1. What can we conclude from the overexpression experiment? 2. Did the overexpre
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Question
1. What can we conclude from the overexpression experiment?
2. Did the overexpression of hnRNP A1 and A2 have any effect on the silencing, as measured by exon 7? Where is the evidence for this in the figure?
3. Did the mutant forms of the silencer differ from the wt in their response to the overexpression?
4. Did the authors make a convincing case that hnRNPA1 and A2 are involved in the activity of this inrronic spicing silencer? Explain.
09 89 100 47 73 64 91 20 3 Figure 5. Effects of hnRNP A1 or A2 overexpression on Exon 7 inclusion in SMNI Minigene Transcripts HEK203 cells were transfected with 5 of the indicated WT or mutant pa-SMN1 plas- mids (described in Figures 3A and 30) The indicated amounts of PCGTT-A1 (A) or pCGTT-A2 (B) expressing N-terminal T7-tagged hnRNP A1 or A2 proteins were cotransfected with the Ni reporters. Two days after transfection, total RNA was collected and radioactive RT-PCR was performed to measure the extent of exon 7 inclusion by native PAGE and phos- phorimage analysis. The expression of tagged hnRNP A1 or A2 was verified by western blotting with monoclonal antibody against the T7 tag. The histograms on the right show the corresponding quantitation from three independent experiments. Emor bars represent the standard deviation.Explanation / Answer
1. Over expression experiment are performed for novel proteins identification and their function study.ex- over expression of proteins can be seen as Down syndrome,cancer in humans.
2. Yes, it have effect .The evidence may be viewd at the bottom specifying the expression of hnRNP A1 or A2 verified by western blotting with monoclonal antibody.
3. No, they don't differ.The mutants when assayed in splicing system with recombinant hnRNP results in recovery of inhibition similar to obtained with WT protein.
4. Spicing silencer cause skipping of exon because of involvement of polypyrimidine tract binding protein.Sequences reduces recognition and use of splice site.A1 is very important intronic spicing enhancer and its regulated by phosphorylation
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