Drosophila FLP/FRT Screens and a model for cancer genetics ...

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

etinoblastoma 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 

w 

FRT m 



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 so there 

are more white cells and the eye is deformed because too many cells are 

made. 

X 

eyFLP w y 

w 


FRT P[w + ]

The last thing that we need to consider now is how the mutant 

screen was carried out, and that is illustrated in the two boxes below. First, 

males hemizygous for white and homozygous for FRT sites on an 

autosome that will be screened are mutagenized with EMS. These males 

are then crossed with females homozygous for white and heterozygous for 

a balancer for the autosome being screened. (Remember that a balancer 

chromosome using has multiple inversions to inhibit the formation of 

viable progeny with a recombinant chromosome, a recessive mutation, 

often a lethal and a dominant mutation—balancers are described on 

pp454-455 and pp817-818.) All of the progeny from this cross will have 

white eyes. Those of interest will contain a mutagenized chromosome 

containing the FRT site and the balancer chromosome, which can de 

identified by the presence of the phenotype caused by the dominant 

mutation on the balancer. 

EMS 

w 

FRT 


or 

w 

w 

mm 


What needs to be done now is to generate animals with the mutagenized 

chromosome, that are also homozygous for the FRT sites and express FLP 

in the eye. To do this, the F1 females are crossed with males that have the 

eyFLP trangene and that are homozygous for the FRP sites and P[w + ]. The 

female progeny from this cross that lack the dominant phenotype caused 

bythe balancer chromosome have eyFLP transgene, are homozygous for 

the FRT sites and have the P[w+] on the nonmutagenized chromosome. If 

the mutagenized chromosome contains a mutation in a tumor suppressor 

gene, then these females should have expansion of white tissue in the eye. 

X 

w 

w 

Balancer 

Balancer 

ff 

Mutagenized chromosome

w 

w 

F2 progeny 

w/o balancer 


Balancer 

Mutagenized chromosome 


w 

Using this approach, Hariharan and his colleagues identified 23 

genes involved in the regulation of proliferation and apoptosis in the eye. 

These genes have homologs in humans, and tumors were screened to see 

of any of them were mutant for the homologs and some were suggesting 

that these genes are also tumor suppressors in humans. 

Problem set G 

1. Integrins are alpha/beta heterodimeric receptors that bind to molecules 

in the extracellular matrix. A primary role of integrins is in adhesion, 

ensuring that cells attach to the matrix, and cell biologists have identified 

several proteins that link the intracellular tail of the beta integrin to the 

actin cytoskeletin. During the development of the Drosophila wing, cells 

on the dorsal and ventral surfaces adhere to the matrix. Mutations in the 

beta integrin gene lethal(1)myospheroid lead to recessive lethality, but 

hypomorphic mutations lead to wing blisters where the portions of the 

dorsal and ventral surfaces separate. From what you know about 

integrins, propose a model for how integrins function in wing 

development. 

You screen for mutations that lead to wing blisters using FLP/FRT 

induced recombination. Describe which genetic tools you will need to 

conduct the screen. 

f 

X 

eyFLP w y 





m 

Mutagenized chromosome

The frequency wing blistering in the hypomorphic mutant of 

lethal(1)myospheroid is 5-10%. Although the mutations you isolated in your 

FLP/FRT screen are recessive, they are dominant enhancers of the 

hypomorpic lethal(1)myospheroid mutant. Why do you think that these 

mutations enhance the severity of the wing blistering phenotype? Do you 

think that they are loss-of-function mutations, and if so describe a genetic 

test that shows the enhancement is caused by a loss of function. 

2. In the Drosophila eye, the R7 photoreceptor cell is specified by signaling 

from R8. You isolate two mutations on chromosome 2 that are missing R7. 

You generate animals that mutant for white gene on the X chromosome. 

They are heterozygous for one chromosome that has one of the mutations 

and a homologous chromosome that has a wild-type white transgene 

integrated in a position that maps near where the mutation lies. These 

animals are irradiated during development and animals with eyes that 

have white patches are analyzed for the presence of mosaic ommatidia. 

Below are the results of mosaic ommatidia that have the R7 cell. Each 

number represents the number of cells that are mutant or wild type for 

white (w). In the 59 ommatidia analyzed for mutant 1, for example, 40 of 

the R1 cells were wild type for white and 19 were mutant. 

Mutant 1 

Cell wild type for w mutant for w 

R1 40 19 

R2 33 26 

R3 32 27 

R4 36 23 

R5 36 23 

R6 47 12 

R7 59 0 

R8 31 28 

Mutant 2 

Cell wild type for w mutant for w 

R1 42 18 

R2 27 33 

R3 32 27 

R4 35 25 

R5 40 20 

R6 39 21 

R7 35 25 

R8 60 0

a) Why are the animals irradiated? 

b) From what you know about R7 specification propose in which cells the 

genes defined by these two mutations act and explain your reasoning. (10 

points) 

3. Ras can exist in two states. A GTP bound form of Ras is active, and a 

GDP bound form is inactive. GEFs stimulate the exchange GDP for GTP 

and thus promote an active Ras. GAPs stimulate the GTPase activity of 

Ras and thus promote the formation of an inactive Ras. Activation of the 

Sev receptor by Boss stimulates the formation of GTP Ras. 

In a screen for dominant suppressors of the R7 phenotype of the sev ts 

strain of flies raised at the nonpermissive or restrictive temperature (the 

temperature at which the flies are lacking R7), investigators identified 

several mutations that resulted in haploinsufficiency of the mutated 

gene(s). Could these mutations be in the GEF or the GAP?

Drosophila FLP/FRT Screens and a model for cancer genetics ...

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