Skip to main content
. 1999 Apr 13;96(8):4627–4632. doi: 10.1073/pnas.96.8.4627

Figure 7.

Figure 7

Disruption of the N-box motif reduces drastically expression of AChE promoter–reporter gene constructs in TA muscle. (A) EMSA using radiolabeled oligonucleotides containing the N-box motif. Note that one specific DNA–protein complex (arrow) was formed when a 24-bp oligonucleotide encompassing the first intronic N-box at position +755 (N int-1) was used. Formation of this protein complex was blocked by competition with a 250-fold molar excess of unlabeled wild-type oligonucleotides (WT oligo). Mutation of the core sequence as shown in C, abolished its protein-binding capacity, as indicated by the inability of the mutant oligonucleotides to compete in formation of specific protein complex. Note also that oligonucleotides containing the two palindromic N-box motifs located in the promoter region (N prom) displayed a weaker affinity for specific protein complexes. Arrowhead indicates the amount of unbound radioactive oligonucleotides present in each sample. (B) The protein complex was supershifted (white arrow) by an additional incubation with antibodies against either GABP α or GABP β. (C) Schematic representation of the nucleotides that were mutated (underlined) in the core region of the first intronic N-box motif at position +755 in NRAP to generate the mutant N-box-NRAP promoter fragment (mN-NRAP). As shown in A, this mutation resulted in a failure to compete in formation of specific DNA–protein complexes. (D) Expression of β-gal in TA muscles injected with reporter plasmids containing either NRAP or mN-NRAP. Note that disruption of this DNA regulatory element essentially abolished (P < 0.001; Student’s t test) expression of the reporter gene, indicating that the N-box is involved in enhancing expression of AChE in muscle. Expression of β-gal was normalized to the activity of a coinjected CAT plasmid used to monitor transduction efficiency. (E) The percentage of synaptic events was also significantly reduced (P < 0.005; Student’s t test) in muscles injected with reporter plasmids containing mN-NRAP. Mean ± SE is shown; a minimum of 10 muscles were analyzed per construct.