mutant screening 234.pdf

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BIOL 233
Don Moerman

1 Mutant ScreensObjectivesTo learn how to perform mutant screens and characterize newlyfound mutants To understand the rationale behind complementation testsIntroductionWhenever a biologist is interested in studying some particular process and heshe wants to find the underlying genes involved in the process a mutant screen is a powerful way to find the genesScientists have devised all different types of mutant screens the simplest are ones in which they look for some easily observable phenotypeWhen the first geneticist working with Caenorhabitus elegans wanted to find mutations in genes involved in the development of the worm nervous system he first thought about what type of mutants he might be likely to see For example if nerves do not properly innervate muscles then the movement of worms could be affectedWith this in mind he looked for mutants that did not move normally what you know as the Unc phenotype uncoordinatedPeople interested in studying mutants which affect the nervous system have to do secondary screens to get rid of mutants that are uncoordinated but do not necessarily affect the nervous systemFor example they would discard any mutants that affect the muscles rather than the nerves because if the muscles dont develop properly you could also see an uncoordinated phenotypeThey could dissect open the worms and look the muscle structure to see if it is normal or not Also they might have a way of looking at the projection patterns of individual nerves with a fluorescent antibody or green fluorescent protein or some other visible marker that is expressed in some individual nerve cells Mutants that changed this nerve projection pattern would be kept see example in Fig 1 below and others discarded Fig 1 Dissected central nervous systems from Drosophila melanogaster labeled with antibodies recognizing a specific set of neurons A is wildtype with the stained axon projections clustering together in a club shape B is a mutant where a number of axons fail to form thistight clubshaped pattern Figure courtesy of J KlenzIf you wanted to screen for mutations that affect petal development in the flowers of Arabidopsis thaliana you would look for mutants lacking petals or with different sizes or shapes of petals However remember you want to make your screen as specific as possible so you would probably not be interested in mutants that change the shape or size of many different organs since then your mutation is not specific to petalsWhen you examine your mutants more closely you might find some that have very small petals but perhaps all the parts of the flower are small and they also have small leavesthis is not a specific mutation for petals so you would probably discard itThere are however many other ways to do mutant screens besides looking for a visual phenotypeFor example one could perform a chemical assay eg sensitivity to nicotine or inability to make the amino acid Leucine etc or one could use PCR primers which amplify a gene of interest and then
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