Problem set I answers1. Feeding C. elegans E. coli that express double stranded RNA from the unc-54 gene,which encodes the major form of muscle specific heavy chain myosin, causes the animalsto be paralyzed because they lack this myosin. You mutagenize wild-type worms withEMS and screen the F2 progeny for worms that are no longer paralyzed when fed thesebacteria. The mutations that cause this resistance are recessive and are also resistant toRNAi for other genes. From what you know about RNAi, what could be the normalfunctions of the genes that are mutated?Since they are involved in implementing RNAi, they could be components involvedin dicer or RISC complex function.2. RNAi in C. elegans can effectively silence gene function though a posttranscriptionalprocess. RNAi can be induced globally in worms simply by feeding the animals bacteriathat express double-stranded RNA representing the gene being silenced. While howRNAi induces mRNA degradation is understood, it is less clear how the effects of RNAispread from cell to cell; for example, from the gut to the remaining cells of the body. Toidentify genes involved in the spreading process, investigators screened for mutantsdefective in RNAi spreading. They mutagenized animals that expressed three transgenes.One transgene expresses GFP in the pharynx, the feeding organ, and the secondexpressed GFP in the body wall muscles, which are used for locomotion. The thirdtransgene expressed double-stranded RNA to GFP in the pharynx. In the absence of thisthird transgene, GFP is expressed in both the pharynx and the body wall muscles; in itspresence, all GFP expression is lost. The investigators then screened for recessivemutations in genes required for RNAi.Describe how the screen was done. Include which generation that was screened, whetherthe animals were crossed or selfed, and the phenotype the investigators were looking for.Describe the difference between the phenotypes of mutants generally defective in RNAi,(for example, in the genes encoding the Risc components) and of mutants defective inspreading of the RNAi effect.The screen was conducted by mutagenizing the strain that carries the threetransgenes and by looking in the F2 (for zygotic genes) or the F3 (for maternal effectgenes) for animals that express GFP. If GFP is expressed in the pharynx but not inthe muscles the mutants would be defective in spreading. Mutants generallydefective in RNAi would express GFP in both tissues.3. C. elegans neurons are particularly insensitive to the effects of RNA interference. Forexample, if you have a strain that expresses Green Fluorescence Protein (GFP) inneurons, feeding bacteria that express double stranded GFP RNA doesn’t have any effecton the levels of GFP. By contrast, feeding these bacteria to animals that normally expressGFP in muscles eliminates this GFP fluorescence. You find this irritating because youwant to carry out an RNAi screen for genes involved in the formation of chemicalsynapses between neurons. You decide that one approach to this problem would be toscreen for mutants with neurons that are sensitive to RNAi.a. Using EMS and the transgenic strain that expresses GFP in neurons, how would youscreen for these mutants?You would screen for mutants that no longer express GFP in the neurons when fedbacteria that express GFP dsRNA.b. You isolate a mutant that has the expected phenotype, and it defines a single gene onchromosome II. You predict that this mutant now allows the canonical RNAi pathway tobe effective in neurons. Describe a simple genetic test of this prediction.Make the double mutant that contains the new mutation on II and a mutation thatdisrupts RNAi. When fed bacteria that express GFP dsRNA, neuronal GFPexpression should still be
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