MOLECULAR-CLONING AND EXPRESSION OF A LEISHMANIA-MAJOR GENE ENCODING A SINGLE-STRANDED DNA-BINDING PROTEIN CONTAINING 9 CCHC ZINC-FINGER MOTIFS

The genes encoding GP63, the major surface glycoprotein of the protozo an parasite Leishmania, are highly conserved across diverse species of Leishmania both within the protein coding region and in the immediate 5'-untranslated region. Located between the 3' trans-spliced leader a cceptor site and the translational initiation codon of the GP63 gene i s an area of conserved hexanucleotide direct repeats (CTCGCC) which va ry in number according to species. To determine whether these repeats represent a site of protein DNA interaction, a Leishmania major lambda gt11 expression library was screened with a radiolabeled synthetic oli godeoxynucleotide probe containing multiple CTCGCC repeats to detect c lones expressing functional DNA-binding proteins. Using this approach a gene was isolated which encodes a sequence-specific DNA-binding prot ein referred to as HEXBP (hexamer-binding protein). Sequence analysis of the HEXBP gene showed that HEXBP contains nine cysteine-rich motifs which are identical to a consensus sequence known as the ''CCHC type' ' zinc finger. This motif is present in a number of nucleic acid-bindi ng proteins including the nucleocapsid protein of retroviruses. In acc ordance with the activity exhibited by other proteins containing the ' 'CCHC'' motif, HEXBP binds to single-stranded nucleic acids as demonst rated by gel mobility shift assays and Southwestern blot assays.