Please outline how the results shown in the gel picture were obtained. What are
ID: 80653 • Letter: P
Question
Please outline how the results shown in the gel picture were obtained. What are we looking at in this picture?TATTO 214G- C 276 be A2. Some cases of hereditary colon cancer result from mutations in an exonic splicing enhancer. As a result of these mutations, exon 3 is skipped and the protein (MLH1) is inactivated. In the experiments shown above, plasmids containing exon3 and its flanking introns were made and then introduced into cells by transfection. Some of the plasmids contained the wild type sequence and others contained mutations in the proposed splicing enhancer. Please outline how the results shown in the gel picture were obtained. What are we looking at in this picture? which nucleotides are important for the correct inclusion of this exon? Please explain as carefully as you can (note that some of the lanes are repeats of each other.)
Explanation / Answer
Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5'
splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the
troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron.
thepicture shows hereditary colon cancer result from mutations in an exonic spiling enhancer. plasmids contain exon 3 and its flanking introns were made and then introduced into cell by transfection.