IS900 TARGETS TRANSLATION INITIATION SIGNALS IN MYCOBACTERIUM-AVIUM SUBSP PARATUBERCULOSIS TO FACILITATE EXPRESSION OF ITS HED GENE

The Mycobacterium avium subsp. paratuberculosis (formerly Mycobacteriu m paratuberculosis) atypical insertion sequence, IS900, encodes a nove l gene on the complementary strand to the putative transposase, p43. T his gene requires a promoter, ribosome binding site (RES) and terminat ion codon to be acquired upon insertion into the M. avium subsp. parat uberculosis genome and hence is designated the hed (host expression-de pendent) gene of IS900. Analysis of IS900 insertion sites suggests tha t this element targets translation initiation signals in M. avium subs p. paratuberculosis, specifically inserting between the RES and start codon of a putative gene sequence. This aligns the hed initiation codo n adjacent to a functional RES and possibly downstream of an active pr omoter, driving expression of bed protein. We have confirmed this uniq ue targeting process by detecting expression of hed in M. avium subsp. paratuberculosis at the level of transcription by reverse transcripti on-PCR. Further, two bed-specific antibodies detected bed translation products in Western blots of protein extracts from M. avium subsp. par atuberculosis. A recombinant form of bed expressed and purified from E scherichia coli will facilitate studies of IS900 transposition and wil l also be assessed as a diagnostic antigen for M. avium subsp. paratub erculosis disease. Implications of IS900 insertion in M. avium subsp. paratuberculosis pathogenicity are discussed.