GEN551 Berg
Paper for 16 Oct 2000
Brunner, D., N. Oellers, J. Szabad, W. Biggs, S. L. Zipursky, and E. Hafen. 1994. A gain-of-function mutation in the Drosophila MAP kinase activates multiple receptor tyrosine kinase signaling pathways. Cell 76: 875-888. The authors screen for mutations that can suppress boss loss of function and identify an activated MAP kinase allele. This gain of function mutation produces phenotypes in other tissues besides the eye.
Questions for Thought
1. What is the logic behind this screen? Why are they looking for dominant mutations? What genes might be mutated by this approach?
What types of changes in a protein might lead to gain of function? How might you distinguish different types of dominant alleles using genetic means?
How does this approach compare to that of Stevenson et al. 1992, in the yeast STE pathway?
2. LOTS of people have screened for dominant wing vein mutations and no one has ever identified an activated MAPK allele. Why were Brunner et al. successful? What aspect of their screen helped them identify mutations in their gene?
Why did they recover only one mutation? Is 70,000 a lot of flies? Why didn't they recover alleles of Ras or sev?
What was the logic behind making double mutants with ora? with sev? That is, what were they testing?
3. What evidence suggests that Sevenmaker is allelic to rolled? Why was this result difficult to prove? How would you have proceeded if you had mapped Sem but no obvious candidate genes resided in the region? Assume you have deficiencies that cover the region and that the entire genome is sequenced.
4. Background: Constitutively active alleles of Ras and Raf have been identified in other systems (Rasv12, RaftorY9). Using promoters from the sevenless gene (sev = sev promoter + enhancers, sE = duplicated sev promoter + enhancers) to drive expression of these gain-of-function mutations specifically in the eye, the authors carry out epistasis experiments to determine the pathway relationships between these genes, rl, and sina. What is the pathway they derive? What is the evidence? How good is the evidence? Do you agree with their conclusion? Figure 4D, 4E: hypomorphic alleles of Raf can suppress rl
Sem! Explain.Imagine that rl mutations only partially suppress the activated Ras phenotypes in the eye. You realize that one explanation of this result is that you haven't completely removed rl function (e.g., sevRas
v12 / + ; rl / +). Since rl is lethal, you decide to make clones in the eye with rl null alleles. Given the pathway defined above and in the paper, what would you predict to see when you make clones of rl null in the eye?What would you predict if you made clones of rl
null in a sevRasv12 background?Imagine two different scenarios.
A. Clones homozygous for rlnull alleles lack R1-R7 cells, due to a requirement for MAPK in the development of all these cells (not just R7). What would you predict for clones of rl
null in a sevRasv12 background? How would you interpret results that showed all ommitidia lacked R1-R6, and only about half of the ommitidia lacked R7?B. Imagine that clones homozygous for rl
null alleles have no phenotype in the eye, and that rl loss of function alleles only weakly suppress activated Ras (e.g., sevRasv12 / + ; rlnull / + is very weakly suppressed). Nevertheless, rlSem can suppress boss loss of function, sev loss of function etc. etc. What could be going on here?5. Background: The torso pathway is very similar to the sevenless pathway, except it acts to establish the terminal regions of the embryo. The pathway is shown in brief outline below:
ligand -> torso RTK -> Ras -> Raf -> rl -> ??? -> induce expression of tailless (tll)
All of these products are loaded into the egg by the female, except tll, whose transcription is activated by the pathway. rl
Sem females produce eggs that look exactly like eggs from females expressing an activated Torso RTK. Why, then, do the authors go through all the trouble of making double mutants with various pathway components? Why not assume rl acts in this pathway, as it does in the eye? (Don't worry about the fact that only 11/68 embryos gave the tll phenotype in double mutants with rlSem; only 1/4 of the embryos should be tll/tll.)6. DER = Drosophila Epidermal growth factor Receptor, now renamed Egfr. The Egfr RTK also uses the Ras, Raf, MAPK pathway. The pathway is used in embryos, eyes, wings and eggs, that is, over and over. One big difference between Egfr and the Tor RTK or Sev RTK is that Egfr has four known ligands. The dominant Elp mutation affects binding of Egfr to only a subset of these ligands, thus explaining the dominant phenotype in eye and wing but not in oogenesis. Nevertheless, if the Ras, Raf, MAPK pathway is being used in all these tissues, wouldn't you expect rl
Sem mutations to affect all the pathways, not just ones from particular ligands? Why don't the authors see dorsalization of the embryo in rlSem mutants? What are the possible mechanisms of rlSem action and how could these factors contribute to differential phenotypes in different tissues?If the same Ras pathway is used in all these tissues, how do cells distinguish between Sev, Tor and Egfr signaling? How could you test these hypotheses?
7. What are the advantages of having gain of function alleles? Disadvantages? Is there a better way to accomplish the goal, obtaining the advantages without the disadvantages?