Analysis of the <i>LmjF</i>.<i>21</i>.<i>0725</i> locus anatomy in wild-type and transfectants of <i>L</i>. <i>major</i>.

(A)In silico physical map of HYG replacement of the endogenous allele is depicted in the cartoon and includes SalI digestion sites (above); expected fragment size of SalI digested DNA is indicated below (interrupted line) and HYG probe fragment is depicted as coarse line above diagram. Southern blotting membrane hybridized with HYG probe is shown below. (B) The in silico physical map of PAC replacement of endogenous allele is depicted in cartoon and includes SalI digestion sites (above); expected fragment size of SalI digested DNA is indicated below (interrupted line) and PAC probe fragment is depicted as coarse line above diagram. The Southern blotting membrane hybridized with PAC probe is shown below. (C) The in silico physical map of endogenous LmjF.21.0725 allele is depicted in cartoon and includes SalI digestion sites (above); the expected 2 fragments of SalI digested DNA are indicated below (interrupted line), and the LmjF.21.0725 probe fragment is depicted as the coarse line above the diagram. The southern blotting membrane hybridized with the LmjF.21.0725 probe is shown below. (D) Map of pxNeoGFP-LmjF21.0725 used for overexpression of LmjF.21.0725, and SalI digestion predicted fragment sizes are indicated below. Leishmania wild-type (WT) and transfectants are indicated in each blot as follows: +/+ = WT; +/PAC = heterozygous line with a PAC cassette replacing one endogenous allele; +/PAC/(+): heterozygous transfectant bearing PAC and plasmid expressing LmjF21.0725 (pxNeoGFP-LmjF21.0725); PAC/HYG/(+) = double replacement transfectant with both selectable markers and plasmid expressing LmjF21.0725 (pxNeoGFP-LmjF.21.0725, addback transfectant). Marker: Lambda DNA-HindIII digested.