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Lecture 32

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McGill University
Biology (Sci)
BIOL 300
Siegfried Hekimi

rd BIOL 300 November 23 2012 Lecture 32 Dr. Shock Many components of the RTK pathway were identified using genetics and the compound eye of Drosophila; this is because mutations in these genes are often viable, and they are very easily seen In all insects, the eye is compound, meaning it’s made up of 600 or so smaller eyes, called ommatidia (each of which function as a separate eye, integrated by the brain) Each ommatidium is made of 8 photoreceptors, labeled as R1  R8; the ligand called Boss (present on R8 cells, don’t need to remember the name) and the RTK Sevenless (called Sev, expressed on R7 cells) both mediate R7 development • While R1  R6 are long and span from the eye all the way inwards, R7 and R8 are stacked on top of one another; R7 being more superficial and R8 being deeper In the wild type, we can see that cross sections of ommatidia stained to be able to see the neurons; we can indeed see all 7 of the neurons (R8 is too deep) When we mutate Boss of Sev, you can see on the bottom photo that we only have 6 receptors, because R7 is not developed • This is easy to see in the eye as a whole because the eye looks messed up when R7 is missing At one point, some components of this pathway were identified (RTK, ligand, Ras and Raf), but we still needed to identify the downstream components which Raf acted on We want to create a screen to identify only the genes acted on by downstream pathways of Ras (much more specific than the random mutagenesis screen we did before) • First, we need to create a constitutively on Raf protein (i.e. through phosphomimetic mutations) and introduce it into the fly • We can see that this produces a rough-looking eye because there are more than 7 of these receptors per ommatidium (because there is too much proliferation) • You can then conduct random mutagenesis on flies, transfect them with the Raf , and then try and find enhancer and suppressor mutants • A suppressor mutation will rescue the wild-type phenotype, this diminishing the effects of the Raf mutation • In this case, the eye would not be as rough, so most of the ommaditia will have 7 photoreceptors • An enhancer would make the rough eye phenotype worse, i.e. going even further away from the wild-type phenotype 1 rd BIOL 300 November 23 2012 Lecture 32 Dr. Shock • In this case, the eye would be even more rough, so each ommatidium will have even more photoreceptors than the RafCA mutant Because the suppressor mutation is able to affect the Raf pathway even though Raf is always on, this means that suppressor mutations must affect components downstream of Raf in the pathway • If we were to mutagenize an upstream component of Raf, like, for example, Ras, it would have no effect because Raf would be turned on anyways, and does not need upstream components in order to be functional • Therefore, mutagenizing upstream components would show no rescue in the wild-type phenotype • Note that the rescued phenotype may not be identical to the wild-type, it may be somewhere between the Raf knockout and the wild-type as complete regulation is not restored An example of a suppressor mutant would be, for example, a heterozygous mutation of MAP or ERK, which are dominant negative • They would be suppressor mutations because they would reduce the function of Raf, thereby restoring the wild-type phenotype However, enhancer mutations would be either upstream or downstream of the pathways • If you produce an overactive Ras by mutagenesis in addition to the already present overactive Raf, this would make the rough phenotype even worse because you would have even more activation of this pathway • Similarly, an overactive downstream component of Raf will be able to carry out its function independent of Raf and other upstream components • An enhancer could even be in a separate pathway which produces the same phenotype We can see the result of such a screen here, where they screened chromosome 2 on Drosophila, and found 2 enhancer mutations and 3 suppressors 2 rd BIOL 300 November 23 2012 Lecture 32 Dr. Shock • Initially, when this screen was conducted, they did not know that the suppressor was ERK, the purpose of this experiment was to find such a component • All the letters underneath the given mutation are different alleles of the same gene; eventually, they mapped and cloned this gene • This is why the protein is called ERK (enhancer for Raf kinase), which was re-named MAPK when they found more pieces to the pathway On the left side of this diagram, we can see a very similar type of screen, but instead of starting with a constitutively on mutant (dominant), we start with a hypomorph, which, for this example, only has 50% of wild-type function • Even though you only end up with 50% gene product for any gene in this pathway in a homozygous hypomorphic mutant, the pathway is still strong enough to produce a functional R7 neuron The idea behind the setup of this experiment is that any mutation in the same pathway detrimental to the pathway, you won’t have enough components to produce R7 cells, and you end up with the rough phenotype • This means the mutation causing this would be a component necessary for the activation of the sev pathway, but you don’t know if the component you identified is upstream or downstream of the initial mutation gene (i.e. sev) • This is because, unlike the previous screen, this mutant is not dominant • For example, if you remove 50% of a ligand, this would be a downstream mutation that destroys functions, and similarly, if you make a Ras null mutant, which would be a downstream mutation which destroys function; both of these pathways will cause the rough phenotype Now, we need to order these components when we find them from screens like this; to do this, we need to carry out genetic assays (like the previous screen) • By using dominant mutations (like constitutively active), we could find out which order the components we found in a screen like the one in this diagram occur • For example, on the right, if we produce a Ras constitutively active and homozygous hypomorhic Sev double mutant, we see that Ras is able to rescue mutation, meaning the Ras is downstream of Sevenless • If Ras were upstream, it wouldn’t matter if it was active or not, it wouldn’t be able to rescue the mutation 3 rd BIOL 300 November 23 2012 Lecture 32
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