You have identified an enhancer trap line (see Figure 14.17) gene...

Question

You have identified an enhancer trap line (see Figure 14.17) generated by P element transposition in Drosophila in which the marker gene from the enhancer trap is specifically expressed in the wing imaginal disc.

How can you identify the gene adjacent to the insertion site of the enhancer trap?

Accepted Answer

Welcome back. Here's our next problem. While performing the I PC R, the genomic DNA is digested with a restriction enzyme that cuts within the P element. But outside of the enhancer trap cassette, what would happen if the restriction enzyme selected cuts inside the enhancer trap cassette? Choice A the enhancer trapped cassette would be amplified more efficiently resulting in a higher yield of the I PC R product choice B. The restriction enzyme would not affect the enhancer trap cassette. And the expression of the marker gene in the wing imaginal disk could still be detected to ac the restriction enzyme would cut the DNA randomly resulting in fragments of varying sizes. Some of which may contain the flanking genomic DNA sequence adjacent to the enhancer trap insertion site. Or choice D, the I PC R product would not contain flanking genomic DNA sequence adjacent to the enhancer trap insertion site, making it difficult to identify the adjacent gene. So the goal of the I PC R is to locate or identify where your enhancer trap inserted into the genome by being able to start inside that sequence and then synthesize the piece of DNA going into the genome. So you have your inserted piece of DNA. I draw a little box here and you're amplifying the DNA from a point within your enhancer trap, going out in either direction into the areas of the genome where it inserted. And we use restriction enzymes to cut the DNA into different pieces and cut once within the P element, but outside of the enhancer trap itself. So our question is asking what would happen if the restriction enzyme cut inside the enhancer trap cassette. Well, if we cut both at that P element and within the enhancer trap cassette, we would end up with a situation. In choice D, we would not contain the flanking genomic DNA. We would have just that little inner part here. And therefore, we would not be able to identify the adjacent gene since we wouldn't be going out into the genomic DNA to see where that enhancer trap integrated into the genome. If we look briefly at our other answers, choice A says that the enhancer trap cassette would be amplified more efficiently. Well, a different um restriction enzyme site, a different location of cutting. The DNA is not going to affect the efficiency of this PC R. So choice A is not our correct answer. Choice. B says this restriction enzyme would not affect the enhancer trap cassette and the expression of the marker gene, the wing disc will still be detected. But again, if we've cut the P element and we've cut within our enhancer trap, we lose the ability to go out into the genome. So that's why choice B is not correct there. And then choice C, the restriction enzyme would cut the DNA randomly resulting in fragments of varying sizes, some of which may contain the flanking genomic DNA sequence. Again, we've now put two sites that cut the DNA within our inserted piece here and don't go out into the genomic DNA. So that's why choice C is not correct. So again, our correct answer here of the effect of the restriction enzyme cutting inside the enhancer trap cassette is choice D that our PC R product would not contain the flanking genomic DNA sequence, making it difficult to identify the adjacent gene. See you in the next video.

You have identified an enhancer trap line (see Figure 14.17) generated by P element transposition in Drosophila in which the marker gene from the enhancer trap is specifically expressed in the wing imaginal disc. How can you identify the gene adjacent to the insertion site of the enhancer trap?

Key Concepts

Enhancer Trap

P Element Transposition

Gene Mapping Techniques

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