Drosophila FLP FRT Screens and a model for cancer genetics Reading Chapter 18 pp637 639 Problem set G So far we have mentioned how hypomorphic mutations vulval development mutants in C elegans and haploinsufficiency in sensitized genetic backgrounds temperature sensitive sev mutant background can identify essential genes involved in development In these cases null or amorphic alleles of these genes result in an early lethality that precludes us from understanding their roles in signaling Another strategy is to make clones of cells that are mutant for an essential gene in the tissue that you are studying As we saw in the last lecture on mosaic analysis the production of clones can be stimulated with X rays Today we will discuss recent advances that use genes and sites that are involved in recombination in yeast to stimulate mitotic recombination in Drosophila We will then discuss how these tolls are used to screen specific chromosomal arms for mutations that disrupt targeting of the photoreceptor cells to the brain The yeast FLP recombinase and FRT sites Where and when mosaic clones are produced during development can be controlled by using tissue specific enhancers to drive recombinase enzymes that recognize specific sites engineered into the genome The yeast 2 um plasmid encodes an enzyme known as the FLP recombinase that recognizes two FRT sites and catalyzes reciprocal exchange between these sites This recombination occurs during replication and results in an internal flipping the orientation of the regions flanking the two sites This flipping results in an addition round of replication and is thought to be important to amplification of the 2 um plasmid which exists in high copy number in yeast The FLP recombinase and FRT sites can also be used to stimulate reciprocal exchange in flies The FRT sites which are recognized and cleaved by FLP recombinase to initiate crossing over between the two sites have been placed near the centromere on all chromosomal arms using P element transposition To stimulate exchange FLP recombinase can be expressed in any tissue to stimulate mitotic recombination We will consider a specific example of the use of FLP recombinase and FRT sites to screen for mutations that disrupt the pattern of photoreceptor axon targeting in the fly brain Tumor suppressor genes Tumor suppressor genes usually encode proteins that inhibit cell proliferation Although more rare Tumor suppressor genes can also encode proteins that inhibit apoptosis Mutated forms of these genes can be inherited dominantly but a second event must occur before the mutant contributes to cancer The wild type copy of the gene must be lost and this can occur by spontaneous mutation chromosomal loss not shown above or mitotic recombination This loss of heterozygosity leads to a cell that is no longer inhibited from proliferating or a surviving cell that is precancerous and normally fated to die The diagram showing how both spontaneous and inherited forms of retinoblastoma occur when mutations lead to a mutant Rb locus In inherited forms of Rb the one mutant allele is inherited but the wild type allele must be lost before retinoblastoma tumor is formed This trait is inherited in a dominant fashion but the at the level of the cell the cancer only forms when both copies are mutant FLP FRT screens for tumor suppressor genes in Drosophila Iswar Hariharan and his coworkers have developed screens to identify tumor suppressor genes in the fly The strategy is to generate mutations in a single chromosome and then to stimulate recombination to generate clones that are homozygous for the mutation If the mutation is in a tumor suppressor gene these cells should proliferate at the expense of cells containing the wild type gene Several tools are used in these screens First the chromosome arm screened contains an FRT site near the centromere inserted into the chromosome using P element mediated transformation Chromosome arms are screened one at a time and the screens will only identify mutations that are distal to the FRT site These sites are where homologous recombination occurs and are necessary to generate clones that are homozygous for the mutation in the tumor suppressor gene Second FLP recombinase is expressed in a particular tissue using a specific promoter This drives recombination in that tissue alone and avoids the possible lethality associated with the loss gene function in other tissues In this case the eyeless promoter is used which drives expression of the recombinase only in the developing eye The eyeless FLP transgene is inserted into a chromosome using P element mediated transformation Finally a cell autonomous marker is used to identify the clones that are homozygous for the mutation These are usually a P element transposed wild type white gene In a white mutant background the P w is placed on the chromosome not containing the mutation In this way white clones that lack the P w transgene will be homozygous for the mutation The rationale for the screen is described in the Figure below The strain is homozygous mutant for white but also has a transgene containing a copy of the wild type white gene P w on the autosome that contains the FRT sites The eyFLP is on the X chromosome and that X is also marked 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 the autosome lacking P w contains a mutation in a tumor suppressor gene labeled m Before recombination the cells are heterozygous for m and hence normal remember that tumor suppressor genes are recessive at the level of the cell they must be homozygous to form a tumor and heterozygous for P w and hence are red In the developing eye FLP is expressed and this stimulates mitotic recombination between the FRT sites Some of these recombination events will lead to cells that are homozygous for the m tumor suppressor mutation These cells on the left will be white and continue to proliferate because of the mutation whereas the cells that aren t homozygous for the mutation will be red and won t divide in an uncontrolled fashion eyFLP w FRT m X w eyFLP w FRT m w FRT m FRT P w eyFLP w y w FRT P w FRT P w 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 or have 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
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