Targeting the L. major lack genes. (A) Physical map showing organization of the L. major lack genes. Restriction enzyme cut sites are indicated: B, BamHI; H, HindIII; K, KpnI; N, NsiI; No, NotI; S, StuI; Sp, SphI; Spe, SpeI; X, XbaI. Gray boxes denote lack1 and lack2 open reading frames. Fragments g and h (triple lines) were used as probes for Southern hybridization analysis. Fragments a–f (heavy lines) were used to make targeting constructs (B, 1–4) and episomal GFP-LACK and GFP expression constructs (B, 5 and 6). (B) Cartoon of the targeting constructs used for generating organisms used in these studies (refer to Materials and Methods). (C) Southern hybridization of L. major transfectant genomic DNAs. Genomic DNAs (10 μg/lane) from untransfected L. major (lane 1) or LHDL transfectants (L. major lack++/−−; lane 2) were digested with KpnI. Genomic DNAs from L. major lack++/−− (lane 3), L. major lack++/−− transfected with L2PDL2 (L. major lack+−/−−; lane 4), and L. major lack++/−− transfected with L1SDL1 (L. major lack−+/−−; lane 5) were double digested with StuI and BamHI. DNAs from L. major lack++/−− transfected with L2PD-LKΔ-L2 (L. major lack++Δ/−) were digested with StuI (lane 6). Blots were either hybridized with fragment h (lanes 1 and 2) or fragment g (lanes 3–6).