Drosophila FLP/FRT Screens and a model for cancer geneticsReading: Chapter 18, pp637-639Problem set GSo far we have mentioned how hypomorphic mutations (vulvaldevelopment mutants in C. elegans) and haploinsufficiency in sensitizedgenetic backgrounds (temperature-sensitive sev mutant background) canidentify essential genes involved in development. In these cases null oramorphic alleles of these genes result in an early lethality that precludesus from understanding their roles in signaling. Another strategy is tomake clones of cells that are mutant for an essential gene in the tissue thatyou are studying. As we saw in the last lecture on mosaic analysis, theproduction of clones can be stimulated with X-rays. Today, we willdiscuss recent advances that use genes and sites that are involved inrecombination in yeast to stimulate mitotic recombination in Drosophila.We will then discuss how these tolls are used to screen specificchromosomal arms for mutations that disrupt targeting of thephotoreceptor cells to the brain.The yeast FLP recombinase and FRT sitesWhere and when mosaic clones are produced during development can becontrolled by using tissue specific enhancers to drive recombinaseenzymes that recognize specific sites engineered into the genome. Theyeast 2 um plasmid encodes an enzyme known as the FLP recombinasethat recognizes two FRT sites and catalyzes reciprocal exchange betweenthese sites. This recombination occurs during replication and results in aninternal flipping the orientation of the regions flanking the two sites. Thisflipping results in an addition round of replication and is thought to beimportant to amplification of the 2 um plasmid, which exists in high copynumber in yeast.The FLP recombinase and FRT sites can also be used to stimulatereciprocal exchange in flies. The FRT sites, which are recognized andcleaved by FLP recombinase to initiate crossing over between the two sites,have been placed near the centromere on all chromosomal arms using Pelement transposition. To stimulate exchange, FLP recombinase can beexpressed in any tissue to stimulate mitotic recombination.We will consider a specific example of the use of FLP recombinaseand FRT sites to screen for mutations that disrupt the pattern ofphotoreceptor axon targeting in the fly brain.Tumor suppressor genesTumor suppressor genes usually encode proteins that inhibit cellproliferation. Although more rare, Tumor suppressor genes can alsoencode proteins that inhibit apoptosis. Mutated forms of these genes canbe inherited dominantly, but a second event must occur before the mutantcontributes to cancer. The wild-type copy of the gene must be lost, and thiscan occur by spontaneous mutation, chromosomal loss (not shown above)or mitotic recombination. This “loss of heterozygosity” leads to a cell thatis no longer inhibited from proliferating or a surviving cell that isprecancerous and normally fated to die. The diagram showing how bothspontaneous and inherited forms of retinoblastoma occur whenmutations lead to a mutant Rb locus. In inherited forms of Rb, the onemutant allele is inherited, but the wild-type allele must be lost beforeretinoblastoma tumor is formed. This trait is inherited in a dominantfashion, but the at the level of the cell the cancer only forms when bothcopies are mutant.FLP/FRT screens for tumor suppressor genes in DrosophilaIswar Hariharan and his coworkers have developed screens to identifytumor suppressor genes in the fly. The strategy is to generate mutations ina single chromosome and then to stimulate recombination to generateclones that are homozygous for the mutation. If the mutation is in a tumorsuppressor gene, these cells should proliferate at the expense of cellscontaining the wild-type gene.Several tools are used in these screens. First, the chromosome armscreened contains an FRT site near the centromere, inserted into thechromosome using P element mediated transformation. Chromosomearms are screened one at a time, and the screens will only identifymutations that are distal to the FRT site. These sites are wherehomologous recombination occurs and are necessary to generate clonesthat are homozygous for the mutation in the tumor suppressor gene.Second, FLP recombinase is expressed in a particular tissue using aspecific promoter. This drives recombination in that tissue alone andavoids the possible lethality associated with the loss gene function inother tissues. In this case the eyeless promoter is used, which drivesexpression of the recombinase only in the developing eye. The eyeless-FLPtransgene is inserted into a chromosome using P-element mediatedtransformation. Finally, a cell autonomous marker is used to identify theclones that are homozygous for the mutation. These are usually a Pelement-transposed wild-type white gene. In a white mutant background,the P[w+] is placed on the chromosome not containing the mutation. Inthis way, white clones that lack the P[w+] transgene will be homozygousfor the mutation.The rationale for the screen is described in the Figure below. Thestrain is homozygous mutant for white but also has a transgene containinga copy of the wild-type white gene (P[w+]) on the autosome that containsthe FRT sites. The eyFLP is on the X chromosome and that X is alsomarked by the yellow mutation. It’s here to mark the X containing eyFLP,but don’t worry about it here. Let’s consider the situation now where theautosome lacking P[w+] contains a mutation in a tumor suppressor gene(labeled m). Before recombination the cells are heterozygous for m andhence normal (remember that tumor suppressor genes are recessive at thelevel of the cell—they must be homozygous to form a tumor) andheterozygous for P[w+] and hence are red. In the developing eye, FLP isexpressed and this stimulates mitotic recombination between the FRTsites. Some of these recombination events will lead to cells that arehomozygous for the m tumor suppressor mutation. These cells (on the left)will be white and continue to proliferate because of the mutation, whereasthe cells that aren’t homozygous for the mutation will be red and won’tdivide in an uncontrolled fashion.This situation leads to the expansion of white cells in a mosaic eye.Below are mosaic fly eyes that don’t contain a tumor suppression (left) orhave mutations in a tumor suppressor gene called sav (middle and right).The allele on the right is more severe than the allele in the middle so thereare more white cells and the eye is
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