LEISHMANIA-MAJOR HEXBP DELETION MUTANTS GENERATED BY DOUBLE TARGETED GENE REPLACEMENT

The Leishmania major single-stranded DNA binding protein HEXBP contain s nine 'CCHC' zinc finger motifs and binds to oligodeoxynucleotides de rived from the antisense strand of the GP63 gene 5' flanking region in gel mobility shift assays and UV-crosslinking assays. In the present study a HEXBP-deficient clone of L. major was generated by double targ eted gene replacement. The two HEXBP alleles were sequentially replace d with genes encoding resistance to the aminoglycoside antibiotics hyg romycin B and G418 and drug-resistant clones were selected by plating on semi-solid drug-containing media. Successful deletion of both copie s of the HEXBP gene implies that HEXBP is a not essential for growth o f Leishmania promastigotes. Characterization HEXBP-deficient promastig otes revealed that HEXBP deficiency had no effect on the abundance of GP63 mRNA and protein in in vitro cultivated promastigotes and that HE XBP-deficient promastigotes were capable of lesion formation in BALB/c mice.