Establishing the Cryptosporidium parvum karyotype by NotI and SfiI restriction analysis and Southern hybridization.

Abstract

The molecular karyotype of the coccidian parasite Cryptosporidium parvum has proven difficult to study because chromosomes of similar sizes migrate together when submitted to pulsed-field gel electrophoresis (PFGE). In the present work, the karyotype was studied by restriction of chromosome-sized DNA with the rare-cutting enzymes NotI and SfiI, followed by PFGE separation of the restriction fragments and Southern hybridization. These experiments showed that the C. parvum karyotype is formed by eight chromosomes, ranging in size from approximately 0.95 to 1.45 million base pairs (Mbp), accounting for a genome size of 9.6Mbp. As a first step towards the construction of a physical map of the C. parvum genome, a total of 20 probes, including 16 genes and the ribosomal DNA (rDNA) sequence, was mapped to intact chromosomes and to their restriction fragments. In this way, all chromosomes, but one, were identified by specific markers. A comparison of mapping data of homologous genes from different species belonging to the phylum Apicomplexa showed differences in the distribution of rDNA sequences and in the chromosomal localization of alpha- and beta-tubulin genes. The variation in genome size among these parasites is also discussed.