T (P < 0.01) (Figure 2A, constructs 1 and 2), whereas deletion of the MCK

T (P < 0.01) (Figure 2A, constructs 1 and 2), whereas deletion of the MCK gene 5'-enhancer results in a greater than 10-fold decrease (P < 0.01). To determine whether the MCK-SIE is critical for MCK gene transcription, it was deleted from the 6.5MCK-CAT construct and the resulting 6.5MCKSIECAT was tested in differentiated skeletal muscle cultures (Figure 2A, construct 3). The deleted construct exhibited a 60 decrease in MIN-101 web transcriptional activity in skeletal myocytes (P < 0.01), demonstrating that, in the context of the 6.5-kb MCK genomic sequence, the MCK-SIE is likely responsible for much of the positive transcriptional activity of MR1.MCK-SIE is active in differentiated skeletal muscle cells when placed 5' of the MCK proximal promoterTo facilitate further analysis of MR1 regulatory functions, subsequent studies were carried out in the context of MR1 placed 5' of the highly conserved MCK proximal promoter (Figure 2B (MR1-proximal promoter-chloramphenicol acetyl transferase (MR1-PP-CAT)), construct 2). This test construct frees MR1 from transcriptional effects of the highly active MCK 5'-enhancer, which could lead toTai et al. Skeletal Muscle 2011, 1:25 http://www.skeletalmusclejournal.com/content/1/1/Page 4 of-1 5'-Enh PP SIE MR13 SIE 4 5'-Enh+A-1256 --MRB1 +740 +904 +-+PP +1721 -358 +7 PP +1721 -358 +7 PP +998 -358 +904 +7 PP +7 PP2 +SIE +904 +MR1BMRMR1B4 -5'EnhFigure 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 (6.5MCKMR1-CAT, #2), the 6.5-kb construct with the MCK-SIE deleted (6.5MCKSIE-CAT, #3) or, for comparison, the 6.5-kb construct with the 5'-enhancer deleted (6.5MCKEnh-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 (MR1SIE-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 ? standard deviation.-1050 -SIE SI E++Tai et al. Skeletal Muscle 2011, 1:25 http://www.skeletalmusclejournal.com/content/1/1/Page 5 ofdampened effects of mutations or deletions within MR1. Importantly, it also avoids potential confounding effects due to cotranscriptional or posttranscriptional events, such as altered splicing efficiency or altered elongation efficiency, which could occur in conjunction with testing MR1 function within its 3' intron 1 location in the native MCK PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/21095114 gene. In agreement together with the decreased activity observed when MR1 is deleted in the six.5-kb sequence (Figure 2A), MR1-PP-CAT exhibits transcriptional activity in skeletal myocyte cultures that is definitely approximately threefold higher than that in the proximal promoter alone (Figure 2B, compare constructs 1 and two). M.