Fig. 2.

Construction of ATI and A26 deletion mutants by homologous recombination: (A) deletion of ORF 149. The arrangement of ORF 148 (RPO132 RNA polymerase subunit), ORF 149 (ATI), ORF 150 (VACV A26 homolog) and ORF 151 (VACV A27 homolog) within the CPXV genome is shown. ORF 149 overlaps ORF 148 by 22 nt; ORF 149 is separated by 44 nt from ORF 150; ORF 150 is separated by 51 nt from ORF 151. The first step was replacement of the region, from 39 nt to the right of the start codon including the ATI promoter to 21 nt preceding the 148 ORF, by the GFP ORF regulated by the P11 late promoter. In the next step, GFP was removed to create the ΔATI deletion mutant, in which the stop codon of ORF 148 was separated from the stop codon of ORF 150 by 5 nt. (B) Deletion of ORF 150. The first step was replacement of the region, from 51 nt upstream of the start codon of ORF 150 including the promoter to the stop codon, by GFP regulated by the P11 late promoter. The GFP ORF was then removed to create the ΔA26 deletion mutant, in which the stop codon of gene 151 was separated from the stop codon of 149 by 44 nt.