The Mycobacterium paratuberculosis atypical insertion sequence, IS900, encodes a novel gene on the complementary strand to the p43 coding sequence. This gene requires a promoter, ribosome binding site (RBS) and termination codon to be acquired upon insertion into the M. paratuberculosis genome, and hence is designated the hed (host expression dependent) gene of IS900. Analysis of IS900 insertion sites suggests that this element targets translation initiation signals in M. paratuberculosis, specifically inserting between the RBS and start codon of a putative gene sequence. This would align the hed initiation codon adjacent to a functional RBS and possibly downstream of an active promoter, driving expression of the Hed protein. A termination codon for hed is not generated by the target site and thus will vary in position for each genomic location of the IS900 element. Expression of hed was detected using two specific antibodies to probe Western blots of protein extracts from M. paratuberculosis. IS900 is unique to M. paratuberculosis and hence any immune response to its derived protein product would be indicative of specific infection. Therefore, recombinant forms of Hed and also p43, expressed and purified from E. coli and baculovirus are being examined as diagnostic antigens in immuno-assays for M. paratuberculosis disease. In conjunction, naked DNA constructs for in vivo expression of Hed and p43 have been used to immunize rabbits for future assessment in M. paratuberculosis vaccine design. IS900 is the major genetic difference between the closely related M. paratuberculosis and M. avium. Consequently, this element has been implicated in the increased pathogenicity of M. paratuberculosis from a M. avium background. The study of IS900 insertion into host gene translation signals and the subsequent post-translational effects of hed expression is beginning to provide a rationale for this element in this evolutionary process. It is hypothesized that the targeting process will result in expression of a number of M. paratuberculosis genes being specifically disrupted after IS900 has "hijacked" its translation signals. Such genes may regulate expression of virulence determinants, resulting in constitutive expression in M. paratuberculosis and conferring an obligate pathogenic status compared to M. avium.