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. Author manuscript; available in PMC: 2008 Dec 3.
Published in final edited form as: Virology. 2008 Jan 31;374(2):361–370. doi: 10.1016/j.virol.2007.12.023

Figure 2. Analysis of the transcription activity of bicistronic genome analogs having genes that overlap by 24 nucleotides.

Figure 2

(A). Schematic representation of genome analog p8(+)NP and the mRNAs that are transcribed from upstream (mRNA1) and downstream (mRNA2) genes separated by the wild-type gene junction. (B). Schematic representation of genome analogs M24-GS and M24-U7, in which the mRNA2 initiation signal is located 24 nts upstream of the mRNA1 termination signal. M24-U7 differs from M24-GS in that it contains an U7 tract upstream of the transcriptional initiation site. Additional template M24-A7 was also generated in which the U7 tract was substituted for A7. (C). These bicistronic genome analogs were expressed in BHK cells, and their RNA synthesis activity compared to that of wild-type template p8(+)NP, which has conventional non-overlapping genes. Actinomyin 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. (D). The RNA synthesis activity of bicistronic templates was also analyzed by primer extension analysis using end-labeled oligonucleotide 1434, which anneals within the 5’ end of mRNA 2, shown schematically in panels A and B. Alignment of the 1434 extension product with the adjacent sequence ladder confirms the mRNA2 product of template M24-U7 is initiated at the overlapped initiation site.