(A). Schematic representation of genome analogs in which the mRNA 2 initiation signal was positioned at increasing distances upstream of the mRNA1 termination signal, up to a distance of 86 nucleotides. Transcription products mRNA1 and mRNA2 are also shown schematically. (B). Bicistronic genome analogs were generated in BHK cells, and their RNA synthesis activity compared to that of wild-type template p8(+)NP, which has conventional nonoverlapping genes. Actinomycin D-resistant RNAs were metabolically labeled with 3H uridine, harvested, treated with RNase H/oligo d(T) and then electrophoresed on a denaturing 1.75% agarose-urea gel. RNAs were visualized by autoradiography and the positions of mRNAs 1 and 2 are marked with arrowheads.