Question 15. You are cloning Gene X from S. cerevisiae into a plasmid. You want
ID: 215294 • Letter: Q
Question
Question 15. You are cloning Gene X from S. cerevisiae into a plasmid. You want to include the sequence 1 kb upstream and downstream from the open reading frame to try to get the gene to express under its own promoter. For this particular region of the genome, there are no other expected AUG codons upstream of the open reading frame for Gene X. To ensure that the pro- tein is expressed, you try to complement the deletion strain for Gene Xwith your plasmid version of Gene X, but it is not comple- menting (not returning to the wild-type phenotype). You are not worried about splicing issues for S. cerevisiae. You suspect that you have a mutation in your cloned Gene X. The sequencing facility is down this week, but you have access to an in vitro trans- lation system in your lab. You translate the wild-type Gene Xand the cloned gene X using the in vitro translation system. You sep- arate your products using SDS-PAGE and stain with Coomassie Brilliant Blue (data below). You expect a small protein, so you use a low molecular weight maker 37 kDa -25 kDa -20 kDa 15 kDa 10 kDa Lane 1: Translation of Gene X Lane 2: Translation of cloned Gene X Lane 3: Molecular weight marker Lane 1 2 3 A. Do you think your cloned gene X has a mutation? If so, what type (missense, nonsense, frameshift)? Explain your answer in terms of the data B. Why do you think your cloned gene is not complementing the deletion strain? Explain your answer in terms of the data.Explanation / Answer
A. From the given gel, it is clear that the cloned gene produces a protein with higher molecular weight compared to the WT protein. This can happen only if it contains a framers ft mutation which alters/increases the length of the protein.Frame shift mutations change the sequence as well as length of a protein.
B. The cloned fragment does not complement the mutant as the protein product is nonfunctional. Frameshift mutation alters the protein sequence so the mutant protein may not exhibit the wt protein function.