M. paratuberculosis (M.ptb) possess the species specific, atypical insertion sequence, IS900 is 1,451bp and was reported to have a single open reading frame (ORF) possessing the translation signals necessary for expression of a protein of 399 amino acids, termed p43. Recently, Murray et al. isolated a promoter sequence, PAN, from M.ptb, located proximal to the 3' end of an IS900 element. PAN could drive expression of a second previously unreported ORF from IS900, encoded on the complementary strand to the p43 gene. We have designated this ORF as hed 900. Intriguingly hed 900 also requires sequences adjacent to, and outside IS900 to enable translation of a protein product. In fact, by virtue of site specific insertion into the sequence 5'AAGGNG*(4-6)NCATG3' (where the asterisk denotes the site of insertion), IS900 appears to target putative translation initiation sequences of host genes, thereby providing the necessary regulatory sequences for hed 900 expression. The termination codon for hed 900 is not generated by the target insertion site, and thus varies in position for each IS900 element. The gene products of IS900 represent specific antigens from M.ptb. As such, we are undertaking a number of approaches to express and purify Hed 900 in order to establish the potential of this protein as an antigen recognized during M.ptb infection in ruminant Johne's disease and in humans during Crohn's disease. Secondly, we are interested in the post-translational effects of hed 900 expression on M.ptb. IS900 has been implicated in the increased virulence of M.ptb from Mycobacterium avium background. A study of IS900 insertion and hed 900 expression may establish a role for this element in this evolutionary process.