Abstract
The detection, in situ or in tissue extracts, of post-translationally modified isoforms of a host-encoded protein, PrP, is diagnostic for scrapie, bovine spongiform encephalopathy (BSE) and the other transmissible spongiform encephalopathies (TSEs). Specific PrP gene mutations and polymorphisms have been linked to the incidence of naturally occuring TSEs in sheep and man, and to predisposition to experimental disease in sheep and mice. Initial restriction fragment length polymorphism (RFLP) analyses of bovine genomic DNA with the enzyme PvuII failed to establish a PrP genotype linkage with the incidence of BSE. Bovine PrP genes were then amplified by the polymerase chain reaction (PCR), cloned, and sequences obtained which agreed with previously published data. Bovine alleles contain a cytosine or thymidine at base 576 (the switch is silent) and encode five or six copies of an octarepeat motif, but no linkage with these polymorphisms was observed from studies of a large dairy herd with multiple BSE cases. Direct automated sequencing from PCR amplified genes was developed and validated by comparing results with manually derived sequences. No new polymorphisms were identified following sequencing of (6) experimental and (6) natural BSE cases, and (6) normal cows. Sequencing the PrP genes of pig and kudu, species also exposed to contaminated feed but with apparent differing susceptibilities, was performed. The pig gene has five copies of the octarepeat motif and encodes a protein of 257 amino acids, with unique residues preceding the attachment of a possible glycosylphosphatidylinositol anchor. The kudu alleles contained five or six octarepeats, encoding proteins of 256 and 264 amino acids respectively, with four amino acid differences between the alleles within the octarepeat region. This novel occurrence may be significantly linked to the kudu's apparent susceptibility. Overall, comparison of the genes from different species demonstrated that primary and predicted secondary structures were highly conserved.