428498.fig.002a
(a)
428498.fig.002b
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428498.fig.002c
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428498.fig.002d
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428498.fig.002e
(e)
Figure 2: Restriction analysis of BACs and BAC-derived viral genomes. (a) EcoRI and HindIII restriction maps illustrate restriction fragments near the right end of BAC-cloned genomes that retain BAC ori. EcoRI T and HindIII V fragments (grey) are designated [17]. Genetic features of this region are illustrated, including the US28, BAC ori cassette, and US29 sequences in pYD-C29, CMV genes US29 to TRS1, and c repeat with single terminal a sequence. (b–e) Restriction digests of virion DNAs made from the vaccine virus mixture (mix) or varS and varL viruses isolated from the vaccine, virions from BAC-derived viruses (V), or circular BAC DNAs (B). Vb indicates TL17 virion DNA that retains the BAC ori; in all other BAC-derived viruses the BAC ori was excised. 4.2 kb XbaI, 5.3 kb EcoRI, and 3.0 kb HindIII fragments are unique to varS genomes, while 2.8  and 3.5 kb BglII fragments are unique to varL genomes. BACs lack 4.5 and 3.5 kb HindIII and 4.4 kb EcoRI fragments due to deletions and a 6.6 kb EcoRI terminal fragment because they are circular. All BACs contain an unexpected 3.4 kb BglII fragment due to a point mutation in the recombination vector. A 4.5 kb BglII fragment that may arise from a single copy of the a sequence is unique to TL17 virus with excised BAC ori.