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Cloning and characterization of a single-stranded DNA-binding protein of Leishmania major Webb, John R.


Protozoan parasites of the genus Leishmania are the etiologic agents of a spectrum of important human diseases collectively referred to as leishmaniasis. The major surface protein on all species of Leishmania is a highly abundant 63 kDa glycoprotein referred to as GP63. GP63 has been characterized as a cell surface protease, however, its exact role in the Leishmania life cycle is not clear. The genes encoding GP63 are arranged in the Leishmania genome as a species-specific combination of direct head to tail tandem repeats and single dispersed gene copies. In the present study, a single repeat unit of the Leishmania donovani GP63 tandem array was cloned and sequenced. Alignment of the L. donovani GP63 gene sequence with the previously determined GP63 gene sequences from two related species, L. major and L. chagasi, revealed that GP63 is highly conserved across species. Consistent with the observed protease activity of GP63, the predicted amino acid sequence of GP63 from all three species contained a conserved motif shared by a number of zinc metalloproteases. In addition, alignment of the untranslated regions of the three GP63 genes revealed that the immediate 5' untranslated region is highly conserved within and across species. This region did not contain any sequences characteristic of higher eukaryotic promoter elements, however, it did contain an area of conserved hexanucleotide direct repeats. To determine whether these direct repeats (CTCGCC )represented a potential site of protein-DNA interaction, a A. gtl 1 expression library ofL. major was screened with a radio labelled oligonucleotide probe to detect clones expressing functional DNA-binding proteins. A gene was isolated which encoded a novel DNA-binding protein, referred to as HEXBP. The deduced amino acid sequence of HEXBP revealed that it is a 28 kDa protein containing nine 'CCHC-type' zinc finger motifs. The CCHC motif, Cys-X2_Cys-X4-His-X4-Cys, is invariant with regards to the number and spacing of cysteine and histidine residues and is shared by a number of nucleic acid-binding proteins. In accordance with the activity exhibited by other CCHC-containing proteins, HEXBP was characterized as a single-stranded nucleic acid-binding protein. Additional analyses indicated that HEXBP bound single-stranded DNA in a sequence specific manner and that the conserved 5' untranslated region of GP63 gene contained multiple HEXBP binding sites. To determine the cellular function of HEXBP, a HEXBP-deficient mutant of L.major was generated using the technique of double homologous gene replacement. Initial characterization of this mutant suggested that HEXBP was not essential for the expression of GP63 by in vitro cultivated promastigotes. Although the HEXBP-deficient mutant did not exhibit any gross phenotypic changes, further characterization will likely provide insight into the function of the HEXBP single-stranded DNA-binding protein. In addition, a plasmid construct was identified that lead to stable transformation of Leishmania when electroporated into promastigotes as an intact circular plasmid. This construct conferred selectable drug-resistance to transfectants and was found to replicate as an extrachromosomal circular concatamer. The construct was modified to produce a functional Leishmania expression vector called pLEX. Initial characterization of the transcriptional regulation of pLEX suggests that it also represents a potentially useful model system for studying the process of polycistronic gene expression in kinetoplastid protozoans.

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