1Genetic pathway analysisReading: lecture notesExtragenic suppressorsInformational suppressors: allelespecific, gene nonspecificBypass suppressors (parallel pathways):allele nonspecific, gene specificBypass suppressors (same pathway):allele nonspecific, gene specificInteraction suppressors: allele specific,gene specificBypass suppressorsallele nonspecific, gene specificSuppressors can be mutations in a:1. Parallel pathway2. Same pathwayBypass suppressor in parallel pathwayAAAGain-of-function mutations in CYC1 arebypass suppressors of loss-of-functionCYC7 mutants.CYC7 cytochrome cCYC1 cytochrome cCYC7CYC1 cytochrome cXcyc7 mutantCYC7CYC1 cytochrome cXCYC1cyc1 mutantTy1Insertion of the transposable elementTy1 in the CYC1 gene causes increasedexpression.2Bypass suppressors can be in distinct process.MaltosepermeaseLactosepermeasemaltose lactoseMaltosepermease-Lactosepermeasemaltose lactoseMaltosepermease -LactosepermeasemutantmaltoselactoseXmaltose lactoseXmaltosemaltoseBypass suppressors that are in thesame pathway are referred to asepistatic suppressors.Two types of regulation:PositiveNegativeBypass suppression in negative regulatory step.We can use epistatic suppressors to ordergenes. e.g., ced apoptosis genes in C.elegansCED-9CED-3CED-4ced-9(lf) animals die because of widespreadapoptosisced-3(lf) or ced-4(lf): all cells than normally diesurviveced-9; ced-3 or ced-9 ced-4 double mutantslive, and all cells that normally die survive.Cells that normally surviveCED-9ONCED-3CED-4OFFCED-9OFFCED-3CED-4ONCells that normally dieBypass suppression in positive regulatory step.The suppressor must be a gain-of-function mutation.3Gain-of-function mutations can be usedto order genes in positive regulatorypathway.Since loss-of-function mutations in ced-3and ced-4 result in a loss of apoptosis,canʼt order genes with these mutations.But can artificially create gain-of-functionced-3 or ced-4 by overexpressingproteins in specific cells.MEC-7 is a ß tubulin expressed in subset ofmechanosensory neurons (e.g., ALM neurons). Usemec-7 promoter to express ced cDNAs.mec-7 promoter mec-7 codingmec-7 promoterced-3 codingmec-7 promoterced-4 codingHigh levels of either CED-3 or CED-4 causes theALM neurons to die.Can now ask whether CED-3 activates CED-4or CED-4 activates CED-3.CED-3 CED-4ORCED-4 CED-3The ALMs die when ced-3 is overexpressedfrom the mec-4 promoter in a ced-4background.ced-4 mutantced-4 ced-3mec-7::ced-3apoptosisXced-4 ced-3mec-7::ced-3; ced-4 mutantapoptosisced-4 ced-3XsurvivalThe ALMs survive when ced-4 is overexpressedfrom the mec-4 promoter in a ced-3background.ced-3 mutantced-4 ced-3mec-7::ced-4apoptosisXced-4 ced-3mec-7::ced-4; ced-3 mutantced-4 ced-3XsurvivalsurvivalModel from epistasisCells that normally surviveCED-9ONCED-4OFFCED-9OFFCED-4ONCells that normally dieCED-3OFFCED-3ON4CED-9 proteintethers CED-4 tothe mitochondrialmembrane!When theapoptoticpathway isactivated CED-4protein moves tothe nuclearmembrane.Check out other examples in lecture notesNote that all of the bypass suppressors, whether inparallel or the same pathway, are predicted to beallele nonspecific.e.g. ced-3 will suppress any loss-of-function alleleof ced-9.Note also that all of the bypass suppressors,whether in parallel or the same pathway, arepredicted to be gene specific.e.g., ced-3 will suppress ced-9 alleles, but notmutant alleles of other genes; for example, thoseinvolved in muscle function.Genetic analysis can be used to order genesX:A xol -1 sdcs her-1 tra-2 fems tra-1 egl-1 ced-9 ced-4 ced-3 XX xol-1 sdcs her-1 tra-2 fems tra-1 egl-1 ced-9 ced-4 ced-3 HSNlives XO xol-1 sdcs her-1 tra-2 fems tra-1 egl-1 ced-9 ced-4 ced-3 HSNdies Genetic pathwayIn malesIn hermaphroditessex determination pathwaycell death pathwayInteraction suppressorsAllele specific, gene specificact1ts/act1tsmutagenGrow at nonpermissive temperatureIsolated sac mutantsact1ts/act1ts; sac6/+ growProtein A Protein Bwild-type phenotypemutant phenotypemutant phenotypewild-type phenotype(suppressed)The suppression of a mutant allele and itssuppressor is reciprocalThe ModelProtein A* Protein BProtein A* Protein B* Protein A Protein B*5Act1p Sac6p Act1*p Sac6pAct1*p Sac6*p Act1p Sac6*pwild type phenotypemutant phenotypemutant phenotypewild-type phenotype(suppressed)The suppression of the act1 and sac-6alleles is reciprocal: a real exampleExtragenic suppressorsInformational suppressors: allelespecific, gene nonspecificBypass suppressors (parallel pathways):allele nonspecific, gene specificBypass suppressors (same pathway):allele nonspecific, gene specificInteraction suppressors: allele specific,gene
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