FIG. 2.
Analysis of 99KO, 99STOP, and 99ΔMyr BAC by Southern hybridization. (A) DNA prepared from three independent blue, ampicillin-resistant colonies of BAC-containing E. coli that had been recombined with the linear Ampr lacZ cassette were digested with HindIII, subjected to electrophoresis in agarose gel, and transferred to nitrocellulose membranes. Hybridization was carried out with a 32P-labeled probe generated by nick translation of DNA containing the Ampr lacZ cassette. Arrow, migration of the approximately 8-kbp HindIII fragment containing the Ampr lacZ cassette. Note that a single fragment hybridized with the probe, indicating a single site of insertion. (B) DNA prepared from E. coli containing the wild-type AD169 BAC (AD169), 99KO BAC, mutant 99STOP BAC, and mutant 99ΔMyr BACs was digested with HindIII and analyzed as described for panel A by Southern hybridization with a 32P-labeled probe generated by nick translation of DNA containing the intact UL99 ORF. Arrow, migration of the HindIII R fragment at approximately 6 kbp. The weak signal in the lane containing the 99KO DNA is secondary to the approximate 100 bp of the 3′ end of the UL99 ORF that remains following this insertion and deletion.