FIG 7.

Linker scanning mutagenesis shows that the 3′ half of region 2 modulates replicon copy number and/or partitioning efficiency. (A) Diagram of the HPV18 central URR and ori. The location of Bgl linker scanning mutations introduced into HPV18 region 2 are indicated by individually numbered white boxes (Bgl 1 to 13). All Bgl linker replicons lack the CER element (Fig. 1B) and are compared to positive controls with and without a CER element. (B) Keratinocyte colonies arising from continuous G418 selection were stained with methylene blue approximately 14 days posttransfection. Neomycin-resistant colonies were counted and averaged from three biological replicates. (C) DNA collected from five passages of transfected cells were analyzed by Southern blotting as described in Fig. 2C. The monomeric, supercoiled form (s/c) of the replicon is indicated. (D) HPV18 genome (left) and replicon (right) copy numbers were measured by qPCR. Error bars represent the standard deviation. The data shown are representative (panels B and C) or an average (panel D) of two or three (panel B) biological replicates. Significance was determined by unpaired t test with unequal variance. n.s. (no significance), P > 0.05.