD₆₀₀ of 1.0, serially diluted 5-fold in dH₂O, and plated on SD medium lacking tryptophan and leucine or yeast two-hybrid selection medium (SD medium lacking tryptophan, leucine, methione, uracil, and histidine and containing 1 mM 3 amino-4,5-triazole (3-AT); 3-AT is a competitive inhibitor of the yeast two-hybrid reporter enzyme His3 that is added to growth medium to inhibit background yeast growth).