Enhancer Traps for Detecting and Cloning Developmental Genes
Molecular genetics provides a streamlined method for the detection of developmentally regulated genes in Drosophila. This regulation can be either with respect to time or with respect to position of the cell. A segment of DNA containing an enhancerless promoter connected to a β-galactosidase gene is inserted into the genome at random locations.Only if the inserted DNA falls near an enhancer will the promoter be activated and will β-galactosidase be synthesized. If the enhancer regulates a developmental gene, then the pattern of β-galactosidase synthesis
Figure 17.8 An enhancer trap vector inserted into chromosomal DNA placesthe β-galactosidase gene under control of a nearby enhancer so that the adjacent DNA can be easily cloned.
will similarly be developmentally regulated. As a result, the pattern in space or time of β-galactosidase synthesis will be the same as that normally followed by the gene under influence of the enhancer. This pattern can be visualized by immersing the embryos in a substrate like X-gal whose product after cleavage by β-galactosidase is insoluble and highly colored.
Once an insertion of interest has been found, subsequent cloning steps can proceed efficiently. If the inserted DNA is a complete E. coli vector and lacks a particular restriction enzyme cleavage site, then a clone can simply be made containing DNA from either side of the insertion. DNA from the insertion strain is cleaved with the enzyme, circularized, and transformed into E. coli. Since the cleavage will occur only in the Drosophila DNA, such DNA will flank the vector sequences, and the recircularized vector will contain some surrounding DNA (Fig. 17.8). Once this has been accomplished, a cDNA library can be screened for sequences also contained on the clone from the enhancer trap. Any such sequences that are expressed with the same pattern as the original β-galactosidase expression pattern are good candidates for developmen-tally regulated genes.
Copyright © 2018-2020 BrainKart.com; All Rights Reserved. Developed by Therithal info, Chennai.