Identification and isolation of Cryptosporidium parvum genes encoding microtubule and microfilament proteins.

Abstract

Microtubules and microfilaments are highly conserved cytoskeletal polymers hypothesized to play essential biomechanical roles in the unusual gliding motility of Apicomplexan zoites and in their invasion of, and development within, host epithelial cells. We have identified and isolated Cryptosporidium parvum genes encoding the microtubule proteins alpha- and beta-tubulin and the microfilament protein actin by screening a lambda gt11 C. parvum genomic DNA library with degenerate oligonucleotide and heterologous cDNA hybridization probes respectively. The alpha- and beta-tubulin genes have been partially sequenced and the deduced peptide sequences show greatest homology with the tubulins of the related parasites, T. gondii and P. falciparum. The complete nucleic acid sequence of the actin gene predicts a 376 amino acid, 42 kDa protein having 85% sequence identity with the P. falciparum actin I and the human gamma-actin proteins. Each of these cytoskeletal protein genes was demonstrated to be of cryptosporidial origin by Southern analyses of C. parvum chromosomes fractionated by pulsed field gel electrophoresis; the cloned alpha- and beta-tubulin genes hybridized with chromosomes of ca. 1,200 and 1,500 kb respectively and the cloned actin gene also hybridized with a 1,200 kb chromosome.