RFLP analysis of pBR- and pBRf-derived viruses and characterization of mini-F loss in pBR-derived viruses. A. BAC DNA or viral DNA prepared from infected Vero cells was digested with XhoI and separated by 0.8% agarose gel electrophoresis. The lanes contain the DNA's from BAC DNA (pBRf, pBR), viruses reconstituted from cloned DNA (vBRf, vBR) or parental CPXV.BR DNA (BR). Changes of the restriction pattern due to the presence and therefore disruption of TK of absence of mini-F sequences are marked with boxes (see text). B. Southern blot analysis after transfer of the gel shown in (A) and using a DIG labeled TK probe. The changes seen with a DIG labeled TK probe show the expected changes due to the disruption of TK with mini-F sequences (pBRf, vBRf), the introduction of an inverse TK sequence (pBR), and the loss of any bacterial sequences in vBR (see text). Sizes of a molecular weight marker (generuler 1-kb plus DNA ladder, Fermentas) are given. C. Development of GFP fluorescence in plaques following removal of mini-F sequences. Initially, all plaques exhibit GFP fluorescence (plaque in left panels), which is then partially lost (plaque in middle panels) or completely absent (plaque in right panels). Identical plaques were photographed under fluorescent light (upper panels) or using phase contrast (lower panels) with a Zeiss Axiovert microscope and a CCD camera (Zeiss).