You have identified an enhancer trap line generated by P element transposition i

ID: 71962 • Letter: Y

Question

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

PART A

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

D. Nothern blot hybridization using P element RNA or cDNA as a probe can indicate the nature of adjacent genes.

How would you show that the expression pattern of the enhancer trap line reflects the endogenous gene expression pattern of the adjacent gene?

Select the 2 correct answers.

A. A genomic DNA library can be created from the line; clones that include the P element and flanking DNA sequences can be identified using P element DNA as a probe. B. Common PCR can be used to amplify sequences flanking the inserted P element. C. Comparing fragments resulting from treating whole-genome DNA from wild-type cells and enhancer trap line cells by a certain restriction endonuclease would indicate the particular site of P element insertion; hence adjacent sequences can be deduced.

D. Nothern blot hybridization using P element RNA or cDNA as a probe can indicate the nature of adjacent genes.

How would you show that the expression pattern of the enhancer trap line reflects the endogenous gene expression pattern of the adjacent gene?

Select the 2 correct answers.

-Expression of the endogenous gene identified in the enhancer trap line could be studied by in situ hybridization. -Expression of the endogenous gene identified in the enhancer trap line could be studied by using GFP or lacZ reporter genes. -Expression of the endogenous gene identified in the enhancer trap line could be studied by creating a construct consisting of the endogenous gene juxtaposed with different endogenous promoters and enhancers; this construct can be further transformed into wild-type Drosophila cells. -Expression of the endogenous gene identified in the enhancer trap line could be studied by creating a knock-out of the endogenous gene and assessing the resulting phenotype. -Expression of the endogenous gene identified in the enhancer trap line could be studied by creating a Drosophila line which lacks the enhancer "trapped" by the P element and comparing the expression of the endogenous gene in that line and in wild-type line by Nothern blotting.

Explanation / Answer

The enhancer trap has a P element transpoton and a marker gene in it. Since the element hijacks enhancer from another gene, the marker gene is expressed. Hence, the correct option is

D. Northern blot hybridization using p element RNA or cDNA as a probe can indicate the nature of adjacent genes

After insertion, the p element used the enhancer of the gene where it got inserted. This gene can be analyzed from the expression pattern of the marker gene, by detection of mRNA or cDNA of the expressed marker gene, and subsequent PCR amplification of the flanking sequences.

Option B. Common PCR can be used to amplify sequences flanking the inserted P element, can also be used after the inital step.

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You have identified an enhancer trap line generated by P element transposition i

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