Cloning, characterization, and comparison of filarial myosin heavy chain genes

Abstract

Filariasis affects over 100 million people worldwide and is classified by the World Health Organization as a major impediment to individual and national development. Screening a recombinant DNA library with human serum resulted in the isolation of a myosin-like antigen that was recognized by sera of nearly all persons residing in a filariasis-endemic area of India.;Using the insert in this clone, I embarked on a genomic walk to confirm the identity of this antigen as myosin, and to characterize and sequence filarial myosin genes in their entirety. The Brugia malayi major body wall myosin heavy chain genomic DNA, 11,766 nucleotides in all, has been sequenced to completion, and its exon-intron organization determined by a combination of homology with the free-living Caenorhabditis elegans and direct sequencing of the B. malayi cDNA. The B. malayi coding sequence consists of 15 exons and encodes a protein of 1957 amino acids. This is 9 amino acids shorter than the homologous unc-54 protein in C. elegans. The two proteins are 75.1% identical. In addition, the complete coding sequence of Onchocerca volvulus myosin was determined from a set of overlapping cDNA clones. This was compared to B. malayi and C. elegans. The two filarial parasites are more closely related to each other than either is to C. elegans. In addition, overlap between O. volvulus clones from different geographical isolates showed near-identity. Only a few single point mutations were found. In addition, the complete myosin gene from the animal filarid B. pahangi was cloned, restriction mapped, and partially sequenced. The B. pahangi sequence is very similar to that of B. malayi, confirming its relationship within the same genus. In fact, the first exon is 100% identical both at the protein and DNA level. The head region and immediate 5{dollar}\sp\prime{dollar} regions of the gene from Wuchereria bancrofti, the primary human parasite, was also cloned and substantially sequenced. Its sequence is also extremely similar to that of B. malayi. Overall, the evolutionary relationships deduced by sequenced comparison parallel and reinforce those established by parasite appearance and disease manifestation.