Molecular cloning and expression of a Leishmania major gene encoding a single-stranded DNA-binding protein containing nine "CCHC" zinc finger motifs

Abstract

The genes encoding GP63, the major surface glycoprotein of the protozoan 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 acceptor site and the translational initiation codon of the GP63 gene is an area of conserved hexanucleotide direct repeats (CTCGCC) which vary 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 oligodeoxynucleotide probe containing multiple CTCGCC repeats to detect clones expressing functional DNA-binding proteins. Using this approach a gene was isolated which encodes a sequence-specific DNA-binding protein 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-binding proteins including the nucleocapsid protein of retroviruses. In accordance with the activity exhibited by other proteins containing the "CCHC" motif, HEXBP binds to single-stranded nucleic acids as demonstrated by gel mobility shift assays and Southwestern blot assays.