IDENTIFICATION OF MYCOBACTERIUM-PARATUBERCULOSIS GENE-EXPRESSION SIGNALS

Mycobacterium paratuberculosis promoter-containing clones were isolate d from a genomic DNA library constructed in the transcriptional-transl ational fusion vector pYUB76. The promoter-containing DNA fragments we re identified in the surrogate host Mycobacterium smegmatis by express ion of the promoterless lacZ reporter gene of pYUB76. The expression s ignals exhibited a wide range of strengths, as indicated by their corr esponding p-galactosidase activities. Eight clones were sequenced and characterized further. Predicted open reading frames and codon usage w ere identified by computer analysis. Database searching for related se quences using the BLAST method revealed no homologies. Transcriptional activity was measured by slot-blot hybridization with steady-state RN A isolated from lacZ(+) M. smegmatis clones. Primer extension analysis identified the transcription start sites within the cloned fragments. The promoter regions characterized in this study were used to establi sh a consensus promoter sequence for M. paratuberculosis. M. paratuber culosis consensus hexanucleotide sequences of TGMCGT and CGGCCS centre d approximately 35 and 10 bp upstream from the transcription startpoin ts do not correspond to the consensus hexanucleotides of Escherichia c oli promoters.