2. MutagenesisScreen:Visual: flower morphology, pigment color, Biochemical pathway mutant auxotrophy: his3- in yeast; arg- in Neurospora; trp1- in plantReporter gene expression (luc bioluminescent protein from firefly)Selection: trp pathway: 5-methylanthranilate ---> 5’methyltrp (toxic)ADH (alcohol dehydrogenase): Allyl alcohol---> acrolein aldehyde (toxic)Lethal mutations: (such as house keeping genes)-maintain as heterozygote-weak hypomorph-conditional such as temperature-sensitive mutants in T4 phaseFig. 7.20 aTryptophan biosynthetic pathwayChorismateAnthranilate (blue fluorescent under UV)trp4 (AS)trp1(PAT)Phosphoribosyl (PR)anthranilatePAI CDRPIndole-3-glycerol phosphateIndole(blue fluorescent under UV)L-tryptophan (amino acid)RD29A promoterfirefly luciferase (Luc) NPT-IIControlPC-LucRD29A-LucBefore stress After stressMutagenizes RD29A-Luc transgenic plants to look for mutants that stop fluorescence under cold stressFig. C.8Lecture 4: Mutant CharacterizationDominant and recessive mutationsComplementation testsEpistasis analysesRead: 594-596; 116-125Fig. 14.1-2; 3.23-26; 3.28Table 1Homework#1 will be posted today!Terminology about different mutationsa) Loss-of-function: Null mutation: complete absence of activityHypomorph: reduced activityb) gain-of-function Hypermorph: increased activityNeomorph: new function of genec) suppressors- compensate for other mutationsd) enhancer- enhances phenotype of a mutationFig. 7.251: null mutation; 2: hypomorphic mutationBoth 1 and 2 are recessiveFig. 7.25ThresholdFor gain offunction3/3 3/wt3: hypermorphic mutation3 is dominantThe underlying nature of recessive or dominant mutationsRecessivehypomorph: reduced level or a protein with a weak functionNull: complete loss of functionDominanthypermorph: increased level or more effective activityneomorphic: new functiondominant-negative: poisonous effecthaploid-insufficientEctopic expressionSemi-dominantFig. 7.26r0: null; r50: hypomorph; R+: wild type1/4 c1/c12/4 c1/+1/4 + /+RecessivecolorlessredreddominantcolorlesscolorlessredDetermine recessive or dominant nature of the mutationc1c1 (Colorless mutant) X +/+ (Red: WT)c1/+(red: recessive)(colorless: dominant)X(self)Determine allelism by complementation testsmale c1c1 c2c2 c3c3 c4c4 c5c5 c6c6female c1c1 c2c2 c3c3 c4c4 c5c5 c6c6Colorless red red colorless red redcolorless colorless red red redcolorless red red redcolorless red redcolorless colorlesscolorlessMaize kernel mutants:c1-c6: colorless, recessivewt: redThree complementation groups:1. c1, c42. c2, c33. c5, c6Pairwise crosses between homozygotes and examine F1 for phenotypeonly applicable for recessive mutations“Complementation group” equals “Gene”If two mutations failed to complement, they are alleles of the same gene they are allelic to each other they belong to the same complementation groupIf two mutations complements each other,they are alleles of different genesthey are not allelic to each otherthey belong to different complementation
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