|Kapur, Vivek - UNIV OF MN|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 28, 2003
Publication Date: N/A
Technical Abstract: The genomes of Mycobacterium avium complex (MAC) bacteria possess a high degree of similarity, but MAC members are capable of infecting a wide range of hosts. In order to better understand the genomic differences underlying variations in pathogenesis among MAC bacteria, we have constructed a whole-genome DNA microarray representing more than 98% of the open reading frames (ORFs) identified from the genomic sequence of M. paratuberculosis, the causative agent of Johne's disease in cattle. A library of PCR products was constructed with primers designed to amplify fragments = 500 bp from each ORF using purified genomic DNA as a template. The PCR products were then checked for quality on agarose gels, cleaned, and arrayed in triplicate onto poly-L-lysine coated glass slides. Genomic DNA from experimental mycobacterial isolates was randomly sheared, labeled with a fluorescent dye and applied to the microarray along with M. paratuberculosis DNA that had been labeled with a spectrally distinct dye. The arrays were then hybridized overnight at 64oC, washed and scanned. Spots in which less than 80% of the pixels were within two standard deviations of the background were discarded along with any ORFs that were not represented by at least two acceptable spots. Ratios of experimental to control DNA were calculated for the remaining ORFs and transformed into log2 for analysis. The software program GACK (Kim et al. Genome Biol. 3:research0065) was utilized to determine the cutoff for classifying ORFs as present or divergent. Our analyses revealed that M. avium subsp. avium (M. avium) and M. avium subsp. silvaticum (M. silvaticum ) had 147 and 219 ORFs classified as divergent, respectively, while M. paratuberculosis clinical isolates from sheep and mink had no ORFs classified as divergent. Many of the divergent ORFs were shared between M. avium and M. silvaticum, which may indicate the presence of less highly conserved regions in MAC genomes. Our studies should help resolve how genome composition and organization influence host specificity and pathogenesis among MAC bacteria.