2. Stop! a. Which of the following best describes why splicing is important for

Question

2. Stop! a. Which of the following best describes why splicing is important for the detection of a nonsense mutation? A. An mRNA that is not spliced cannot be exported from the nucleus. B. An mRNA that is not spliced will not have exon junction complex proteins associated with it. C. An mRNA that is not spliced cannot be bound by the ribosome. b. The Salmon gene is GFP tagged in a human tissue culture cell line. One of the cells in the line acquires a nonsense mutation in the Salmon gene. i. Which of the below methods would allow one to identify the presence of the nonsense mutation? Select all that apply. A. Northern blot B. Western blot C. RNA Seo D. Immunoprecipitation ii. For the above technique(s) that you selected, describe the experimental result that would be observed from them assuming there is a nonsense mutation. ii If you were detecting the nonsense mutation through the GFP tag, does it matter if the GFP gene was added at the 5' end of the Salmon gene versus the 3' end? YES or NO. Which of the statements below best describes your selection. A. As long as the promoter is unaffected, protein will still be produced. B. Anything downstream of the nonsense mutation will not be translated. C. Anything downstream of the nonsense mutation will not be transcribed. D. As long as there is no mRNA splicing occurring, exon junction complexes will not impact the process

Explanation / Answer

a) The option which best describes why splicing is important for the detection of a nonsense mutation is 'option c'. As splicing is very much needed for the RNA to get transported from nucleus to the cytoplasm, but until and unless the ribosomes bind to the RNA in the cytoplasm, we will not get a protein. Once the translation is completed, then only we can see, whether the protein obtained is a functional one or a non-functional truncated protein. If it is a non-functional truncated protein, then only we can conclude that a nonsense mutation has occurred.

b) (i) The methods which would allow one to identify the presence of the nonsense mutation are Western Blotting, RNA seq. and Immunoprecipitation.

(ii) In Western blot, we can see the proteins and can compare the sizes of the wild type GFP tagged Salmon gene protein and the mutated one having the nonsense mutation. The mutated protein will be of different and shorter in size than the wild type protein.

Similarly with Immunoprecipitation technique we can precipitate the wild type GFP tagged Salmon gene protein with its appropriate antibody, whereas we cannot precipitate the mutated protein with same antibody which may not bind to the mutated protein.

With RNA sequencing we can detect the small changes in RNA transcribed from the mutated gene. Thus, we will be able to detect the nonsense mutation by comparing it with the RNA sequence transcribed from wild type gene.

Whereas with technique like Northern Blot which only detects the presence and size of RNA, will fail to detect a point mutation in the gene. The resolution of this type of gel will not allow one to detect a small addition or deletion of RNA.

(iii) YES, it matters. As (B) Anything downstream of the nonsense mutation will not be translated.

So, we have to tag GFP at the 5' end, and not at the 3' end, as, if there is a nonsense mutation in the coding region of the gene the translation will stop there releasing the truncated protein. As translation proceeds from 5' end to 3' end, if we add the GFP to the 3' end, it may not get translated if there is a nonsense mutation in the coding region.

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