FIG. 2.

(A) Schematic diagram of pT7CAT-5375. The thin line indicates the vector sequence, and the triangle on the line represents the location of the T7 RNA polymerase promoter. The thick line indicates the PSIV sequence. The open and shaded boxes show the CAT gene and PSIV capsid-coding region, respectively. The nucleotide positions in the PSIV genome are indicated above the line. The EcoRI site used to linearize the plasmid is also shown. (B) Cap influence on in vitro translation of the RNAs transcribed from pT7CAT-5375. Capped and uncapped RNAs were translated in a rabbit reticulocyte lysate with or without a cap analog, m⁷GTP. To examine the electrophoretic mobility of the CAT protein, uncapped RNA from pT7CAT, which contains only a CAT gene, was also translated. Each product was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% polyacrylamide), blotted onto a polyvinylidene difluoride membrane, and then detected by enhanced chemiluminescence. The positions of the translation products (CAT and capsid protein) and molecular mass markers are indicated on the right and left of the panel, respectively. The 55-kDa bands observed in lanes 3 to 5 were thought to be insufficiently denatured proteins translated from the second cistron (see Materials and Methods).