Concept explainers
You have synthesized an enhancerless GFP reporter
gene in which the jellyfish GFP cDNA is placed
downstream of a basal promoter that functions in
mice. You will now fuse this enhancerless reporter to
the three types of sequences listed below (x–z).
a. Which of the three types of sequences would you
use for which of the three listed purposes (i–iii)? In
each case, explain how the particular fusion would
address the particular use.
Types of sequences fused to the reporter:
x. random mouse genome sequences
y. known mouse kidney-specific enhancer
z. fragments of genomic DNA surrounding
the transcribed part of a mouse gene
Uses:
i. to identify a gene’s enhancer(s)
ii. to express GFP tissue-specifically
iii. to identify genes expressed in neurons
b. Which of the sequences (x–z) would you fuse to a
particular mouse gene of interest in order to express the protein product of the gene ectopically,
that is, in a tissue in which the gene is not usually
expressed? Why might you want to do this experiment in the first place?
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- 1.Your goal is to design a strategy to create thisfinal pDHFR plasmid for fusion protein expressionfrom the materials available:You have an empty pET21a expression vector and another vectorthat contains the fusion protein nucleotide sequence (pBluescript + GST-DHFR-His). (#) indicatesbp location ofenzyme cut: a.EcoRIonly, HindIII only, NotIonly, EcoRI and HindIII, EcoRI and NotI, or HindIII and NotI? b.Briefly explain the rationale for your selection. Please connect your rationale to the need for compatible ends without any further manipulation and required directionality of the DHFR fusion protein coding sequence relative to the T7 promoter that will drive its expression. c.What is the final size(kb)of your desired pDHFR plasmid ligation product based onyour design? d. Complete the chart providedto indicate thesize of all fragments(in bp)resultingfrom complete digestion of each plasmid givenyour strategy, andthe fragment(in kb)from each you would isolate for ligation.Please mind…arrow_forwarda. Would you expect a cell to continue or to stopdividing at a nonpermissive high temperature if itis a temperature-sensitive Ras mutant whose protein product is fixed in the GTP-bound form atnonpermissive temperature?b. What would you expect if you had a temperaturesensitive mutant in which the Ras protein staysin the GDP-bound form at high temperature?arrow_forwardMechanisms that inhibit TE activity include theproduction of transposition repressors throughalternative splicing and the action of _________ thatblock TE transcription.arrow_forward
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