1Lecture 3 Mutagens and MutagenesisA. Physical and Chemical mutagensB. TransposonsC. T-DNARead: 460-464 Figs: 7.12; 7.14; 7.20; 5.21; C8; 1.11; 13.20; 13.22; 13.23; 13.24;13.25A. ScreenB. SelectionC. Lethal mutations 1. Mutagens2. Mutagenesis2Mutagen treatment greatly increases the mutation rateExposure to X-ray, UV lightChemical treatment: base analogs 5’-bromouracil (=T or rarely C) hydroxylating agent (add OH-group to C) alkylating agent such as E!MS (ethylmethane sulfonate) deaminating agent such as nitrous acid intercalating agent such as Acridine OrangeTransposons that insert into a gene and disrupt the normal reading frame 1. Mutagens3Fig. 7.12a1Chemical Mutagens4Fig. 7.12a25Fig. 7.12b156Fig. 7.12b267Fig. 7.12c18Fig. 7.12c29Transposon (transposable element) as a mutagen(1) RetrotransposonsCopia DrosophilaTy1 YeastLINEs HumanSINEs (Alu) Human(2) TransposonsAc/Ds MaizeP-element DrosophilaTransposon: DNA segment that can move from one position to another10Fig. 13.231011Fig. 13.24aP-element in Drosophila12Fig. 13.24b1213Fig. 13.25Transposons can insert into different regions of a gene14Ac: activator, autonomous, 4.6 kb long, enocdes a3.5 kb transcript of transposaseDs: dissociation, non autonomous-both Ac and Ds have 11 bp inverted repeats at the ends,which function in the transposase recognition-an 8 bp direct repeat generated from the host genome--footprintTransposaseAc/Ds are Maize transposonsAc/Ds can function in other plants15Fig. 13.22Ac/Ds in Corn Kernels16Fig. 13.20Barbara McClintock171. T-DNA (transfer DNA) from Agrobacterium tumefaciens Agrobacterium causes crown-gall diseases in plants. This tumor-inducing (Ti) ability is linked to the Ti-plasmid. During the infection, a segment of the Ti-plasmid, the T-DNA, is transferred into the plant cell and integrated into the plant genome.NPT-IILBRBTransposon mutagenesis facilitates gene cloningT-DNA for Plant transposon-tagging18Ti-plasmidT-DNAvirAgrobacteria Plant Cellhost chromosomenucleusvir: vir region (vir = virulence)Ti: tumor-inducing plasmid Principles of gene transfer from Agrobacteria into plant cells192. 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 phase (p220-221)20WT Arabidopsis flowerap2-2 floral mutant21sex-determination mutant (tsl2 =tassel seed 2)22Fig. 1.112223Fig. 7.142324Fig. 5.212425Fig. 7.20 a26Tryptophan 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)27RD29A promoterfirefly luciferase (Luc) NPT-IIControlPC-LucRD29A-LucBefore stress After stressMutagenizes RD29A-Luc transgenic plants to look for mutants that stop fluorescence under cold stress28Fig.
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