Fig. 6.

The δ-subunit gene enhancers increase promotion from the transcriptional start site of the enkephalin gene. C2C12 myotubes were transfected with MEK pXP2, δ −47/−1 MEK pXP2, or δ −81/−32 MEK pXP2 along with MCKCAT. Cells were harvested 48 hr after transfection, and RNA was isolated for mapping the transcriptional start site by RNase protection assays. The diagram at thetop illustrates the organization of the transfected constructs containing the δ-subunit gene enhancer 5′ of the MEK promoter, which flanks the transcriptional start site of the enkephalin gene (indicated by the arrow). The arrow above the δ-enhancer is meant to indicate potential transcriptional start sites. The thick line below the top diagram indicates the region of DNA represented in the antisense RNA probe. Illustratedbelow the probe are possible RNase-resistant products that are expected to be generated depending on whether transcription begins at the enkephalin or δ-gene start sites. The bottom halfof the figure shows results from an RNase protection assay. RNA isolated from C2C12 cells transfected with the MEK pXP2, δ −47/−1 MEK pXP2, or δ −81/−32 MEK pXP2 constructs, and cotransfected with MCKCAT DNA, was probed with the 865 nt probe and a 270 bp CAT probe. Protection of a product at 800 nt indicates promoter activity originating from the MEK start site, which is increased by the presence of the δ-sequences. Protection of the 270 nt probe was used to assay MCKCAT expression to account for transfection variations.