Fig. 2. Targeted gene replacement of the lmexlpg1 alleles. (A) Restriction map of the lmexlpg1 locus. Restriction sites relevant for Southern blot analysis, the resistance genes and the primer-binding sites for the construction of gene deletion cassettes and for the generation of DIG-labelled probes are indicated. (B) Southern blot analysis of restriction enzyme-digested L.mexicana chromosomal DNA (5 µg) resolved on a 0.7% agarose gel using a labelled lmexlpg1 gene probe. Lane 1, HindIII; lane 2, NcoI; lane 3, KpnI; lane 4, PvuII; lane 5, PstI; lane 6, SalI; lane 7, SacI; lane 8, SacII; lane 9, SphI; lane 10, XhoI. The size of DNA standards is indicated in kb. (C and D) Southern blot analysis of L.mexicana Δlmexlpg1 mutants. Genomic DNA was digested with PstI, separated on a 0.7% agarose gel, blotted and probed with a labelled lmexlpg1 5′-UTR fragment (C) or an lmexlpg1 ORF probe (D). Lanes 1, L.mexicana Δlmexlpg1, clone I/8D; lanes 2, L.mexicana wild type. The asterisk depicts a background spot due to antibody aggregates.