Recent observations show that the insertion sequence IS900, from Mycobacterium paratuberculosis, has targeted translation initiation signals. This is part of a process which results in expression of a previously cryptic open reading frame now designated the hed (host expression dependent) gene. There are five main RFLP patters observed in M. paratuberculosis in which many bands are conserved across strains, even where bands differ significantly the genetic locus may still be the same. We have isolated a 14 of a predicted 18 loci, each of which contain a copy of IS900. Promoter and open reading frame analysis is being performed on each of these genetic loci. One locus, IDL3, contains the PAN promoter. Examination of downstream sequence will reveal the gene regulated by this promoter. Another locus, IDL5, has produced a blue colony, on lacZa complementation screening, indicating an open reading frame fusion from an open reading frame in the IS900 flanking sequence. In vitro transcription-translation of another locus, IDL22 shows expression of a high molecular weight protein. Disruption of gene expression from loci such as IDL3, IDL5 and IDL22 is likely to be responsible for some of the phenotypic differences seen between M. paratuberculosis and other members of the M. avium complex. One or more loci may be linked to the obligate pathogen phenotype. This could provide a unique opportunity to identify genes involved in regulation of virulence and pathogenicity.