Figure 2

MR1 is a positive regulator of MCK transcription. (A) MM14 skeletal myocytes were cotransfected with an MCK enhancer-alkaline phosphatase (AP) reference plasmid and test gene plasmids containing the chloramphenicol acetyl transferase (CAT) reporter gene driven by the full-length 6.5-kb MCK construct (6.5MCK- CAT, #1), the 6.5-kb construct with MR1 deleted (6.5MCK ΔMR1-CAT, #2), the 6.5-kb construct with the MCK-SIE deleted (6.5MCK ΔSIE-CAT, #3) or, for comparison, the 6.5-kb construct with the 5'-enhancer deleted (6.5MCK ΔEnh-CAT, #4). Test construct activities are represented as the average values of relative CAT over AP activity normalized to the activity of 6.5MCK- CAT. (B) MR1 is composed of regions that promote transcription in MM14 cultures. Constructs containing the "full-length" MR1 (MR1-PP-CAT, #2), a construct lacking the MCK-SIE (MR1ΔSIE-PP-CAT, #3) or just the MCK-SIE (SIE-PP-CAT, #4) were generated to test the functional activity of the MCK-SIE. Activities of these test constructs were normalized to activities of the proximal promoter alone (PP-CAT, #1). The activity of the 5'-enhancer (5'Enh-PP-CAT, #5) is provided for comparison. Each experiment was performed in at least twelve plates in three separate experiments, and activities are averages of those experiments. Error bars represent ±1 standard deviation.