FIG. 4.
Southern hybridizations between individual M. avium cDNA clones and cDNA probes prepared from M. avium grown for 48 and 110 h in human macrophages and from M. avium grown in Middlebrook 7H9 broth-OADC-0.05% Tween 80. At the time the original stock cDNA mixtures were prepared (cDNA mixtures 4-1, 5-1, and 6-1 [described in Table 1]) and at 6 (cDNA mixtures 4-2, 5-2, and 6-2 [Table 1]) and 11 (cDNA mixtures 4-3, 5-3, and 6-3 [Table 1]) months later, samples were removed from each cDNA mixture and prepared by three rounds of SCOTS. Labeled probes were prepared from each SCOTS-derived mixture by incorporation of digoxigenin-11-dUTP with PCR DIG Probe Synthesis kits (Roche Molecular Biochemicals) and hybridized to 12 individual cDNA clones that had originally been identified from the cDNA mixture prepared from M. avium grown for 48 h in macrophages (cDNA mixture 5-1 [Table 1]). DNA fragments from atpB, clpC902, lsr2, and c25, genes that were expected to be expressed under all of the growth conditions studied, were included in each hybridization assay as positive controls. Hybridization and wash conditions were as described in Materials and Methods.