Figure 5

Detection of re-initiation events by Western Blot. BTH102 were transformed with empty pREAL, or pREAL ligated to the WildType, Nonsense or Frameshift fragments of the brca1 gene. Transformants were cultured, centrifuged, and the protein pellets were resolved on 10% SDS-PAGE. The membrane with the transferred proteins was probed with a monoclonal antibody for fragment T18. The corresponding insert sequences are shown at the right. PCR primer sequences are shown in gray, stop codons in red, and translation initiation codons in green. Lane1: BTH102 transformed with empty pREAL. A 45 kDa band is observed corresponding to the fused T25 and T18 fragments. Lane 2: BTH102 transformed with pREAL/WT (wild type exon 11 of brca1). The upper band of 59kDa corresponds to the T25-insert-T18 protein. Two smaller fragments can be observed, likely produced by translation re-initiation events from two downstream and in-frame ATG codons. The lowest band (21 kDa) corresponds to translation re-initiation of fragment T18. Lane 3: BTH102 transformed with pREAL/FS (exon 11 of brca1 with a frame shift mutation that introduces a PSC). Notice the absence of the complete 59 kDa fragment due to the presence of a TAA stop codon. The presence of two smaller fragments can be explained by two in frame initiation codons (GTG and ATG). The lowest band corresponds to translation re-initiation of fragment T18. Lane 4: BTH102 transformed with pREAL/NS (exon 11 of brca1 carrying a nonsense mutation). The absence of the full-length 59 kDa fragment is caused by the TAA codon included in the primer sequence. One smaller fragment is observed, consistent with an initiation ATG codon. Again, the lowest band corresponds to fragment T18.