We will meet on Sunday (3/13) @ 7pm in SFH 250 to go over our answers to these questions, as well as come up with questions for the SOS paper (and potentially the map based cloning paper).
[Remember Daylight Savings on Sunday!]
Assignments:
Claire - #1
Natalie - #2
Dan - #3
Sam - #4
Kat - #5
Vilma - #6
Questions:
1) You design a mutagenesis screen for Drosophila sensitive to UV light, but are unable to decipher any other wavelength spectrum. You successfully find a single animal in the F1 generation that is blind to everything except UV and call it sunburn (SBN) with allele uv1. Morphologically, the ommatidia seem fine, but a reporter gene driven by promoter of target of sevenless signaling cascade is activated in R1-R7. You map this mutation close to a region that is 30kb and notice a point mutation in a putative gene that is largely uncharacterized. This gene is closely linked to the pigment marker P(GFP+). Lay out how you would determine that this is the gene of interest.
You conduct another mutagenesis screen of sbn(uv1) and find F2 flies that can see in both UV and normal light conditions. Is the suppressor intragenic or extragenic? What are potential characteristics of both types of suppressors recovered in this screen?
After part 1, you now know that the null allele sbn(spf45) has phenotype that it does not make R7 cells, you determine that sbn(spf45);Sos(JC2) has R7 cells in a third of the ommatidia. What do you now know?
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2) Previously in your lab, you screened C. elegans after an EMS mutagenesis and found a mutation that causes a motor phenotype in which the worms crawl in circles, eventually boring their way through the plate media. You call this phenotype “dizzy”. You want to further explore this mutation and find suppressors of the dizzy phenotype.
a) How would you perform a screen for suppressors of dizzy?�
You find 2 potential suppressor mutations (mutation E & mutation F) of dizzy.
b) How could you determine whether your suppressor mutations are dominant or recessive? (include crosses)
c) How could you determine whether your suppressors are intragenic or extragenic (include crosses and genotype and phenotype ratios)
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f) Assume that ABCD are all dominant mutations (and are non lethal). How would create a quadruple mutant?
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4) Normally: the presence of ligand Y activates a pathway resulting in production of amino acid X. You have isolated a missense mutation in a gene that results in constitutive activation of a pathway encoding amino acid X, without needing activation by Y. You also have a null version of the protein that results in no production of amino acid X. You conduct a secondary screen and find a dominant suppressor of the missense phenotype.
a. How could you differentiate between a bypass suppressor and an allele-specific suppressor?
b. If it’s a bypass suppressor, how could you establish if the suppressor you have is involved in that gene pathway? (and is not a gain-of-function neomorph)
c. What if you can’t delete the gene of the suppressor (null mutants are recessive lethal)?
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5)
You have found a new genetic mutant in yeast called repel (RP1) in which alpha type yeast secrete pheromones that scare away opposite sex types. RP1 mutants are 35% less likely to successfully mate compared to wild-type. Double mutant yeast RP1/EV1 (repel 1 and evade 1) results in 60% decrease in mating compared to wild-type. Triple mutant RP1/EV1/GA1 (repel 1, evade1, go away 1) results in 95% decrease in mating and are considered sterile. This mutations seems to be an ON switch with a gain of new function, which is the scaring away of opposite sex types instead of attracting or having pheromones shut off. Suppose the mutation screen for this phenotype has been saturated. How would you conduct a modifier screen to isolate suppressors or enhancers of this ON switch? How would you perform a genetic selection for this phenotype? How would you determine whether these suppressors are involved in the same pathway?
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6) A set of discussion / broad questions
a) In the ethylene response paper, the suppressor screen was able to identify loss-of-function alleles in genes with redundant functions. Could this same screen have worked if the original dominant mutation was in an essential gene without any redundant function
b) If you have a new genetic mutant, who has a saturated mutant screen, how would you go about conducting a suppressor/enhancer screen
c) Are suppressor screens or double/triple/quadruple mutants only useful in situations where there are redundant mutants?
