FIG. 3.
(A) Diagram of UL44 mutants generated by shuttle mutagenesis. Initially, the entire UL44 ORF of BAC20 was deleted. This deletion mutant was used as the parental clone to reintroduce various constructs of UL44. (B, BamHI site; P, PstI site). The resulting mutants are given: BAC20 with a complete deletion of UL44 (20ΔgC); 20ΔgC in which the deletion was repaired (20ΔgC-R); 20ΔgC in which the UL44 sequence was reinserted with a defective start codon (20ΔMgC [the ACG depicts the replacement of ATG and the introduction of a defective start codon]); BAC20 in which UL44 is under the transcriptional control of the human cytomegalovirus immediate-early promoter (PCMV) (20ExpgC); BAC20 harboring UL44 with a mutated start codon driven by PCMV (20ExpΔMgC). (B) The resulting mutant BAC clones were analyzed by 0.8% agarose gel electrophoresis and ethidium bromide staining, and restriction patterns were compared with that of parental BAC20 DNA. DNA was isolated from E. coli, digested with the restriction enzyme BamHI, and separated. Lane 1, BAC20; lane 2, 20kanΔgC; lane 3, 20ΔgC; lane 4, 20ΔgC-R; lane 5, 20ΔMgC; lane 6, 20ExpgC; lane 7, 20ExpΔMgC. The 1-kb ladder (Invitrogen) was used as the size marker (lanes M). Asterisks indicate bands that arose as a consequence of mutagenesis and correspond exactly to calculated fragment sizes.