Figure 2. Southern blot analysis of Himar1 transformants of E. chaffeensis.

Genomic DNA from Himar1 transposon E. chaffeensis mutants was assessed by DNA blot analysis using a spectinomycin resistance gene (aad) probe following digestions with different restriction enzymes (N, Nde I; S, Spe I; Bs, Bsrg I; E, EcoR V; B, Bgl II and Hind III). A) E. chaffeensis genomic DNA was recovered from the mutant organisms of mCherry plasmid transformed (m); GFPuv plasmid transformed (1^st experiment) (g) grown in ISE6 tick cells (I) or DH82 macrophages (D). B) Genomic DNAs from the mCherry and GFPuv (1^st experiment) mutants propagated in DH82 cultures was also assessed at two different randomly selected harvest times (separated about 1 month apart) to evaluate the stability of the transformants over time. The lanes mD1 and mD2 represent two different days when mCherry mutants were harvested, and gD1 and gD2 represent different harvest dates for GFPuv mutants. C) Second GFPuv plasmid transformed mutants grown in DH82 culture and assessed by DNA blot analysis (GD). Genomic locations for the DNA fragments indicated by the arrows were established by sequence analysis. Values shown on the left side of panels represent DNA size markers.